Use of valeric acid for delaying senescence or assessing senescence state

Abstract

The invention relates to the field of biological pharmacy. Specifically, the invention relates to application of valeric acid or a composition containing valeric acid as a medicine for delaying or reversing senescence and preventing and / or treating senescence-related diseases. In addition, the invention also relates to valeric acid or a valeric acid-containing marker combination for evaluating the degree of aging of a subject and / or the risk of the subject to suffer from an aging-related disease.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceuticals. Specifically, the present invention relates to the use of valeric acid or a composition containing valeric acid as a drug for delaying or reversing aging, preventing and / or treating aging-related diseases. In addition, the present invention also relates to valeric acid or a marker combination containing valeric acid for assessing the aging degree of a subject and / or the risk of a subject suffering from an aging-related disease. Background Art

[0002] Population aging and senescence issues pose huge challenges to human health and medical security worldwide. How to analyze aging and further intervene in aging has become an important direction to tackle in the current field of aging research.

[0003] At present, the screening of small molecule drugs for aging intervention is mainly focused on the screening of existing small molecule drug libraries (such as FDA drug libraries, natural product libraries, etc.). The main process is: 1) Obtaining small molecule drug libraries: purchasing or synthesizing candidate compound libraries for screening through existing drug databases (the range of candidate compounds can be predicted and narrowed by bioinformatics); 2) Drug screening: using candidate compound libraries to intervene in cell aging models (natural passage aging or construction of premature aging cell lines). By setting multiple concentration gradients, treatment time and other conditions for all drugs in the compound library, candidate small molecules (multiple) and optimal treatment conditions that have a delaying effect on cell aging are obtained; 3) Verification of drug intervention effects: using the obtained candidate small molecules to intervene in the cell aging model under appropriate conditions, and testing multiple aging-related indicators (such as aging-related secretory phenotypes, corresponding cell functions, etc.), verifying the effects of the drugs, and obtaining effective compounds and corresponding conditions that can intervene in cell aging.

[0004] However, current research lacks aging intervention drug screening based on endogenous aging-related metabolite data of the Chinese population, resulting in the following disadvantages: 1) Large workload and low systematicity / efficiency in drug screening: Existing large-scale drug libraries contain a huge number of small molecule species. Screening all drugs requires a lot of material, human and time resources, and there is a high possibility of omissions and lack of systematicity; 2) It is difficult to verify the safety of exogenous drugs: The pharmacokinetics and toxicology verification of exogenous drugs are complex, requiring a longer period and more studies to prove their safety and conditions of use (dose, time, drug interactions, etc.); 3) Lack of analysis of aging-related metabolite changes based on the healthy Chinese population: The natural aging process is affected by multiple factors such as race, environment, and lifestyle habits. Analysis data based on foreign populations or disease populations are difficult to obtain aging-related metabolite change patterns that are consistent with the healthy Chinese population. Summary of the invention

[0005] Based on a large number of analyses of changes in endogenous metabolites associated with aging in healthy Chinese people, the inventors of this application have discovered the law of endogenous metabolites that decay with aging, and based on the law and a large number of experimental screenings, human endogenous metabolites with the function of intervening in cell aging have been obtained. Based on the above research, a method or use of valeric acid (or a composition comprising valeric acid and myristic acid and / or piperine) for delaying or reversing aging, preventing and / or treating aging-related diseases is provided. In addition, the application also provides a method or use of valeric acid (or a marker combination comprising valeric acid and myristic acid and / or piperine) for assessing the degree of aging of a subject and / or the risk of a subject suffering from an aging-related disease.

[0006] Therefore, in one aspect, the present application provides the use of valeric acid in the preparation of a medicament, wherein the medicament is used to delay or reverse aging in a subject, and / or, prevent and / or treat aging-related diseases.

[0007] In certain embodiments, the medicament further comprises myristic acid and / or piperine.

[0008] In certain embodiments, there is a synergistic effect of delaying or reversing aging between valeric acid and myristic acid and / or piperine.

[0009] In certain embodiments, there is a synergistic effect between valeric acid and myristic acid and / or piperine in preventing and / or treating aging-related diseases.

[0010] In certain embodiments, the medicament is for slowing or reversing cellular senescence in a subject.

[0011] In certain embodiments, the medicament is used to delay or reverse one or more of the following states of cellular senescence in a subject:

[0012] (1) Reduced cell proliferation;

[0013] (2) weakened cell migration ability;

[0014] (3) The ability of cells to form tubes is weakened;

[0015] (4) upregulation of cellular β-galactosidase activity;

[0016] (5) The expression of genes related to cell senescence is upregulated.

[0017] In certain embodiments, the cell is a vascular endothelial cell.

[0018] In certain embodiments, the cell is a cardiovascular cell.

[0019] In certain embodiments, the cell is an arterial endothelial cell.

[0020] In certain embodiments, the aging-related genes include but are not limited to p21, IGF1, and IL-1β.

[0021] In certain embodiments, the subject is a mammal.

[0022] In certain embodiments, the subject is human, preferably Asian.

[0023] In certain embodiments, the subject is Southeast Asian or East Asian (eg, Chinese, Japanese, Korean, Mongolian).

[0024] In certain embodiments, the valeric acid is n-valeric acid.

[0025] In another aspect, the present application provides a method for delaying or reversing aging in a subject, and / or preventing and / or treating aging-related diseases, the method comprising administering an effective amount of valeric acid to a subject in need thereof.

[0026] In certain embodiments, the method further comprises administering (eg, sequentially or simultaneously) myristic acid and / or piperine to the subject.

[0027] In certain embodiments, the methods are used to slow or reverse cellular senescence in a subject.

[0028] In certain embodiments, the methods are used to slow or reverse one or more of the following states of cellular senescence in a subject:

[0029] (1) Reduced cell proliferation;

[0030] (2) weakened cell migration ability;

[0031] (3) The ability of cells to form tubes is weakened;

[0032] (4) upregulation of cellular β-galactosidase activity;

[0033] (5) The expression of genes related to cell senescence is upregulated.

[0034] In certain embodiments, the cell is a vascular endothelial cell.

[0035] In certain embodiments, the cell is a cardiovascular cell.

[0036] In certain embodiments, the cell is an arterial endothelial cell.

[0037] In certain embodiments, the aging-related genes include but are not limited to p21, IGF1, and IL-1β.

[0038] In certain embodiments, the subject is a mammal.

[0039] In certain embodiments, the subject is human, preferably Asian.

[0040] In certain embodiments, the subject is Southeast Asian or East Asian (eg, Chinese, Japanese, Korean, Mongolian).

[0041] In certain embodiments, the valeric acid is n-valeric acid.

[0042] On the other hand, the present application provides a method for delaying or reversing cell senescence, comprising the step of contacting valeric acid with the cells.

[0043] In some embodiments, the method comprises contacting the cells with 5-50 ng / ml (e.g., 5-40 ng / ml, 5-30 ng / ml, 5-20 ng / ml, 5-15 ng / ml, 5-10 ng / ml, 10-50 ng / ml, 10-40 ng / ml, 10-30 ng / ml, 10-20 ng / ml, 10-15 ng / ml, 10 ng / ml) of valeric acid.

[0044] In some embodiments, the method comprises contacting the cells with 5-50 ng / ml (e.g., 5-40 ng / ml, 5-30 ng / ml, 5-20 ng / ml, 5-15 ng / ml, 5-10 ng / ml, 10-50 ng / ml, 10-40 ng / ml, 10-30 ng / ml, 10-20 ng / ml, 10-15 ng / ml, 10 ng / ml) of valeric acid for 3-7 days (e.g., 3-6 days, 3-5 days, 4-7 days, 4-6 days, 5-7 days, 5-6 days, 5 days).

[0045] In certain embodiments, the method further comprises contacting the cell with myristic acid and / or piperine. In certain embodiments, the method comprises contacting the cell with valeric acid and myristic acid and / or piperine simultaneously. In certain embodiments, the method comprises contacting the cell with valeric acid and myristic acid and / or piperine sequentially in any order.

[0046] In certain embodiments, the method comprises contacting the cell with 0.01-10 μM (e.g., 0.01-8 μM, 0.01-5 μM, 0.05-10 μM, 0.05-8 μM, 0.05-5 μM) of myristic acid and / or 0.1-20 μM (e.g., 0.5-20 μM, 1-20 μM) of piperine.

[0047] In certain embodiments, the method comprises contacting the cells with 0.01-10 μM (e.g., 0.01-8 μM, 0.01-5 μM, 0.05-10 μM, 0.05-8 μM, 0.05-5 μM) of myristic acid for 3-7 days (e.g., 3-6 days, 3-5 days, 4-7 days, 4-6 days, 5-7 days, 5-6 days, 5 days).

[0048] In certain embodiments, the method comprises contacting the cells with 0.1-20 μM (e.g., 0.5-20 μM, 1-20 μM) piperine for 3-7 days (e.g., 3-6 days, 3-5 days, 4-7 days, 4-6 days, 5-7 days, 5-6 days, 5 days).

[0049] In some embodiments, the method comprises contacting the cells with 5-50 ng / ml (e.g., 5-40 ng / ml, 5-30 ng / ml, 5-20 ng / ml, 5-15 ng / ml, 5-10 ng / ml, 10-50 ng / ml, 10-40 ng / ml, 10-30 ng / ml, 10-20 ng / ml, 10-15 ng / ml, 10 ng / ml) of valeric acid and 0.01-10 μM (e.g., 0.01-8 μM, 0.01-5 μM, 0.05-10 μM, 0.05-8 μM, 0.05-5 μM) of myristic acid for 3-7 days (e.g., 3-6 days, 3-5 days, 4-7 days, 4-6 days, 5-7 days, 5-6 days, 5 days).

[0050] In some embodiments, the method comprises contacting the cells with 5-50 ng / ml (e.g., 5-40 ng / ml, 5-30 ng / ml, 5-20 ng / ml, 5-15 ng / ml, 5-10 ng / ml, 10-50 ng / ml, 10-40 ng / ml, 10-30 ng / ml, 10-20 ng / ml, 10-15 ng / ml, 10 ng / ml) of valeric acid and 0.1-20 μM (e.g., 0.5-20 μM, 1-20 μM) of piperine for 3-7 days (e.g., 3-6 days, 3-5 days, 4-7 days, 4-6 days, 5-7 days, 5-6 days, 5 days).

[0051] In certain embodiments, the method comprises treating the cells with 5-50 ng / ml (e.g., 5-40 ng / ml, 5-30 ng / ml, 5-20 ng / ml, 5-15 ng / ml, 5-10 ng / ml, 10-50 ng / ml, 10-40 ng / ml, 10-30 ng / ml, 10-20 ng / ml, 10-15 ng / ml, 10 ng / ml ) valeric acid, 0.01-10 μM (e.g., 0.01-8 μM, 0.01-5 μM, 0.05-10 μM, 0.05-8 μM, 0.05-5 μM) myristic acid and 0.1-20 μM (e.g., 0.5-20 μM, 1-20 μM) piperine are contacted and cultured for 3-7 days (e.g., 3-6 days, 3-5 days, 4-7 days, 4-6 days, 5-7 days, 5-6 days, 5 days).

[0052] In certain embodiments, the methods are used to delay or reverse one or more of the following states of cellular senescence:

[0053] (1) Reduced cell proliferation;

[0054] (2) weakened cell migration ability;

[0055] (3) The ability of cells to form tubes is weakened;

[0056] (4) upregulation of cellular β-galactosidase activity;

[0057] (5) The expression of genes related to cell senescence is upregulated.

[0058] In certain embodiments, the cell is a vascular endothelial cell.

[0059] In certain embodiments, the cell is a cardiovascular cell.

[0060] In certain embodiments, the cell is an arterial endothelial cell.

[0061] In certain embodiments, the aging-related genes include but are not limited to p21, IGF1, and IL-1β.

[0062] In certain embodiments, the methods are used for therapeutic purposes.

[0063] In certain embodiments, the methods are used for non-therapeutic purposes.

[0064] In certain embodiments, the methods are performed in vitro.

[0065] In certain embodiments, the valeric acid is n-valeric acid.

[0066] In another aspect, the present application provides the use of a reagent capable of detecting valeric acid levels in the preparation of a detection reagent for assessing the degree of aging of a subject and / or the risk of the subject suffering from an aging-related disease.

[0067] In certain embodiments, the detection reagents further comprise a reagent capable of detecting myristic acid levels and / or a reagent capable of detecting piperine levels.

[0068] In certain embodiments, the detection reagent measures the level of valeric acid, myristic acid, or piperine by an in vitro detection method.

[0069] In certain embodiments, the in vitro detection method is selected from mass spectrometry (eg, quadrupole time-of-flight mass spectrometry), chromatography (eg, high performance liquid chromatography, hydrophilic interaction liquid chromatography), or a combination thereof.

[0070] In certain embodiments, the assessment comprises the steps of: (1) determining the level of valeric acid in a sample from the subject; (2) comparing the level to a reference value; and (3) assessing the degree of aging of the subject and / or the subject's risk of developing an aging-related disease.

[0071] In certain embodiments, the sample is selected from a blood sample, such as whole blood, serum, or plasma.

[0072] In certain embodiments, the reference value is the valeric acid level (or average level) in the same type of sample of individuals (or a population) who are in the same chronological age group as the subject and in a normal aging state.

[0073] In certain embodiments, the extent of aging includes normal aging, accelerated aging, and / or delayed aging.

[0074] In certain embodiments, when the level of valeric acid in the sample from the subject is lower than the reference value, it indicates that the subject is in a state of accelerated aging and / or the subject has a higher risk of suffering from an aging-related disease.

[0075] In certain embodiments, the assessment comprises the steps of: (1) determining the levels of valeric acid and myristic acid and / or piperine in a sample from the subject; (2) comparing the levels to their respective corresponding reference values; and (3) assessing the degree of aging of the subject and / or the risk of the subject suffering from an aging-related disease.

[0076] In certain embodiments, the sample is selected from a blood sample, such as whole blood, serum, or plasma.

[0077] In certain embodiments, the reference value is the level (or average level) of valeric acid, myristic acid, or piperine in the same type of sample from individuals (or a population) who are in the same chronological age group as the subject and in a normal aging state.

[0078] In certain embodiments, the extent of aging includes normal aging, accelerated aging, and / or delayed aging.

[0079] In certain embodiments, when the level of valeric acid in the sample from the subject is lower than its corresponding reference value, and the level of myristic acid and / or the level of piperine in the sample from the subject is lower than its corresponding reference value, it indicates that the subject is in a state of accelerated aging and / or the subject has a higher risk of suffering from an aging-related disease.

[0080] In certain embodiments, the subject is a mammal.

[0081] In certain embodiments, the subject is human, preferably Asian.

[0082] In certain embodiments, the subject is Southeast Asian or East Asian (eg, Chinese, Japanese, Korean, Mongolian).

[0083] In certain embodiments, the valeric acid is n-valeric acid.

[0084] On the other hand, the present application provides a method for assessing the degree of aging of a subject and / or the subject's risk of developing an aging-related disease, wherein the method comprises the following steps: (1) determining the level of valeric acid in a sample from the subject; (2) comparing the level with a reference value; and (3) assessing the degree of aging of the subject and / or the subject's risk of developing an aging-related disease.

[0085] In certain embodiments, the sample is selected from a blood sample, such as whole blood, serum, or plasma.

[0086] In certain embodiments, the reference value is the valeric acid level (or average level) in the same type of sample of individuals (or a population) who are in the same chronological age group as the subject and in a normal aging state.

[0087] In certain embodiments, the extent of aging includes normal aging, accelerated aging, and / or delayed aging.

[0088] In certain embodiments, when the level of valeric acid in the sample from the subject is lower than the reference value, it indicates that the subject is in a state of accelerated aging and / or the subject has a higher risk of suffering from an aging-related disease.

[0089] In certain embodiments, the subject is a mammal.

[0090] In certain embodiments, the subject is human, preferably Asian.

[0091] In certain embodiments, the subject is Southeast Asian or East Asian (eg, Chinese, Japanese, Korean, Mongolian).

[0092] In certain embodiments, the valeric acid is n-valeric acid.

[0093] On the other hand, the present application provides a method for assessing the degree of aging of a subject and / or the risk of the subject suffering from an aging-related disease, wherein the method comprises the following steps: (1) determining the levels of valeric acid and myristic acid and / or piperine in a sample from the subject; (2) comparing the levels with their respective corresponding reference values; and (3) assessing the degree of aging of the subject and / or the risk of the subject suffering from an aging-related disease.

[0094] In certain embodiments, the sample is selected from a blood sample, such as whole blood, serum, or plasma.

[0095] In certain embodiments, the reference value is the level (or average level) of valeric acid, myristic acid, or piperine in the same type of sample from individuals (or a population) who are in the same chronological age group as the subject and in a normal aging state.

[0096] In certain embodiments, the extent of aging includes normal aging, accelerated aging, and / or delayed aging.

[0097] In certain embodiments, when the level of valeric acid in the sample from the subject is lower than its corresponding reference value, and the level of myristic acid and / or the level of piperine in the sample from the subject is lower than its corresponding reference value, it indicates that the subject is in a state of accelerated aging and / or the subject has a higher risk of suffering from an aging-related disease.

[0098] In certain embodiments, the subject is a mammal.

[0099] In certain embodiments, the subject is human, preferably Asian.

[0100] In certain embodiments, the subject is Southeast Asian or East Asian (eg, Chinese, Japanese, Korean, Mongolian).

[0101] In certain embodiments, the valeric acid is n-valeric acid.

[0102] In another aspect, the present application also provides a method for screening drugs, comprising:

[0103] (1) Provide biological samples and divide them into test group and control group;

[0104] (2) exposing the test group biological samples to the candidate drug;

[0105] (3) analyzing the levels of valeric acid in biological samples from the test group and comparing them with those of the control group;

[0106] wherein the control group is not exposed to the candidate drug (for example, the control group is not treated, or the control group is exposed to a control agent without an active ingredient);

[0107] If the level of valeric acid in the biological sample of the test group is higher than that of the control group, it indicates that the candidate drug has the potential to delay or reverse aging, and / or prevent and / or treat aging-related diseases.

[0108] In certain embodiments, step (3) further comprises: analyzing the level of myristic acid and / or piperine in the biological sample of the test group and comparing it with that of the control group.

[0109] In certain embodiments, if the level of valeric acid in the biological sample of the test group is higher than that of the control group, and the level of myristic acid and / or piperine is higher than that of the control group, it indicates that the candidate drug has the potential to delay or reverse aging in the subject, and / or prevent and / or treat aging-related diseases.

[0110] In certain embodiments, the valeric acid is n-valeric acid.

[0111] In another aspect, the present application also provides a reagent combination, which comprises:

[0112] (i) a reagent capable of detecting valeric acid levels; and,

[0113] (ii) a reagent capable of detecting the level of myristic acid, and / or a reagent capable of detecting the level of piperine.

[0114] In certain embodiments, the detection reagent measures the level of valeric acid, myristic acid, or piperine by an in vitro detection method.

[0115] In certain embodiments, the in vitro detection method is selected from mass spectrometry (eg, quadrupole time-of-flight mass spectrometry), chromatography (eg, high performance liquid chromatography, hydrophilic interaction liquid chromatography), or a combination thereof.

[0116] In certain embodiments, the valeric acid is n-valeric acid.

[0117] In another aspect, the present application also provides a pharmaceutical composition comprising:

[0118] (i) valeric acid,

[0119] (ii) myristic acid and / or piperine, and

[0120] (iii) pharmaceutically acceptable carriers and / or excipients.

[0121] In certain embodiments, the valeric acid is n-valeric acid.

[0122] Definition of terms

[0123] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the virology, biochemistry, and immunology laboratory operation steps used herein are conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0124] When the terms "for example," "such as," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0125] The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0126] As used herein, the term "aging" has the meaning commonly understood by those skilled in the art, and is a common biological phenomenon in the growth and development of the body. Aging can be considered as a process of body function decline accompanied by aging, which is usually accompanied by a continuous decline in physical health and an increase in the risk of aging-related diseases, functional disorders and death.

[0127] As used herein, the term "cellular senescence" refers to a physiological process of cells that exists in a specific state. In certain embodiments, cellular senescence can be caused by various cell stresses, such as DNA damage, oxidative stress, or exposure to exogenous toxins. Cell senescence is not equivalent to cell death. Senescent cells remain metabolically active for a period of time and may also show some obvious changes, with typical characteristics such as cell cycle arrest, senescence-related secretory phenotype, macromolecular damage, and metabolic disorders.

[0128] As used herein, the term "aging-related diseases" refers to aging-related diseases whose risk of occurrence increases significantly with aging. In certain embodiments, the aging-related diseases include, but are not limited to, neurodegenerative diseases (such as Alzheimer's disease), diabetes, heart disease, cardiovascular and cerebrovascular diseases, hypertension, hyperlipidemia, osteoporosis, osteoarthritis, chronic kidney disease, cancer, and vision loss.

[0129] As used herein, unless the context clearly indicates otherwise, the compounds described herein have the meanings generally understood by those skilled in the art, for example, the compounds used herein and their corresponding CAS numbers are as follows: n-valeric acid (CAS: 109-52-4), myristic acid (CAS: 544-63-8), piperine (CAS: 94-62-2). The compounds can be obtained by separation and purification from organisms or the environment, or by artificial preparation.

[0130] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with a subject and an active ingredient, and is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19 th ed.Pennsylvania:Mack Publishing Company,1995), and include but are not limited to: pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, preservatives. For example, pH regulators include but are not limited to phosphate buffers. Surfactants include but are not limited to cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include but are not limited to sodium chloride. Preservatives include but are not limited to various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Agents for maintaining osmotic pressure include but are not limited to sugars, NaCl and the like. Agents for delaying absorption include but are not limited to monostearate and gelatin. Diluents include but are not limited to water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc. Stabilizers have the meanings generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredients in the drug, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), etc.

[0131] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease or condition or symptom (e.g., aging, aging-related diseases) in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical result. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partial or complete), whether detectable or undetectable.

[0132] As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human.

[0133] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., aging, aging-related diseases) refers to an amount sufficient to prevent, prevent, or delay the occurrence of a disease (e.g., aging, aging-related diseases); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent the disease and its complications in patients who already have the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, and the like.

[0134] Advantageous Effects of the Invention

[0135] The markers for assessing the aging state of the body provided in the present application (valeric acid or a marker combination comprising valeric acid and myristic acid and / or piperine) are obtained based on the analysis of the changes in endogenous metabolites associated with aging in healthy Chinese people and are screened, and can more specifically and accurately assess the aging state of the Chinese population. In addition, the valeric acid (or myristic acid, piperine) provided in the present application for delaying or reversing aging, preventing and / or treating aging-related diseases is a natural component of human metabolites, has better safety in use, and has significant clinical application value.

[0136] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0137] Figure 1 : Endogenous metabolites that change with age.

[0138] Figure 2 :The level of n-valeric acid in plasma decreases significantly with age.

[0139] Figure 3 :Myristic acid in plasma decreases significantly with age.

[0140] Figure 4 :The level of piperine in plasma decreases significantly with age.

[0141] Figure 5 :Crystal violet staining analysis of HAECs treated with gradient concentrations of small molecules.

[0142] Figure 6 :Valeric acid reduces the SA-β-Gal positive cell rate in HAECs (scale bar is 50 μm).

[0143] Figure 7 :Myristic acid reduces the SA-β-Gal positive cell rate in HAECs (scale bar is 50 μm).

[0144] Figure 8 :Piperine reduces the SA-β-Gal positive cell rate in HAECs (scale bar is 50 μm).

[0145] Fig. 9 :Valeric acid improves the migration ability of HAECs (scale bar is 100 μm).

[0146] Fig.10 : Myristic acid enhances the migration ability of HAECs (scale bar: 100 μm).

[0147] Fig.11 : Piperine improves the migration ability of HAECs (scale bar is 100 μm).

[0148] Fig.12 :Valeric acid can enhance the cell tube-forming ability of HAECs (scale bar is 200 μm).

[0149] Fig.13 : Myristic acid can enhance the cell tube-forming ability of HAECs (scale bar is 200 μm).

[0150] Fig.14 :Piperine can enhance the cell tube-forming ability of HAECs (scale bar is 200 μm). DETAILED DESCRIPTION

[0151] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0152] Example 1: Volunteer recruitment and sample collection

[0153] The "Preliminary Screening Questionnaire" was published through the WeChat Mini Program to collect basic information of potential volunteers who are interested in joining this study based on the inclusion criteria, and to screen volunteers who meet the inclusion criteria; the inclusion criteria are as follows:

[0154] 1.1 Enrollment criteria

[0155] 1.1.1 Aged 18 or above, with no major bad habits;

[0156] 1.1.2 The applicant should be in good health and have not undergone any major surgery within the past six months. Routine conditions related to the normal aging process are permitted, including:

[0157] 1.1.2.1 Hypertension, well controlled (taking no more than 3 medications);

[0158] 1.1.2.2 Osteoporosis, osteopenia and / or osteoarthritis;

[0159] 1.1.2.3 Benign prostatic hypertrophy;

[0160] 1.1.2.4 Cataract, glaucoma, macular degeneration;

[0161] 1.1.2.5 Dyslipidemia;

[0162] 1.1.2.6 Hypothyroidism;

[0163] 1.1.2.7 Prediabetes or abnormal fasting blood glucose (fasting blood glucose 100-126 mg / dL).

[0164] 1.1.3 The subjects can communicate well with the researchers and cooperate with all tasks during the study;

[0165] 1.1.4 Subjects must voluntarily sign a written informed consent.

[0166] 1.2 Exclusion criteria

[0167] 1.2.1 Age does not meet the inclusion criteria;

[0168] 1.2.2 The participant is currently undergoing other clinical trials or is receiving any research drug or device for treatment within 30 days prior to enrollment;

[0169] 1.2.3 The investigator reports a history or current diagnosis of significant chronic illness, including:

[0170] 1.2.3.1 Any cancer (except polycythemia vera, basal cell or squamous cell skin cancer);

[0171] 1.2.3.2 Coronary artery disease / myocardial infarction / clinically significant congestive heart failure;

[0172] 1.2.3.3 Stroke / transient ischemic attack;

[0173] 1.2.3.4 Deep vein thrombosis / pulmonary embolism;

[0174] 1.2.3.5 Serum creatinine>1.5mg / dl (male);

[0175] 1.2.3.6 Poorly controlled hypertension (significant hypertension (systolic blood pressure > 160 mmHg, or diastolic blood pressure > 100 mmHg) despite treatment);

[0176] 1.2.3.7 History of active liver disease or metabolic acidosis;

[0177] 1.2.3.8 Chronic kidney disease / hemodialysis treatment, history of severe renal impairment and / or

[0178] eGFR≤45ml / min / 1.73m2;

[0179] 1.2.3.9 Severe autoimmune / inflammatory diseases, such as rheumatoid arthritis, lupus, Crohn's disease, etc.;

[0180] 1.2.3.10 Neurological diseases such as dementia, such as Alzheimer's / Parkinson's, etc.;

[0181] 1.2.3.11 Diabetes (hemoglobin A1C>6.5% or fasting blood glucose>126 mg / dL or taking diabetes medication or insulin treatment);

[0182] 1.2.3.12 Recent (within 3 months) cardiovascular disease events (myocardial infarction, coronary artery intervention, coronary artery bypass grafting);

[0183] 1.2.3.13 Infectious diseases such as HIV, hepatitis, tuberculosis, etc.

[0184] 1.2.4 Currently taking the following medications regularly:

[0185] 1.2.4.1 Chemotherapy drugs (such as tamoxifen, doxorubicin, mitoxantrone, bleomycin);

[0186] 1.2.4.2 Antiplatelet drugs (e.g. clopidogrel / Plavix, dipyridamole / Angeleno, ticlopidine / Ticlopidine, excluding aspirin);

[0187] 1.2.4.3 Cholinesterase inhibitors for Alzheimer's disease (donepezil / alicept).

[0188] 1.2.5 Continuous alcohol or drug abuse;

[0189] 1.2.6 Unable to provide informed consent;

[0190] 1.2.7 The researcher believes that the physical factors of the participants may have an adverse effect on the research process or results.

[0191] 1.3 Termination criteria

[0192] 1.3.1 Failure to follow the diet, work and rest schedule and physical examination requirements in accordance with the research content;

[0193] 1.3.2 Experiencing abnormal physical conditions such as cold or fever during the study;

[0194] 1.3.3 Accidental injury or physical discomfort during the research process;

[0195] 1.3.4 Comply with the subject’s subjective wishes to terminate the program.

[0196] Volunteers who meet the inclusion criteria and are confirmed to be included in the group will sign an informed consent form and join the volunteer WeChat group for management and information communication. Within one month before the start of the project, volunteers need to maintain a normal schedule, eat a proper diet, avoid strenuous exercise, and refrain from excessive smoking or drinking. The day before the project, volunteers will receive a unified standardized diet (menu see Table 1) at the implementation hospital and will no longer be allowed to take other foods.

[0197] On the day of sample collection and physical examination, volunteers are required to wear clothes and shoes that are convenient for movement, are not allowed to wear makeup, and must bring identification documents (such as ID cards, etc.) to the hospital for physical examination and plasma sample collection.

[0198] Plasma was collected using EDTA collection tubes. After obtaining the blood sample (approximately 5 ml), it was centrifuged (400 g, 15 min, 4°C) and packaged according to 500 uL / tube, about 6 tubes / case, quickly frozen in liquid nitrogen, and transferred to a -80 refrigerator for freezing.

[0199] Table 1 Unified and standardized dietary menu

[0200]

[0201] Example 2: Plasma metabolome analysis

[0202] Plasma samples were thawed at 4°C, and 100 μL of the sample was mixed with 400 μL of ice-cold methanol / acetonitrile (volume ratio 1:1) to remove proteins. The mixture was centrifuged at 14,000 g for 15 min at 4°C, and the supernatant was removed. For LC-MS analysis, the sample was reconstituted in 100 μL of acetonitrile / water (volume ratio 1:1) solvent. The analysis was performed using high-performance liquid chromatography (UHPLC) (1290 Infinity LC from Agilent Technologies) coupled to a quadrupole time-of-flight mass spectrometer (AB Sciex TripleTOF 6600). For hydrophilic interaction liquid chromatography (HILIC) separation, the samples were analyzed using a 2.1 mm × 100 mm ACQUIY UPLC BEH 1.7 μm column (Waters, Ireland). For the first MS / MS acquisition, the instrument was set to acquire over the m / z range of 60–1000 Da, and the product ion scan time was set to 0.2 s / spectrum. The second MS / MS acquisition was performed in the m / z range of 25-1000Da with a time of 0.05 sec / spectrum. Product ion scans were performed using information-dependent acquisition (IDA) with high sensitivity mode selected. Briefly, the dissociation potential (DP) was fixed at ±60V and isotopes within the 4Da range were excluded. The collision energy (CE) was fixed at 35V, ±15eV. Ten candidate ions were monitored per cycle.

[0203] After obtaining the metabolome data, the mass spectrometry raw data were first converted into MzXML files using ProteoWizard MSConvert (version 3.0.6428); then, peak screening was performed using XCMS software (version 3.14.1), with the key parameters including centWave m / z = 10 ppm, peakwidth = c(10, 60), prefilter = c(10, 100); the screened peaks were then grouped, with the key parameters including bw = 5, mzwid = 0.025, minfrac = 0.5; then, CAMERA software (version 3.6) was used to annotate isotopes and adducts, retaining only extracted ion features with non-zero values ​​exceeding 50% in all samples; metabolite species were identified by comparing m / z values ​​(<10 ppm) with mass spectra from a database (from Sino-Science New Life Sciences); the raw intensity values ​​were divided by the median value of each sample to obtain a standardized metabolite abundance matrix.

[0204] The analysis obtained the change pattern of endogenous metabolites accumulated or decreased with age ( Figure 1), and found that valeric acid, myristic acid, and piperine decreased significantly with aging in healthy people ( Figure 2-4 ).

[0205] Example 3: Screening of concentrations of valeric acid, myristic acid, and piperine for delaying endothelial cell senescence

[0206] 3.1 Cell culture and drug treatment

[0207] Human aortic endothelial cells (HAECs) were purchased from Lonza (CC-2535). HAECs were cultured on collagen (Advanced BioMatrix, 5005) (in 12-well plates, 3000 cells / well) and cultured in EGM-2 medium (Lonza, CC-3126) containing 1% penicillin-streptomycin (Lonza, CC-3126) and 0.01% antimicrobial (Invivogen, ant-mpt). For valeric acid treatment, valeric acid (soluble in the culture medium, set concentration gradient: 10, 50, 100, 200 ng / ml) was added each time the culture medium was changed, and the control group was not treated; for myristic acid treatment, myristic acid (in DMSO, set concentration gradient: 0.05, 0.2, 1, 5 μM) was added each time the culture medium was changed, and 0.01% DMSO was added to the control group; for piperine treatment, piperine (in DMSO, set concentration gradient: 1, 5, 20, 50 μM) was added each time the culture medium was changed, and 0.01% DMSO was added to the control group. No mycoplasma contamination was observed during cell culture.

[0208] 3.2 Crystal violet staining analysis

[0209] After two weeks of culture, the cells were fixed with paraformaldehyde (4%) for 15 min, washed with PBS, and then added with crystal violet dye, and stained at room temperature for 15 h. The excess dye and floating color were then washed away with running water, and the cells were left to air dry before being photographed.

[0210] The results showed that valeric acid, myristic acid and piperine could promote the proliferation of HAECs within a certain concentration range ( Figure 5 ).

[0211] Example 4: Analysis of HAECs cell senescence delayed by valeric acid, myristic acid and piperine 4.1 SA-β-Gal staining analysis

[0212] After removing the culture medium, the cells treated in step 3.1 of Example 3 were washed twice with PBS buffer and fixed with 2% formaldehyde and 0.2% glutaraldehyde fixation buffer at room temperature for 5 minutes. After washing twice with PBS buffer again, SA-β-Gal staining solution containing 1 mg / ml X-gal (Amresco, 0428) was added to the culture dish. The cells were stained at 37°C and stored in the dark for 12 hours. Photos were taken using a digital microscope camera (Olympus). The proportion of SA-β-Gal positive cells was quantitatively analyzed by ImageJ software (version 1.48v).

[0213] The results showed that valeric acid, myristic acid and piperine could reduce the SA-β-Gal positive cell rate of HAECs of the same generation (13th generation) Figure 6-8 ).

[0214] 4.2 Cell migration assay

[0215] HAECs treated in step 3.1 of Example 3 were cultured in 12-well plates, and the migration ability of HAECs was evaluated using a scratch test. The cells were cultured to 100% density and scratched using a 10 μl pipette tip. Photos were taken 8 hours later. ImageJ was used to analyze the cell migration rate at the same location.

[0216] The results showed that valeric acid, myristic acid and piperine could enhance the cell migration ability of HAECs of the same generation (13th generation). Figure 9-11 ).

[0217] 4.3 In vitro tube formation experiment

[0218] HAECs treated in step 3.1 of Example 3 were plated at 5×10 4 The cells were added to a 24-well plate pre-coated with Matrigel (Corning) at a density of 1.5 ng / mL. Cells were cultured using incucyte s3 (Essen Bioscience) and photographed after 12 h. The number of tubular structures / nodes and branch length were analyzed using ImageJ-Angiogenesis Analyzer.

[0219] The results showed that valeric acid, myristic acid and piperine could improve the cell tube-forming ability of HAECs of the same generation (generation 13). Figure 12-14 ).

[0220] The above conclusions suggest that valeric acid, myristic acid and piperine can delay the aging of human arterial endothelial cells in vitro, and delay the decline of migration and tube formation associated with aging.

[0221] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. Use of valeric acid in the preparation of medicines, wherein: The drug is used to delay or reverse aging in a subject, and / or prevent and / or treat aging-related diseases.

2. The use of claim 1, wherein The medicament further comprises myristic acid and / or piperine.

3. The use of claim 1 or 2, wherein The drug is used to delay or reverse cellular senescence in a subject; Preferably, the drug is used to delay or reverse one or more of the following states of cellular senescence in a subject: (1) Reduced cell proliferation; (2) weakened cell migration ability; (3) The ability of cells to form tubes is weakened; (4) upregulation of cellular β-galactosidase activity; (5) The expression of genes related to cell senescence is upregulated.

4. The use of claim 3, wherein The cells are vascular endothelial cells.

5. A method for delaying or reversing cell senescence, comprising the step of contacting valeric acid with the cells.

6. The method of claim 5, wherein The method further comprises contacting the cell with myristic acid and / or piperine.

7. The method of claim 5 or 6, wherein The method is used to delay or reverse one or more of the following states of cellular senescence: (1) Reduced cell proliferation; (2) weakened cell migration ability; (3) The ability of cells to form tubes is weakened; (4) upregulation of cellular β-galactosidase activity; (5) The expression of genes related to cell senescence is upregulated.

8. The method of any one of claims 5 to 7, wherein The cells are vascular endothelial cells.

9. Use of a reagent capable of detecting valeric acid levels in the preparation of a detection reagent for assessing the degree of aging of a subject and / or the risk of the subject suffering from an aging-related disease.

10. The use according to claim 9, wherein The detection reagents further comprise a reagent capable of detecting the level of myristic acid and / or a reagent capable of detecting the level of piperine.

11. The use according to claim 9 or 10, wherein The detection reagent determines the level of valeric acid, myristic acid or piperine by an in vitro detection method; Preferably, the in vitro detection method is selected from mass spectrometry (eg, quadrupole time-of-flight mass spectrometry), chromatography (eg, high performance liquid chromatography, hydrophilic interaction liquid chromatography), or a combination thereof.

12. The use according to any one of claims 9 to 11, wherein The assessment comprises the steps of: (1) determining the level of valeric acid in a sample from the subject; (2) comparing the level to a reference value; and (3) assessing the subject's degree of aging and / or the subject's risk of developing an aging-related disease; Preferably, the sample is selected from a blood sample, such as whole blood, serum or plasma; Preferably, the reference value is the valeric acid level (or average level) in the same type of sample of individuals (or groups) who are in the same chronological age group as the subject and in a normal aging state; Preferably, the degree of aging includes normal aging, accelerated aging, and / or delayed aging; Preferably, when the level of valeric acid in the sample from the subject is lower than the reference value, it indicates that the subject is in a state of accelerated aging and / or the subject has a higher risk of suffering from an aging-related disease.

13. The use according to any one of claims 9 to 11, wherein The assessment comprises the following steps: (1) determining the levels of valeric acid and myristic acid and / or piperine in a sample from the subject; (2) comparing the levels with their respective corresponding reference values; and (3) assessing the subject's degree of aging and / or the subject's risk of developing an aging-related disease; Preferably, the sample is selected from a blood sample, such as whole blood, serum or plasma; Preferably, the reference value is the level (or average level) of valeric acid, myristic acid or piperine in the same type of sample of an individual (or population) who is in the same chronological age group as the subject and in a normal aging state; Preferably, the degree of aging includes normal aging, accelerated aging, and / or delayed aging; Preferably, when the level of valeric acid in the sample from the subject is lower than the corresponding reference value, and the level of myristic acid and / or the level of piperine in the sample from the subject is lower than the corresponding reference value, it indicates that the subject is in a state of accelerated aging and / or the subject has a higher risk of suffering from an aging-related disease.

14. A method for screening drugs, comprising: (1) Provide biological samples and divide them into test group and control group; (2) exposing the test group biological samples to the candidate drug; (3) analyzing the levels of valeric acid in biological samples from the test group and comparing them with those of the control group; wherein the control group is not exposed to the candidate drug (for example, the control group is not treated, or the control group is exposed to a control agent without an active ingredient); If the level of valeric acid in the biological sample of the test group is higher than that of the control group, it indicates that the candidate drug has the potential to delay or reverse aging, and / or prevent and / or treat aging-related diseases.

15. The method of claim 14, wherein The step (3) further comprises: analyzing the levels of myristic acid and / or piperine in the biological samples of the test group and comparing them with those of the control group; Preferably, if the level of valeric acid in the biological sample of the test group is higher than that of the control group, and the level of myristic acid and / or piperine is higher than that of the control group, it indicates that the candidate drug has the potential to delay or reverse aging in the subject, and / or prevent and / or treat aging-related diseases.

16. A reagent combination comprising: (i) a reagent capable of detecting valeric acid levels; and, (ii) a reagent capable of detecting the level of myristic acid, and / or a reagent capable of detecting the level of piperine.

17. A pharmaceutical composition comprising: (i) valeric acid, (ii) myristic acid and / or piperine, and (iii) pharmaceutically acceptable carriers and / or excipients.

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