Use of hesperidin-7-o-glucoside and derivatives thereof in the delay of aging

Abstract

The present application relates to the use of hesperetin-7-O-glucoside and derivatives thereof in the delay of aging. Provided is the use of hesperetin-7-O-glucoside or a pharmaceutically or food acceptable salt thereof, or a tautomer, stereoisomer, enantiomer or diastereomer 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 the delay of aging and / or the extension of lifespan.

Description

Technical Field

[0001] This application relates to the pharmaceutical field, specifically to the use of hesperidin-7-O-glucoside and its derivatives. Background Technology

[0002] In the most ideal scenario, lifespan is primarily determined by the rate of aging. Aging is controlled by interconnected processes occurring at the tissue, cellular, molecular, and genetic levels. With age, the structure and function of various tissues and organs undergo irreversible degenerative changes, such as protein degradation, tissue atrophy, decreased metabolic rate, and abnormal calcium metabolism. Accompanying this increase are age-related diseases such as Alzheimer's disease, Parkinson's disease, cardiovascular diseases, and cancer, which are growing exponentially worldwide. With the continuous increase in the number and proportion of the elderly population, and the growing demand for anti-aging and lifespan extension, research into the mechanisms of human aging and the development of anti-aging drugs, within the limits determined by genetic characteristics, is of significant social importance in slowing down the aging process and extending lifespan.

[0003] *Caenorhabditis elegans* is a classic model organism for aging-related research, possessing advantages such as a short lifespan, clear genetic background, simple physiological structure, and ease of culture. Nematodes share many similarities with higher organisms at the cellular and molecular levels, such as mechanisms like apoptosis and signal transduction. Most importantly, nematodes exhibit distinct aging characteristics, including decreased motility, reduced reproductive capacity, and changes in body morphology (such as muscle atrophy and intestinal degeneration), making them easy to study, observe, and quantify.

[0004] There is a need in this field to develop substances that can extend lifespan, and developing such substances based on Caenorhabditis elegans is a feasible approach. Summary of the Invention

[0005] Based on the results of Caenorhabditis elegans, the applicant has for the first time discovered the efficacy of hesperidin-7-O-glucoside and its derivatives in prolonging lifespan, as well as their mechanism of action.

[0006] Using nematodes as an anti-aging model has the following advantages: Since 60%-80% of nematode genes are highly conserved with human genes, and of the 12 signal transduction pathways discovered to date, nematodes possess 12 of them. Therefore, this application utilizes *C. elegans* as a model organism for validating anti-aging drugs. The rich genetic resources of nematodes can be utilized, and suitable mutants can be selected according to research objectives to study aging and anti-aging mechanisms. In fact, several major theories of aging mechanisms have been proven in nematodes. Therefore, drugs that have anti-aging effects on nematodes are generally considered to have the same efficacy in humans.

[0007] Caenorhabditis elegans has been used for lifespan analysis for 30 years. Due to its unique advantages, it has become the preferred model for aging research. The nematode has a short generation cycle, typically around 3 days, and a lifespan of around 3 weeks. This makes the experiments reproducible and stable. To ensure the reliability of experimental methods and the accuracy of results, and to obtain more accurate and reliable information for drug screening, repeated experiments are necessary. Because of these unique advantages, Caenorhabditis elegans has become the preferred model for aging research. Therefore, nematodes can be used to evaluate the anti-aging effects of compositions, and thus determine whether these compositions 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 found in humans and other organisms, whose main function is to regulate cellular energy metabolism and maintain energy homeostasis. It can activate or inhibit multiple cellular metabolic pathways by phosphorylating a series of substrate proteins. AMPK activation can delay the aging process through mechanisms such as regulating cellular energy metabolism and inhibiting oxidative stress and inflammatory responses, and has important anti-aging significance. The vha-3 (ok1501) strain represents the lysosomal acidification and lysosomal autophagy pathway. Reduced expression of vha-3 damages the V-ATPase proton pump on the lysosomal membrane, affecting lysosomal acidification and autophagy functions. 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 functional pathway; knockout of this gene leads to damage to the mitochondrial respiratory chain. atg-7 (bp422) represents the autophagy pathway; ATG7 is an essential protein for the autophagy binding system and autophagosome formation, playing a crucial regulatory role in autophagosome formation.

[0009] The structure of hesperidin-7-O-glucoside is shown below:

[0010] .

[0011] In one aspect, this application provides the use of hesperidin-7-O-glucoside or a pharmaceutically acceptable salt thereof, or tautomers, stereoisomers, enantiomers or non-corresponding isomers thereof, or a prodrug or metabolite thereof, or a natural source of said hesperidin-7-O-glucoside, salt, isomer, prodrug or metabolite, in the preparation of compositions or formulations for delaying aging and / or prolonging life.

[0012] In some embodiments, the composition or formulation is used to delay aging and / or prolong lifespan by regulating 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 prolong lifespan by activating the AMPK signaling pathway, the TOR signaling pathway, mitochondrial function, lysosomal acidification, and / or autophagy pathway.

[0014] In some embodiments, the composition or formulation is used to delay aging or prolong the lifespan of humans or animals.

[0015] In some embodiments, the composition or formulation is a culture medium.

[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 a powder, tablet, capsule, pill, injection, sustained-release formulation, ointment, or cream.

[0018] In some embodiments, the composition or formulation may also include pharmaceutically acceptable additives, carriers, or excipients.

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

[0020] On the other hand, this application provides the use of hesperidin-7-O-glucoside or a pharmaceutically acceptable salt thereof, or tautomers, stereoisomers, enantiomers or non-corresponding isomers thereof, or a prodrug or metabolite thereof, or a natural source of said hesperidin-7-O-glucoside, salt, isomer, prodrug or metabolite in the preparation of compositions or formulations for regulating 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 used on humans or animals.

[0022] In some embodiments, the composition or formulation is a culture medium.

[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 a powder, tablet, capsule, pill, injection, sustained-release formulation, ointment, or cream.

[0025] In some embodiments, the composition or formulation may also include pharmaceutically acceptable additives, carriers, or excipients.

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

[0027] On the other hand, this application provides the use of hesperidin-7-O-glucoside or a pharmaceutically acceptable salt thereof, or tautomers, stereoisomers, enantiomers or non-corresponding isomers thereof, or a prodrug or metabolite thereof, or a natural source of said hesperidin-7-O-glucoside, salt, isomer, prodrug or metabolite, in the preparation of compositions or formulations for activating the AMPK signaling pathway, the TOR signaling pathway, mitochondrial function, lysosomal acidification and / or autophagy pathway.

[0028] In some embodiments, the composition or formulation is used on humans or animals.

[0029] In some embodiments, the composition or formulation is a culture medium.

[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 a powder, tablet, capsule, pill, injection, sustained-release formulation, ointment, or cream.

[0032] In some embodiments, the composition or formulation may also include pharmaceutically acceptable additives, carriers, or excipients.

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

[0034] On the other hand, this application provides compositions for expressing anti-aging and / or life-extending effects, comprising hesperidin-7-O-glucoside or a pharmaceutically acceptable salt thereof, or a tautomer, stereoisomer, enantiomer, or non-corresponding isomer thereof, or a prodrug or metabolite thereof, wherein the hesperidin-7-O-glucoside or a pharmaceutically acceptable salt thereof, or a tautomer, stereoisomer, enantiomer, or non-corresponding isomer thereof, or a prodrug or metabolite thereof, is derived from a plant. In some embodiments, the plant may be pomelo, mandarin orange, tangerine, orange peel, lemon, bergamot, sour orange, tangerine peel, citron, bitter orange, trifoliate orange, immature bitter orange, etc.

[0035] In some embodiments, the composition is used on humans or animals.

[0036] In some embodiments, the composition is a culture medium.

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

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

[0039] In some embodiments, the composition also includes pharmaceutically acceptable additives, carriers, or excipients.

[0040] In some embodiments, the composition contains hesperidin-7-O-glucoside in a unit dose of 0.001-1000 mg. Attached Figure Description

[0041] Figure 1 The survival curve of Caenorhabditis elegans is shown.

[0042] Figure 2 The average lifespan statistics of Caenorhabditis elegans are shown.

[0043] Figures 3A-3I Survival curves of Caenorhabditis elegans under different conditions are shown. Detailed Implementation

[0044] To facilitate understanding of this disclosure, certain terms are defined below. Additional definitions for the following terms and other terms are set forth throughout this specification.

[0045] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly specifies otherwise.

[0046] In this document, the terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., meaning “including but not limited to”).

[0047] In this document, “and / or” means and includes any and all possible combinations of one or more of the associated listed items. For example, “the composition contains A and / or B” can be interpreted as the composition contains A, the composition contains B, or the composition contains both A and B.

[0048] All numerical names, such as pH, temperature, time, concentration, and molecular weight, including ranges, are approximate values, which are appropriately changed in increments of 1.0 or 0.1, or optionally in variations of + / - 15%, 10%, 5%, or 2% (+) or (-). 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 exemplary only, and their equivalents are known in the art. When referring to measurable values ​​such as amount or concentration, the term "about" as used herein means 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" or "containing" and is inclusive of endpoints or open-ended, and does not exclude additional undescribed elements or method steps. "Comprising" is a technical term used in the language of claims, meaning that the stated element is present, but other elements may be added and still form a construction or method within the scope of the claims.

[0050] In this document, the term "administration" is intended to mean the delivery of a substance to a subject, such as an animal or a human. Administration can be performed in a single dose, continuously or intermittently throughout the treatment. Methods for determining the most effective manner and dosage of administration are known to those skilled in the art and will vary depending on factors such as the composition used for treatment, the purpose of treatment, and the age, health, or sex of the subject being treated. In some embodiments, administration may be performed single or multiple times, with the dosage level and pattern chosen by a physician, or, in the case of pets and other animals, by a veterinarian.

[0051] In this document, the term “treatment” includes: (1) suppressing symptoms, disease or condition, i.e., preventing, reducing or delaying the development of a 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 disappearance of at least one of symptoms, disease or condition or its clinical or subclinical symptoms.

[0052] In this article, the term “improvement” refers to the improvement of symptoms related to the disease, and may refer to the improvement of at least one parameter that measures or quantifies the symptom.

[0053] In this document, the term “prevention” of a symptom, disease, or condition includes: preventing, delaying, or reducing the incidence and / or likelihood of developing at least one clinical or subclinical symptom of a symptom, disease, or condition in a subject who may have or be susceptible to the symptom, disease, or condition but has not yet experienced or exhibited clinical or subclinical symptoms of the symptom, disease, or condition.

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

[0055] The term "pharmaceutical composition" generally refers to a formulation that exists in a form that allows for the effective biological activity of the active ingredient and does not contain any additional ingredients that would have unacceptable toxicity to the subject to which the composition will be administered. The composition is sterile. A "sterile" composition is either sterilized or free of all live microorganisms and their spores.

[0056] Exemplary carriers used in the pharmaceuticals of this application include saline, buffered saline, glucose, and water. Exemplary excipients used in the compositions of this application include fillers, binders, disintegrants, coating agents, adsorbents, anti-adhesion agents, flow aids, preservatives, antioxidants, flavoring agents, coloring agents, sweeteners, solvents, co-solvents, buffers, chelating agents, viscosity-improving agents, surfactants, diluents, wetting agents, carriers, preservatives, emulsifiers, stabilizers, and tension modifiers. Those skilled in the art know how to select suitable excipients to prepare the compositions or formulations of this application. Generally, the selection of suitable excipients depends in particular 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 components for administering the compositions or formulations of this application to treat or prevent a related disease. The components of the kit may be contained in separate vials (i.e., kits with separate portions) or provided in a single vial. The kit may contain reagents such as buffers, protein stabilizing agents, signal generation systems (e.g., fluorescence signal generation systems), antibodies, control proteins, and test containers. The kit may also contain instructions for performing the methods. In some embodiments of this application, the kit may also include an administration device capable of administering the pharmaceutically active ingredient (e.g., hesperidin-7-O-glucoside of this application) to a subject in a suitable manner.

[0058] The term "prodrug," also known as a prodrug, drug precursor, or predrug, refers to a compound that can be converted in vivo to the compound of this application or a pharmaceutically acceptable salt, hydrate, or solvate of that compound. This conversion can occur through various mechanisms (e.g., metabolic or chemical processes), such as hydrolysis in the blood, gastrointestinal tract, or lungs. A discussion of the uses of prodrugs is provided in the following: T. Higuchi and W. Stella, "Prodrugs as Novel Delivery Systems," Vol. 14 of the ACS 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 metabolic processes.

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

[0061] The hesperidin-7-O-glucoside or its pharmaceutically acceptable salt, or its tautomers, stereoisomers, enantiomers, or non-corresponding isomers, or its prodrugs or metabolites, in this application may be derived from plants, such as plant extracts, plant powders (e.g., citrus fruit powder), juices, and infusions. Plants may include grapefruit, mandarin orange, tangerine, orange, pomelo, dried tangerine peel, lemon, bergamot, sour orange, tangerine peel, orange peel, citron, bitter orange, trifoliate orange, and immature bitter orange.

[0062] In this application, natural sources of hesperidin-7-O-glucoside and its derivatives may include citrus plants, such as, but not limited to, *Citrus maxima*: pomelos, yuzu, red-fleshed pomelos, grapefruits; *Citrus aurantium*: citron, bergamot, lime, bitter orange; *Citrus japonica*: kumquat, tangerine peel, round kumquat, long-leaf kumquat; as well as citrus fruits, pomelos, dried tangerine peel, lemons, bergamot, bitter orange, tangerine peel, citron, bitter orange blossom, trifoliate orange, trifoliate orange fruit, etc. These natural sources can also be used for the purposes of hesperidin-7-O-glucoside described herein.

[0063] This application provides the use of hesperidin-7-O-glucoside or a pharmaceutically acceptable salt thereof, or tautomers, stereoisomers, enantiomers or non-corresponding isomers thereof, or a prodrug or metabolite thereof, or a natural source of said hesperidin-7-O-glucoside, salt, isomer, prodrug or metabolite, in the preparation of compositions or formulations for delaying aging and / or prolonging life.

[0064] In some embodiments, the composition or formulation is used to delay aging and / or prolong lifespan by regulating the expression of the aak-2 gene.

[0065] In some embodiments, the composition or formulation is used to delay aging and / or prolong lifespan by regulating the expression of the vha-3 gene.

[0066] In some embodiments, the composition or formulation is used to delay aging and / or prolong lifespan by regulating the expression of the rsks-1 gene.

[0067] In some embodiments, the composition or formulation is used to delay aging and / or prolong lifespan by regulating the expression of the clk-1 gene.

[0068] In some embodiments, the composition or formulation is used to delay aging and / or prolong lifespan by regulating the expression of the atg-7 gene.

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

[0070] In this paper, "regulating" gene expression can mean either suppressing or enhancing gene expression.

[0071] In some embodiments, the composition or formulation is used to delay aging and / or prolong lifespan by activating the AMPK signaling pathway.

[0072] In some embodiments, the composition or formulation is used to delay aging and / or prolong lifespan by activating the TOR signaling pathway.

[0073] In some implementations, the composition or formulation is used to delay aging and / or prolong lifespan by activating mitochondrial function.

[0074] In some embodiments, the composition or formulation is used to delay aging and / or prolong lifespan by activating lysosomal acidification.

[0075] In some embodiments, the composition or formulation is used to delay aging and / or prolong lifespan by activating the autophagy pathway.

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

[0077] In some embodiments, the composition or formulation is used to delay aging or prolong the lifespan of humans or animals.

[0078] In some embodiments, the composition or formulation is a culture medium.

[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 a powder, tablet, capsule, pill, injection, sustained-release formulation, ointment, or cream.

[0081] In some embodiments, the composition or formulation may also include pharmaceutically acceptable additives, carriers, or excipients.

[0082] In some embodiments, the composition or formulation contains hesperidin-7-O-glucoside in unit doses 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, and 900 mg.

[0083] On the other hand, this application provides the use of hesperidin-7-O-glucoside or a pharmaceutically acceptable salt thereof, or tautomers, stereoisomers, enantiomers or non-corresponding isomers thereof, or a prodrug or metabolite thereof, or a natural source of said hesperidin-7-O-glucoside, salt, isomer, prodrug or metabolite in the preparation of compositions or formulations for regulating the expression of the aak-2, vha-3, rsks-1, clk-1 and / or atg-7 genes.

[0084] In some embodiments, the composition or formulation is used on humans or animals.

[0085] In some embodiments, the composition or formulation is a culture medium.

[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 a powder, tablet, capsule, pill, injection, sustained-release formulation, ointment, or cream.

[0088] In some embodiments, the composition or formulation may also include pharmaceutically acceptable additives, carriers, or excipients.

[0089] In some embodiments, the composition or formulation contains hesperidin-7-O-glucoside in unit doses 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, and 900 mg.

[0090] On the other hand, this application provides the use of hesperidin-7-O-glucoside or a pharmaceutically acceptable salt thereof, or tautomers, stereoisomers, enantiomers or non-corresponding isomers thereof, or a prodrug or metabolite thereof, or a natural source of said hesperidin-7-O-glucoside, salt, isomer, prodrug or metabolite, in the preparation of compositions or formulations for activating the AMPK signaling pathway, the TOR signaling pathway, mitochondrial function, lysosomal acidification and / or autophagy pathway.

[0091] In some embodiments, the composition or formulation is used on humans or animals.

[0092] In some embodiments, the composition or formulation is a culture medium.

[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 a powder, tablet, capsule, pill, injection, sustained-release formulation, ointment, or cream.

[0095] In some embodiments, the composition or formulation may also include pharmaceutically acceptable additives, carriers, or excipients.

[0096] In some embodiments, the composition or formulation contains hesperidin-7-O-glucoside in unit doses 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, and 900 mg.

[0097] To make the objectives, technical solutions, and advantages of this application clearer, the application is further described below in conjunction with specific embodiments and accompanying drawings. The advantages and features of this application will become clearer with the description. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional conditions in the art, such as those described in Sambrook and Russeii et al., Molecular Cloning: A Laboratory Manual (Third Edition) (2001), CSHL Press, or according to the manufacturer's recommendations. Unless otherwise stated, the experimental materials and reagents used in the following embodiments are commercially available.

[0098] Example

[0099] Material preparation

[0100] Nematode growth medium (NGM) plates were prepared as follows: 3.5 g NaCl, 2.0 g peptone, and 25 g agar powder were weighed and added to 800 mL of deionized water. After thorough dissolution, the mixture was autoclaved (121℃, 20 min). After cooling to 55℃, 1 mL of 1 mol / L CaCl2, 1 mL of 1 mol / L MgSO4, and 1 mL of 5 mg / L cholesterol were added in a clean bench. Then, 25 mL of 1 mol / L potassium phosphate solution (pH = 6.0) was added, and the volume was adjusted to 1 L with ddH2O. The mixture was thoroughly mixed and quickly dispensed into petri dishes.

[0101] FUdR plates were prepared as follows: Weigh 3.5 g NaCl, 2.0 g peptone, and 25 g agar powder, add 800 mL of deionized water, dissolve thoroughly, and autoclave (121℃, 20 min). After cooling to 55℃, add 1 mL of 1 mol / L CaCl2, 1 mL of 1 mol / L MgSO4, 1 mL of 5 mg / L cholesterol, and 5 mL of 10 mg / mL 5-fluoro-2'-deoxyguanosine (FUdR) in a clean bench, bringing the final FUdR concentration to 50 μg / mL. Then add 25 mL of 1 mol / L potassium phosphate solution (pH = 6.0) and bring the volume to 1 L with ddH2O. Mix well and quickly dispense into petri dishes to obtain the final FUdR plate.

[0102] NGM and FUdR plates were sealed in film and stored at 4°C.

[0103] Prepare M9 buffer as follows: Weigh 0.25 g MgSO4·7H2O, 3.0 g KH2PO4, 6.0 g Na2HPO4, and 5.0 g NaCl, respectively, and add ddH2O to bring the volume to 1 L. Filter the buffer using a 0.45 μm filter membrane in a clean bench, aliquot the solution into sterile containers, and store at room temperature for later use.

[0104] Prepare bleach buffer as follows: Mix 10 mL NaClO (4%), 10 mL NaOH (1M) and 20 mL ddH2O thoroughly, then transfer to a brown centrifuge tube and store at room temperature away from light.

[0105] Example 1. The promoting effect of hesperidin-7-O-glucoside on the lifespan of nematodes.

[0106] Step 1: Culture medium preparation.

[0107] Prepare a 20 mg / mL stock solution of hesperidin-7-O-glucoside using DMSO, and dilute it to a working solution of 10 μg / mL using E. coli OP50. Prepare NGM plates coated with a mixture of hesperidin-7-O-glucoside solution and OP50 E. coli (working solution); NGM plates containing only OP50 E. coli; FUdR (Full Form Restriction Nematode Oviposition Medium) plates coated with a mixture of hesperidin-7-O-glucoside solution and OP50 E. coli; and FUdR plates containing only OP50 E. coli. Prepare a common nematode strain N2.

[0108] Step 2: Synchronization of nematodes.

[0109] Cultured nematodes on NGM plates for 2-3 days to obtain plates rich in nematode eggs, provided that OP50 is not depleted. Using a pipette, 1-2 mL of M9 buffer was added to the NGM plate and the plate was repeatedly rinsed to ensure that no nematode eggs remained on the plate. The liquid containing nematode eggs was then transferred 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 cap, and shake for 3-5 min. The time should be controlled to ensure that the entire nematode is not visible under the microscope, but also to avoid prolonged soaking of the eggs and inactivation. 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] The final 100 μL of liquid was pipetted several times to ensure even distribution of the precipitate, then transferred to the vicinity of the bacterial growth on a new NGM plate prepared in step one. After drying, the plate was inverted and placed in an incubator at a constant temperature of 20°C.

[0112] Step 3: After the nematodes are cultured to the L4 stage, 50 nematodes are selected and placed in the corresponding fresh FUdR plates, with 3 replicates for each feeding group.

[0113] Step 4: Count the number of dead nematodes daily. If there is no reaction after touching, the nematode is dead. Continue until all nematodes in the plate are dead.

[0114] The results are as follows Figure 1 and Figure 2 As shown. The results show that compared to cultures without the addition of hesperidin-7-O-glucoside ( Figure 1 Displayed as the Vehicle group; Figure 2 N2 nematodes (shown as the control group) showed significantly prolonged lifespan when cultured with hesperidin-7-O-glucoside.

[0115] Example 2. Mechanism by which hesperidin-7-O-glucoside enhances the lifespan of nematodes.

[0116] Step 1: Gene editing of Caenorhabditis elegans was performed using CRISPR / Cas 9 technology to knock down the following nematode genes, resulting in the following gene knockdown strains: daf-16 (mu86), eat-2 (sd1116), aak-2 (gt33), vha-3 (ok1501), glp-1 (e2144), rsks-1 (ok1255), clk-1 (qm30), and atg-7 (bp422).

[0117] Prepare heat-sterilized Escherichia coli (HK E. coli) and spread them 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 germline stem cell Notch signaling pathway; 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; and the HK E. coli plating (adding HK E. coli plating to the NGM and FUdR plating in Example 1) represents the microbial host interaction pathway.

[0119] Step 2: Following the steps in Example 1, prepare NGM and FUdR plates, and culture the nematode eggs on the NGM plates. Simultaneously, culture the unedited N2 (wild-type) nematode strain on NGM plates coated with HK E. coli until the L4 stage. Then, select 50 nematodes from each group and place them on the corresponding FUdR plates for incubation. Three replicates of nematodes from each feeding group were used.

[0120] Step 3: Count the number of dead nematodes daily. If there is no reaction after touching, the nematode is dead. Continue until all nematodes in the plate are dead.

[0121] The results are as follows Figures 3A-3I As shown, where Figures 3A to 3H The images show the Caenorhabditis elegans strain 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 Survival curves (Figure 1); Figure 3I Survival curves of Caenorhabditis elegans strain N2 fed with heat-killed Escherichia coli (HK E. coli) are shown.

[0122] The results showed that Figure 3C , Figure 3D , Figure 3F , Figure 3G , Figure 3HThe lack of difference in the survival curves indicates that the effect of hesperidin-7-O-glucoside on the lifespan of *C. elegans* disappeared after the corresponding pathway was blocked. This suggests that the lifespan extension of *C. elegans* by hesperidin-7-O-glucoside is regulated by the aak-2, vha-3, rsks-1, clk-1, and atg-7 genes, meaning that the lifespan extension of *C. elegans* by hesperidin-7-O-glucoside depends on the AMPK signaling pathway, lysosomal acidification, the TOR signaling pathway, mitochondrial function, and autophagy.

[0123] Although this application has been described with reference to illustrative embodiments, those skilled in the art will understand that various other changes, omissions, and / or additions can be made without departing from the spirit and scope of this application, and that elements of the described embodiments can be substituted with substantially equivalents. Furthermore, many modifications can be made without departing from the scope of this application to adapt particular situations or materials to the teachings of this application. Therefore, this application is not intended to be limited to the specific embodiments disclosed for carrying out this application, but rather is intended to include all embodiments falling within the scope of the appended claims.

Claims

1. Use of hesperidin-7-O-glucoside in the preparation of formulations for life extension.

2. The use according to claim 1, wherein the formulation is used for humans or animals.

3. The use according to claim 1 or 2, wherein the formulation is in liquid, semi-solid or solid form.

4. The use according to claim 1 or 2, wherein the formulation is in gel or lyophilized form.

5. The use according to claim 1 or 2, wherein the formulation is a drug.

6. The use according to claim 5, wherein the dosage form of the drug is powder, tablet, capsule, pill, injection or ointment.

7. The use according to claim 5, wherein the formulation further comprises a pharmaceutically acceptable excipient.

8. The use according to claim 1 or 2, wherein the formulation further comprises a pharmaceutically acceptable additive or carrier.

9. The use according to claim 1 or 2, wherein the formulation comprises hesperidin-7-O-glucoside in a unit dose of 0.001-1000 mg.

10. The use according to claim 1 or 2, wherein the hesperidin-7-O-glucoside is derived from citrus.

11. The use according to claim 1 or 2, wherein the hesperidin-7-O-glucoside is derived from dried tangerine peel.

12. The use according to claim 1 or 2, wherein the hesperidin-7-O-glucoside is derived from one or more of bitter orange blossom, bitter orange peel, and bitter orange fruit.

13. The use according to claim 1 or 2, wherein the hesperidin-7-O-glucoside is derived from one or more of grapefruit, lemon, bergamot, lime, and citron.

14. The use according to claim 1 or 2, wherein the hesperidin-7-O-glucoside is derived from Citrus reticulata peel.

15. The use according to claim 1 or 2, wherein the hesperidin-7-O-glucoside is derived from orange peel.

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