Application of hesperetin-7-O-glucoside and derivatives thereof in delaying senescence

Abstract

The invention relates to an application of hesperetin-7-O-glucoside and a derivative thereof in delaying senescence. Provided are hesperetin-7-O-glucoside, or a pharmaceutically or food acceptable salt thereof, or a tautomer, stereoisomer, enantiomer or diastereoisomer thereof, or a prodrug or metabolite thereof, or a natural source of the hesperetin-7-O-glucoside, salt, isomer, prodrug or metabolite in preparation of a composition or preparation for delaying senescence and / or prolonging life.

Description

Technical Field

[0001] This application relates to the field of food or medicine, and specifically to the uses of hesperetin-7-O-glucoside and its derivatives. Background Art

[0002] In the most ideal situation, the length of life is mainly determined by the aging rate. Aging is controlled by interrelated processes occurring at the tissue, cellular, molecular, and genetic levels. With the increase of age, irreversible degenerative changes gradually occur in the structure and function of various tissues and organs of the body, such as protein degradation, tissue atrophy, reduced metabolic rate, and abnormal calcium metabolism, etc.; with the passage of time, age-related diseases such as Alzheimer's disease, Parkinson's disease, cardiovascular and cerebrovascular diseases, and cancer, etc. show exponential growth globally. With the continuous increase of the elderly population base and proportion, as well as the continuous increase of people's demand for anti-aging and life extension, conducting research on the mechanism of human aging and developing anti-aging drugs to delay the aging rate of humans within the limit determined by genetic characteristics and extend the lifespan of the body has important social significance.

[0003] Caenorhabditis elegans is a classic model organism for conducting aging-related research, with advantages such as a short life cycle, clear genetic background, simple physiological structure, and easy cultivation. C. elegans has many similarities with higher organisms at the cellular and molecular levels, such as mechanisms of apoptosis, signal transduction, etc. Most importantly, the aging characteristics of C. elegans are obvious, including decreased locomotor ability, reduced reproductive ability, and changes in body morphology (such as muscle atrophy, intestinal degeneration, etc.), which are easy to study, observe, and quantify.

[0004] There is a need in this field to develop substances with the effect of extending lifespan, and conducting research based on C. elegans is a feasible solution. Summary of the Invention

[0005] Through the results reflected by C. elegans, the applicant has first discovered the efficacy of hesperetin-7-O-glucoside and its derivatives in extending lifespan, as well as their mechanism of action.

[0006] Using C. elegans as an anti-aging model has the following advantages: Since 60%-80% of the genes of C. elegans are highly conserved with the relevant genes of humans, and among the signal transduction pathways discovered so far, C. elegans has 12 of them, so this application uses C. elegans as a model organism to verify anti-aging drugs. The rich genetic resources of C. elegans can be utilized to select appropriate mutants according to the research purpose to study the aging and anti-aging mechanisms. In fact, several major theories of the aging mechanism have been proven in C. elegans. Therefore, drugs with anti-aging effects on C. elegans are usually also considered to have the same efficacy on humans.

[0007] Caenorhabditis elegans has been used in lifespan analysis for 30 years. Due to its unique advantages, it has become the first-choice model for aging research. The generation cycle of C. elegans is short, usually about 3 days, and its lifespan is short, usually about 3 weeks. This makes it possible to achieve repeat stability in experiments. To ensure the reliability of experimental methods, the accuracy of experimental results, and to obtain more accurate and credible information in drug screening, repeated experiments are necessary. Due to the above unique advantages of C. elegans, it has become the first-choice model for aging research. Therefore, C. elegans can be used to evaluate the anti-aging effect of a composition, and further to determine whether the composition can be used to prepare anti-aging drugs.

[0008] The nematode gene knockdown mutant strain aak-2(gt33) represents the AMPK signaling pathway. AMPK is a protein kinase widely present in humans and other organisms, and its main function is to regulate cellular energy metabolism and maintain energy balance. It can activate or inhibit multiple cellular metabolic pathways by phosphorylating a series of substrate proteins. The activation of AMPK can delay the aging process by regulating cellular energy metabolism, inhibiting oxidative stress and inflammatory responses, etc., and has important anti-aging significance. The vha-3(ok1501) strain represents the lysosome acidification and lysosomal autophagy pathway. The reduced expression of vha-3 impairs the V-ATPase proton pump on the lysosomal membrane, affecting the acidification and autophagy functions of lysosomes. The rsks-1(ok1255) strain represents the TOR signaling pathway. RSKS-1 is the only nematode homolog of human S6-kinase (S6K) and plays a key role in protein synthesis, lifespan, autophagy, and mTOR signaling. The clk-1(qm30) strain represents the mitochondrial function pathway. The knockout of this gene leads to damage to the mitochondrial respiratory chain. The atg-7(bp422) represents the autophagy pathway. ATG7 is a protein essential for the autophagy conjugation system and autophagosome formation, and it plays an important regulatory role in the formation of autophagosomes.

[0009] The structure of hesperetin-7-O-glucoside is shown as follows:

[0010] 。

[0011] In one aspect, the present application provides the use of hesperetin-7-O-glucoside or a pharmaceutically or food-acceptable salt thereof, or a tautomer, stereoisomer, enantiomer or diastereoisomer thereof, or a prodrug or metabolite thereof, or a natural source of the hesperetin-7-O-glucoside, salt, isomer, prodrug or metabolite in the preparation of a composition or formulation for delaying aging and / or extending lifespan.

[0012] In some embodiments, the composition or formulation is used to delay aging and / or extend lifespan by modulating the expression of the aak-2, vha-3, rsks-1, clk-1, and / or atg-7 genes.

[0013] In some embodiments, the composition or formulation is used to delay aging and / or extend lifespan by activating the AMPK signaling pathway, the TOR signaling pathway, mitochondrial function, lysosomal acidification, and / or the autophagy pathway.

[0014] In some embodiments, the composition or formulation is used to delay the aging of a human or animal or extend the lifespan of a human or animal.

[0015] In some embodiments, the composition or formulation is a food, feed, culture medium, health food, cosmetic, or skin care product.

[0016] In some embodiments, the composition or formulation is in liquid, gel, lyophilized, semi-solid, or solid form.

[0017] In some embodiments, the composition or formulation is a drug. In some embodiments, the dosage form of the drug is powder, tablet, capsule, pill, injection, sustained release agent, ointment, or cream.

[0018] In some embodiments, the composition or formulation further comprises a pharmaceutically or food acceptable additive, carrier, or excipient.

[0019] In some embodiments, the composition or formulation contains hesperetin-7-O-glucoside in a unit dose of 0.001 - 1000 mg.

[0020] In another aspect, the present application provides the use of hesperetin-7-O-glucoside or a pharmaceutically or food acceptable salt thereof, or a tautomer, stereoisomer, enantiomer, or diastereoisomer thereof, or a prodrug or metabolite thereof, or a natural source of the hesperetin-7-O-glucoside, salt, isomer, prodrug, or metabolite in the preparation of a composition or formulation for modulating the expression of the aak-2, vha-3, rsks-1, clk-1, and / or atg-7 genes.

[0021] In some embodiments, the composition or formulation is for human or animal use.

[0022] In some embodiments, the composition or formulation is a food, feed, culture medium, health food, cosmetic, or skin care product.

[0023] In some embodiments, the composition or formulation is in liquid, gel, lyophilized, semi-solid, or solid form.

[0024] In some embodiments, the composition or formulation is a drug. In some embodiments, the dosage form of the drug is powder, tablet, capsule, pill, injection, sustained release formulation, ointment or cream.

[0025] In some embodiments, the composition or formulation further comprises a pharmaceutically or food acceptable additive, carrier or excipient.

[0026] In some embodiments, the composition or formulation contains hesperetin-7-O-glucoside in a unit dose of 0.001 - 1000 mg.

[0027] In another aspect, the present application provides the use of hesperetin-7-O-glucoside or a pharmaceutically or food acceptable salt thereof, or a tautomer, stereoisomer, enantiomer or diastereoisomer thereof, or a prodrug or metabolite thereof, or a natural source of the hesperetin-7-O-glucoside, salt, isomer, prodrug or metabolite in the preparation of a composition or formulation for activating the AMPK signaling pathway, TOR signaling pathway, mitochondrial function, lysosomal acidification and / or autophagy pathway.

[0028] In some embodiments, the composition or formulation is for human or animal use.

[0029] In some embodiments, the composition or formulation is food, feed, culture medium, health food, cosmetic or skin care product.

[0030] In some embodiments, the composition or formulation is in liquid, gel, lyophilized, semi-solid or solid form.

[0031] In some embodiments, the composition or formulation is a drug. In some embodiments, the dosage form of the drug is powder, tablet, capsule, pill, injection, sustained release formulation, ointment or cream.

[0032] In some embodiments, the composition or formulation further comprises a pharmaceutically or food acceptable additive, carrier or excipient.

[0033] In some embodiments, the composition or formulation contains hesperetin-7-O-glucoside in a unit dose of 0.001 - 1000 mg.

[0034] On the other hand, the present application provides a composition for expressing anti-aging and / or extending lifespan, which comprises hesperetin-7-O-glucoside or a pharmaceutically or food-acceptable salt thereof, or a tautomer, stereoisomer, enantiomer or diastereoisomer thereof, or a prodrug or metabolite thereof, and the hesperetin-7-O-glucoside or a pharmaceutically or food-acceptable salt thereof, or a tautomer, stereoisomer, enantiomer or diastereoisomer thereof, or a prodrug or metabolite thereof is derived from a plant. In some embodiments, the plant can be pomelo, tangerine, mandarin, citrus, huyou, dried tangerine peel, lemon, fingered citron, lime, trogopterus dung, orange peel, citron, bitter orange, fructus aurantii immaturus, fructus aurantii, etc.

[0035] In some embodiments, the composition is for human or animal use.

[0036] In some embodiments, the composition is food, feed, culture medium, health food, cosmetic or skin care product.

[0037] In some embodiments, the composition is in the form of liquid, gel, lyophilized, semi-solid or solid.

[0038] In some embodiments, the composition is a drug. In some embodiments, the dosage form of the drug is powder, tablet, capsule, pill, injection, sustained release agent, ointment or paste.

[0039] In some embodiments, the composition further comprises a pharmaceutically or food-acceptable additive, carrier or excipient.

[0040] In some embodiments, the composition contains hesperetin-7-O-glucoside in a unit dose of 0.001-1000 mg. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Shows the survival curve of Caenorhabditis elegans.

[0042] Figure 2 Shows the statistical results of the average lifespan of Caenorhabditis elegans.

[0043] Figure 3A - Figure 3I Shows the survival curves of Caenorhabditis elegans under different conditions. DETAILED DESCRIPTION

[0044] To make the present disclosure easier to understand, certain terms are first defined below. Additional definitions of the following terms and other terms are set forth throughout the specification.

[0045] As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0046] In this text, the terms "comprising", "having", "including", and "containing" shall be construed as open-ended terms (i.e., meaning "including but not limited to").

[0047] In this text, "and / or" means and includes any and all possible combinations of one or more of the associated listed items. For example, a composition comprising A and / or B can be interpreted as the composition comprising A, the composition comprising B, or the composition comprising A and B.

[0048] All numerical names, such as pH, temperature, time, concentration, and molecular weight, including ranges, are approximate values, which are appropriately incremented by 1.0 or 0.1, or optionally changed (+) or (-) by variations of + / - 15%, 10%, 5%, 2%. It should be understood that all numerical names are preceded by the term "about". It should also be understood that the reagents described herein are merely exemplary, and their equivalents are known in the art. When referring to measurable values such as amounts or concentrations, etc., the term "about" as used herein means including variations within 20%, 10%, 5%, 1%, 0.5%, or 0.1% of the specified amount.

[0049] As used herein, the term "comprising" is synonymous with "including", "containing", and is inclusive of endpoints or is open-ended, and does not exclude additional unrecited elements or method steps. "Comprising" is a technical term used in claim language, meaning that the recited elements are present, but other elements may be added and still form a construction or method within the scope of the claim.

[0050] In this text, the term "administer" is intended to mean delivering a substance to a subject, such as an animal or a human. Administration can be carried out in a single dose, continuously, or intermittently throughout the course of treatment. Methods for determining the most effective mode of administration and dose are known to those skilled in the art and will vary depending on factors such as the composition used for treatment, the treatment objective, and the age, health, or gender of the subject being treated. In some embodiments, single or multiple administrations can be carried out, and the dose levels and patterns are selected by a physician, or in the case of pets and other animals, by a veterinarian.

[0051] In this text, the term "treat" includes: (1) inhibiting a condition, disease, or disorder, i.e., preventing, reducing, or delaying the development of the disease or its recurrence or the development of at least one of its clinical or subclinical symptoms; or (2) alleviating a disease, i.e., causing the regression of at least one of the condition, disease, or disorder or its clinical or subclinical symptoms.

[0052] In this text, the term "ameliorate" refers to the improvement of symptoms related to a disease and can refer to the improvement of at least one parameter that measures or quantifies the symptom.

[0053] As used herein, the term "preventing" a condition, disease or disorder includes preventing, delaying or reducing the incidence and / or likelihood of the appearance of at least one clinical or subclinical symptom of a condition, disease or disorder developing in a subject who may have or be susceptible to the condition, disease or disorder but has not yet experienced or exhibited a clinical or subclinical symptom of the condition, disease or disorder.

[0054] The term "pharmaceutically acceptable" means a carrier, excipient or diluent that is suitable, within the scope of sound medical judgment, for contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0055] The term "pharmaceutical composition" generally refers to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to whom the composition is to be administered. The composition is sterile. A "sterile" composition is sterilized or free of all living microorganisms and their spores.

[0056] Exemplary carriers for the drugs used in this application include saline, buffered saline, glucose and water. Exemplary excipients for the compositions used in this application include fillers, binders, disintegrants, coating agents, adsorbents, anti-adhesion agents, glidants, preservatives, antioxidants, flavoring agents, coloring agents, sweetening agents, solvents, co-solvents, buffers, chelating agents, viscosity imparting agents, surfactants, diluents, wetting agents, carriers, diluents, preservatives, emulsifying agents, stabilizers and tonicity modifiers. Those skilled in the art know how to select suitable excipients to prepare the compositions or preparations of this application. Generally, the selection of suitable excipients depends particularly on the active agent used, the disease to be treated and the desired dosage form of the composition.

[0057] The term "kit" generally refers to a packaged product containing the components for administering the compositions or preparations of this application to treat or prevent related diseases. The components of the kit may be contained in separate vials (i.e., a kit with separate parts), or provided within a single vial. The kit may contain reagents such as buffers, protein stabilizing reagents, signal generating systems (e.g., fluorescent signal generating systems), antibodies, control proteins, and test containers. The kit may also contain instructions for carrying out the method. In some embodiments of this application, the kit may also contain an administration device that is capable of administering the pharmaceutically active ingredient in the kit (e.g., hesperetin-7-O-glucoside of this application) to a subject in a suitable manner.

[0058] The term "prodrug" is also known as pro-drug, drug precursor, precursor drug, etc., and refers to a compound that can be converted in vivo to the compound of the present application or a pharmaceutically acceptable salt, hydrate or solvate of the compound. This conversion can occur through various mechanisms (e.g., metabolic or chemical processes), for example, by hydrolysis in the blood, gastrointestinal tract or lung. A discussion of prodrug uses is provided in the following: T. Higuchi and W. Stella, "Prodrugs as Novel Delivery Systems," Vol. 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

[0059] The term "metabolite" refers to any substance produced from another substance through metabolism or a metabolic process.

[0060] In the present application, the term "natural source" refers to a natural substance containing a certain compound and its physically processed products, which can be of plant, animal, microbial or mineral origin. For example, microbial products, plant extracts, plant powders (such as citrus fruit powder), juice products, and soaking solutions, etc.

[0061] In the present application, hesperetin-7-O-glucoside or a pharmaceutically or food-acceptable salt thereof, or a tautomer, stereoisomer, enantiomer or diastereoisomer thereof, or a prodrug or metabolite thereof can be derived from plants, such as plant extracts, plant powders (such as citrus fruit powder), juice products, and soaking solutions, etc. The plants can be pomelo, orange, mandarin, citrus, huyou, dried tangerine peel, lemon, fingered citron, bitter orange, citri grandis epicarpium, tangerine peel, citron, daidai flower, fructus aurantii, fructus aurantii immaturus, etc.

[0062] In the present application, the natural sources of hesperetin-7-O-glucoside and its derivatives can include citrus plants, such as but not limited to pomelo (Citrus maxima) varieties: Shatian pomelo, honey pomelo, red-fleshed pomelo, grapefruit; bitter orange (Citrus aurantium) varieties: citron, fingered citron, lime, daidai bitter orange; mandarin (Citrus japonica): kumquat, luofu kumquat, round kumquat, long-leaved kumquat; and citrus, huyou, dried tangerine peel, lemon, fingered citron, bitter orange, citri grandis epicarpium, tangerine peel, citron, daidai flower, fructus aurantii, fructus aurantii immaturus, etc. These natural sources can also be used for the uses of hesperetin-7-O-glucoside in this article.

[0063] The present application provides the use of hesperetin-7-O-glucoside or a pharmaceutically or food acceptable salt thereof, or a tautomer, stereoisomer, enantiomer or diastereoisomer thereof, or a prodrug or metabolite thereof, or a natural source of the hesperetin-7-O-glucoside, salt, isomer, prodrug or metabolite in the preparation of a composition or preparation for delaying aging and / or extending lifespan.

[0064] In some embodiments, the composition or preparation is used for delaying aging and / or extending lifespan by regulating the expression of the aak-2 gene.

[0065] In some embodiments, the composition or preparation is used for delaying aging and / or extending lifespan by regulating the expression of the vha-3 gene.

[0066] In some embodiments, the composition or preparation is used for delaying aging and / or extending lifespan by regulating the expression of the rsks-1 gene.

[0067] In some embodiments, the composition or preparation is used for delaying aging and / or extending lifespan by regulating the expression of the clk-1 gene.

[0068] In some embodiments, the composition or preparation is used for delaying aging and / or extending lifespan by regulating the expression of the atg-7 gene.

[0069] In some embodiments, the composition or preparation is used for delaying aging and / or extending lifespan by regulating the expression of the aak-2, vha-3, rsks-1, clk-1 and / or atg-7 genes.

[0070] As used herein, "regulating" the expression of a gene can be inhibiting gene expression or enhancing gene expression.

[0071] In some embodiments, the composition or preparation is used for delaying aging and / or extending lifespan by activating the AMPK signaling pathway.

[0072] In some embodiments, the composition or preparation is used for delaying aging and / or extending lifespan by activating the TOR signaling pathway.

[0073] In some embodiments, the composition or preparation is used for delaying aging and / or extending lifespan by activating mitochondrial function.

[0074] In some embodiments, the composition or preparation is used for delaying aging and / or extending lifespan by activating lysosomal acidification.

[0075] In some embodiments, the composition or preparation is used for delaying aging and / or extending lifespan by activating the autophagy pathway.

[0076] In some embodiments, the composition or formulation is used to delay aging and / or extend lifespan by activating the AMPK signaling pathway, the TOR signaling pathway, mitochondrial function, lysosomal acidification, and / or the autophagy pathway.

[0077] In some embodiments, the composition or formulation is used to delay the aging of a human or animal or extend the lifespan of a human or animal.

[0078] In some embodiments, the composition or formulation is a food, feed, culture medium, health food, cosmetic, or skin care product.

[0079] In some embodiments, the composition or formulation is in liquid, gel, lyophilized, semi-solid, or solid form.

[0080] In some embodiments, the composition or formulation is a drug. In some embodiments, the dosage form of the drug is powder, tablet, capsule, pill, injection, sustained release agent, ointment, or cream.

[0081] In some embodiments, the composition or formulation further comprises a pharmaceutically or food acceptable additive, carrier, or excipient.

[0082] In some embodiments, the composition or formulation contains hesperetin-7-O-glucoside in a unit dose of 0.001 - 1000 mg, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 mg of hesperetin-7-O-glucoside.

[0083] In another aspect, the present application provides the use of hesperetin-7-O-glucoside or a pharmaceutically or food acceptable salt thereof, or a tautomer, stereoisomer, enantiomer, or diastereoisomer thereof, or a prodrug or metabolite thereof, or a natural source of the hesperetin-7-O-glucoside, salt, isomer, prodrug, or metabolite in the preparation of a composition or formulation for regulating the expression of aak-2, vha-3, rsks-1, clk-1, and / or atg-7 genes.

[0084] In some embodiments, the composition or formulation is for use in a human or animal.

[0085] In some embodiments, the composition or formulation is a food, feed, culture medium, health food, cosmetic, or skin care product.

[0086] In some embodiments, the composition or formulation is in liquid, gel, lyophilized, semi-solid, or solid form.

[0087] In some embodiments, the composition or formulation is a drug. In some embodiments, the dosage form of the drug is powder, tablet, capsule, pill, injection, sustained release formulation, ointment or cream.

[0088] In some embodiments, the composition or formulation further comprises a pharmaceutically or food acceptable additive, carrier or excipient.

[0089] In some embodiments, the composition or formulation contains hesperetin-7-O-glucoside in a unit dose of 0.001 - 1000 mg, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 mg of hesperetin-7-O-glucoside.

[0090] In another aspect, the present application provides the use of hesperetin-7-O-glucoside or a pharmaceutically or food acceptable salt thereof, or a tautomer, stereoisomer, enantiomer or diastereoisomer thereof, or a prodrug or metabolite thereof, or a natural source of the hesperetin-7-O-glucoside, salt, isomer, prodrug or metabolite in the preparation of a composition or formulation for activating the AMPK signaling pathway, TOR signaling pathway, mitochondrial function, lysosomal acidification and / or autophagy pathway.

[0091] In some embodiments, the composition or formulation is for human or animal use.

[0092] In some embodiments, the composition or formulation is food, feed, culture medium, health food, cosmetic or skin care product.

[0093] In some embodiments, the composition or formulation is in liquid, gel, lyophilized, semi-solid or solid form.

[0094] In some embodiments, the composition or formulation is a drug. In some embodiments, the dosage form of the drug is powder, tablet, capsule, pill, injection, sustained release formulation, ointment or cream.

[0095] In some embodiments, the composition or formulation further comprises a pharmaceutically or food acceptable additive, carrier or excipient.

[0096] In some embodiments, the composition or formulation contains hesperetin-7-O-glucoside in a unit dose of 0.001 - 1000 mg, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 mg of hesperetin-7-O-glucoside.

[0097] In order to make the objectives, technical solutions, and advantages of this application clearer and more understandable, the following further describes this application in combination with specific embodiments and drawings. The advantages and features of this application will become clearer as the description progresses. It should be understood that these embodiments are only used to illustrate this application and not to limit the scope of this application. For the experimental methods without specific conditions indicated in the following embodiments, they are carried out according to the conventional conditions in the art, for example, the conditions described in Molecular Cloning: A Laboratory Manual (Third Edition) (2001), Cold Spring Harbor Laboratory Press, by Sambrook and Russel et al., or according to the conditions recommended by the manufacturer. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are all commercially available.

[0098] Example

[0099] Material preparation

[0100] Prepare nematode growth medium (NGM) plates in the following way: Weigh 3.5 g of NaCl, 2.0 g of peptone, and 25 g of agar powder, add 800 mL of deionized water, fully dissolve and then autoclave (121 °C, 20 min). After cooling to 55 °C, add 1 mL of 1 mol / L CaCl2, 1 mL of 1 mol / L MgSO4, 1 mL of 5 mg / L cholesterol in a laminar flow hood, then add 25 mL of 1 mol / L potassium phosphate solution (pH = 6.0), and make up the volume to 1 L with ddH2O. After mixing evenly, quickly dispense it into culture dishes to obtain the plates.

[0101] Prepare FUdR plates in the following way: Weigh 3.5 g of NaCl, 2.0 g of peptone, and 25 g of agar powder, add 800 mL of deionized water, fully dissolve and then autoclave (121 °C, 20 min). After cooling to 55 °C, add 1 mL of 1 mol / L CaCl2, 1 mL of 1 mol / L MgSO4, 1 mL of 5 mg / L cholesterol, 5 mL of 10 mg / mL 5-fluoro-2'-deoxyuridine (FUdR) in a laminar flow hood, with the final concentration of FUdR being 50 μg / mL, then add 25 mL of 1 mol / L potassium phosphate solution (pH = 6.0), and make up the volume to 1 L with ddH2O. After mixing evenly, quickly dispense it into culture dishes to obtain the plates.

[0102] Package the NGM plates and FUdR plates with sealing film and store them at 4 °C.

[0103] Prepare the M9 buffer as follows: Weigh 0.25 g of MgSO4·7H2O, 3.0 g of KH2PO4, 6.0 g of Na2HPO4, and 5.0 g of NaCl respectively, then add ddH2O to make the volume up to 1 L. Filter it through a 0.45 μm filter membrane in a laminar flow hood, dispense it into sterile containers, and store it at room temperature for later use.

[0104] Prepare the bleach buffer as follows: Mix 10 mL of NaClO (4%), 10 mL of NaOH (1M), and 20 mL of ddH2O evenly, then transfer it to a brown centrifuge tube and place it in the dark at room temperature.

[0105] Example 1. Promotion of the lifespan of nematodes by hesperetin-7-O-glucoside.

[0106] Step 1: Medium preparation.

[0107] Prepare a 20 mg / mL stock solution of hesperetin-7-O-glucoside using DMSO, and dilute it to a 10 μg / mL working solution using Escherichia coli OP50. Make NGM plates coated with a mixture of hesperetin-7-O-glucoside solution and OP50 Escherichia coli (working solution); NGM plates with only OP50 Escherichia coli; plates of the nematode egg-laying medium (FUdR) coated with a mixture of hesperetin-7-O-glucoside solution and OP50 Escherichia coli; and FUdR plates with only OP50 Escherichia coli. Prepare the common nematode strain N2.

[0108] Step 2: Synchronization of nematodes.

[0109] Cultivate the nematodes on NGM plates for 2 - 3 days. On the premise that OP50 is not exhausted, obtain plates rich in eggs. Pipette 1 - 2 mL of M9 buffer onto the NGM plates and rinse repeatedly to ensure that the eggs do not remain on the plates as much as possible. Transfer the liquid containing the eggs to a 1.5 mL centrifuge tube.

[0110] Centrifuge at 4,000 rcf for 1 min and discard the supernatant. Add 500 μL of bleach buffer to the remaining 100 μL of liquid, tighten the tube cap, and shake for 3 - 5 min. Control the time to ensure that no whole nematodes can be seen under the microscope, and at the same time, the eggs are not soaked for too long and inactivated. Centrifuge at 4,000 rcf for 0.5 min and discard the supernatant. Add 1 mL of M9 buffer to the remaining 100 μL of liquid to resuspend the precipitate, centrifuge at 4,000 rcf for 1 min, discard the supernatant, and repeat this step once more.

[0111] For the finally reserved 100 μL of liquid, after pipetting and blowing several times to evenly distribute the precipitate, transfer it to near the bacterial lawn of the new NGM plate prepared in Step 1. After it dries, invert the plate and place it in an incubator for cultivation. The incubator is a constant-temperature incubator at 20°C.

[0112] Step 3: After cultivating the eggs into nematodes at the L4 stage, pick 50 nematodes respectively and place them in the corresponding fresh FUdR plates, with 3 replicates for the nematodes in different feeding groups.

[0113] Step 4: Count the number of dead nematodes every day. A nematode is considered dead if it shows no response after being touched until all the nematodes on the plate are dead.

[0114] The results are as Figure 1 and Figure 2 shown. The results show that compared with the N2 nematodes cultured without adding hesperetin-7-O-glucoside ( Figure 1 shown as the Vehicle group; Figure 2 shown as the control group), the cultivation with hesperetin-7-O-glucoside significantly prolonged the lifespan of the nematodes.

[0115] Example 2. Mechanism of hesperetin-7-O-glucoside in enhancing the lifespan of nematodes.

[0116] Step 1: Use the CRISPR / Cas 9 technology to perform gene editing on Caenorhabditis elegans to knockdown the following nematode genes and obtain the corresponding gene knockdown strains: daf-16(mu86), eat-2(sd1116), aak-2(gt33), vha-3(ok1501), glp-1(e2144), rsks-1(ok1255), clk-1(qm30), atg-7(bp422).

[0117] Prepare heat-killed Escherichia coli (HK E. coli) and spread it onto NGM and FUdR plates respectively.

[0118] Among them, the daf-16(mu86) strain represents the insulin / IGF-1 / FOXO signaling pathway; the eat-2(sd1116) strain represents the calorie restriction pathway; the aak-2(gt33) strain represents the AMPK signaling pathway; the vha-3(ok1501) strain represents the lysosomal acidification and autophagy pathway; the glp-1(e2144) strain represents the Notch signaling pathway of germline stem cells; the rsks-1(ok1255) strain represents the TOR signaling pathway; the clk-1(qm30) strain represents the mitochondrial function pathway; the atg-7(bp422) strain represents the autophagy pathway; the HK E. coli spread plate (adding HK E. coli spread on the basis of the NGM plate and FUdR plate in Example 1) represents the microbial-host interaction pathway.

[0119] Step 2: According to the steps in Example 1, prepare the NGM plate and FUdR plate, and culture the above-mentioned nematode eggs on the NGM plate; at the same time, culture the unedited strain N2 (wild type) on the NGM plate spread with HK E. coli until the L4 stage, then pick 50 worms respectively and place them on the corresponding FUdR plate, and culture them in an incubator. Each nematode in different feeding groups has three replicates.

[0120] Step 3: Count the number of dead nematodes every day. A nematode is considered dead if it shows no response after being touched. Keep counting until all the nematodes on the plate are dead.

[0121] The results are as Figure 3A - Figure 3I shown, where Figure 3A to Figure 3H respectively show the survival curves of Caenorhabditis elegans strains daf-16(mu86) ( Figure 3A ), eat-2(sd1116) ( Figure 3B ), aak-2(gt33) ( Figure 3C ), vha-3(ok1501) ( Figure 3D ), glp-1(e2144) ( Figure 3E ), rsks-1(ok1255) ( Figure 3F ), clk-1(qm30) ( Figure 3G ), atg-7(bp422) ( Figure 3H Figure); Figure 3I shows the survival curve of Caenorhabditis elegans strain N2 fed with heat-killed Escherichia coli HK E. coli.

[0122] The results show that Figure 3C , Figure 3D , Figure 3F , Figure 3G , Figure 3HThe survival curves of the figures were indistinguishable, indicating that after blocking the corresponding pathways, the effect of hesperetin-7-O-glucoside on the lifespan of Caenorhabditis elegans also disappeared accordingly. Thus, it is shown that the extension of the lifespan of Caenorhabditis elegans by hesperetin-7-O-glucoside is regulated by the aak-2, vha-3, rsks-1, clk-1, and atg-7 genes, that is, the extension of the lifespan of Caenorhabditis elegans by hesperetin-7-O-glucoside depends on the AMPK signaling pathway, lysosomal acidification, the TOR signaling pathway, mitochondrial function, and the autophagy pathway.

[0123] Although the present application has been described with reference to the illustrative embodiments, those skilled in the art will understand that various other changes, omissions, and / or additions can be made and elements of the embodiments can be substituted with substantially equivalent ones without departing from the spirit and scope of the present application. Additionally, many modifications can be made to adapt a particular situation or material to the teachings of the present application without departing from the scope of the present application. Therefore, it is not intended to limit the present application to the particular embodiments disclosed for carrying out the present application, but rather the present application is intended to cover all embodiments falling within the scope of the appended claims.

Claims

1. Use of hesperetin-7-O-glucoside in the preparation of a composition or preparation for delaying aging and / or prolonging life.

2. The use according to claim 1, wherein the composition or preparation is used to regulate aak-2 , vha-3 , rsks-1 , clk-1 and / or atg-7 Gene expression to delay aging and / or extend lifespan.

3. The use according to claim 1, wherein the composition or preparation is used to delay aging and / or prolong life by activating the AMPK signaling pathway, the TOR signaling pathway, mitochondrial function, lysosomal acidification and / or the autophagy pathway.

4. The use according to any one of claims 1 to 3, wherein the composition or formulation is for use in humans or animals.

5. The use according to any one of claims 1 to 3, wherein the composition or preparation is a food, a feed, a culture medium, a health food, a cosmetic or a skin care product.

6. The use according to claim 5, wherein the composition or preparation is in the form of liquid, semi-solid or solid.

7. The use according to claim 5, wherein the composition or preparation is in the form of a gel or freeze-dried product.

8. The use according to any one of claims 1 to 3, wherein the composition or formulation is a medicament.

9. The use according to claim 8, wherein the dosage form of the drug is powder, tablet, capsule, pill, injection or ointment.

10. The use according to any one of claims 1 to 3, wherein the composition or preparation further comprises a pharmaceutically or food acceptable additive, carrier or excipient.

11. The use according to any one of claims 1 to 3, wherein the composition or preparation comprises hesperetin-7-O-glucoside in a unit dose of 0.001-1000 mg.

12. The use according to any one of claims 1 to 3, wherein the hesperidin-7-O-glucoside is derived from one or more of citrus, grapefruit, tangerine peel, lemon, bergamot, sour orange, tangerine peel, citron, daidaihua, fructus aurantii, and immature bitter orange.

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