Use of queuine in Anti-aging

Abstract

The present disclosure provides the use of Queuine, or a pharmaceutically acceptable salt thereof, or a composition comprising the same or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for delaying and / or mitigating aging of a subject or prolonging the lifetime of a subject.

Description

Use of queuine in anti-aging

[0001] This application claims priority to Chinese Patent Application CN202411295149.7 filed on September 15, 2024 and Chinese Patent Application CN202511184895.3 filed on August 22, 2025. TECHNICAL FIELD

[0002] The present disclosure belongs to the field of medicine, and specifically relates to the use of Queuine, or a pharmaceutically acceptable salt thereof, or a composition comprising the same or the pharmaceutically acceptable salt thereof in the preparation of a medicament for delaying and / or improving aging of a subject or prolonging the life of a subject. BACKGROUND

[0003] Aging is a complex physiological process, usually manifested as gradual decline in organ function, and increase in disease incidence and mortality risk. As of the end of 2023, there are 210 million people aged 65 and above in China, accounting for 15.4% of the total population. The process of population aging is rapid, and a series of health and social problems that follow are gradually highlighted. It is reported that the global population aged 65 and above will reach 1.6 billion by 2050. Therefore, delaying biological aging or prolonging healthy life is a timeless theme for mankind and one of the hotspots of scientists' research.

[0004] As early as 2013, Carlos et al. systematically summarized the characteristics of aging and proposed various markers of aging, including genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient-sensing, mitochondrial dysfunction, stem cell exhaustion, and altered intercellular communication, disabled macroautophagy, chronic inflammation, and dysbiosis (Lopez-Otin, C., et al. (2023). Hallmarks of aging: An expanding universe. Cell, 186(2), 243-278.). These characteristics are closely related and interdependent, and have great significance for understanding the occurrence, development, and intervention of the aging process.

[0005] Epigenetic alterations are one of the important markers of aging. Currently, research has mainly focused on epigenetic alterations associated with aging, including changes in DNA methylation levels, abnormalities in histone post-translational modifications, and chromatin remodeling. At the same time, RNA epigenetic regulation has gradually become a new field of concern in recent years, as an important part of epigenetics, it may play a key role in the aging process. Recent research has revealed the potential impact of RNA modifications such as RNA methylation in aging, but the relationship between specific types of RNA modifications, such as tRNA modifications, and aging remains to be further studied, and many issues remain unresolved.

[0006] tRNA is an important participant in protein translation and is also the most abundant RNA species containing epigenetic modifications. Epigenetic modifications on tRNA are of great significance for the regulation of translation rate and the guarantee of translation accuracy. According to statistics, more than 100 human diseases are closely related to mutations of tRNA modification enzymes, including cancer, cardiovascular disease, genetic birth defects, metabolic disease, nervous system disease and mitochondrial-related defects. It is striking that some tRNA modification enzymes are involved in the regulation of cell aging and lifespan. Therefore, it is speculated that tRNA modification may play a crucial role in the aging and longevity of the body, and the regulation of modification changes at the molecular level may have the effect of delaying aging. In this context, the study of tRNA modification seems to provide new insights into the extension of lifespan. However, the research on the related molecular mechanisms is still in its infancy, especially in the intervention of tRNA modification and the extension of lifespan.

[0007] Queuine (CAS No: 72496-59-4, its hydrochloride salt CAS No: 69565-92-0) is a 7-deazaguanosine derivative, which is directly involved in the synthesis of various tRNA epigenetic modifications, including Queuosine (mainly present in tRNA-His and tRNA-Asn), gal-Queuosine (galQ, mainly present in tRNA-Tyr) and man-Queuosine (manQ, mainly present in tRNA-Asp). These modifications play an important regulatory role in the function of tRNA, which in turn affects the regulation of physiological and pathological states. However, there is still a lack of research on the role and mechanism of these Queuine-related tRNA modifications, especially manQ and galQ, in the aging process. SUMMARY

[0008] The purpose of the present disclosure is to provide a new way for anti-aging by supplementing Queuine, or its pharmaceutically acceptable salt, or a composition comprising the same or the pharmaceutically acceptable salt thereof, for delaying and / or improving aging or extending lifespan, in view of the current situation of drugs for delaying and / or improving aging of a subject or extending lifespan of a subject.

[0009] In one aspect, the present disclosure provides a use of Queuine, or its pharmaceutically acceptable salt, or a composition comprising the same or the pharmaceutically acceptable salt thereof, in the preparation of a medicament for delaying and / or improving aging of a subject or extending lifespan of a subject.

[0010] In another aspect, the present disclosure provides a method of delaying and / or improving aging in a subject or prolonging lifespan in a subject, comprising administering to the subject an effective amount of Queuine, or a pharmaceutically acceptable salt thereof, or a composition comprising the same or the pharmaceutically acceptable salt thereof.

[0011] In some embodiments, the aging is selected from one or more of cellular aging, organ aging, and physiological system aging.

[0012] In some embodiments, the cellular aging is selected from one or more of epithelial cells, connective tissue cells, muscle cells, neural cells, blood cells, germ cells, stem cells, immune cells, sensory cells (such as cone cells, rod cells, and hair cells, etc.), and glandular cells.

[0013] In some embodiments, the organ aging is selected from one or more of heart, liver, spleen, lung, kidney, brain, thymus, pancreas, skin, bone, etc.

[0014] In some embodiments, the system aging is selected from one or more of motor system, nervous system, endocrine system, cardiovascular system, lymphatic system, respiratory system, digestive system, urinary system, reproductive system, immune system, integumentary system, and sensory system.

[0015] In some embodiments, the subject is selected from mammals and non-mammals.

[0016] In some embodiments, the subject is at least about 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 years old.

[0017] In some embodiments, the medicament comprises at least about 300 ng of the Queuine or the salt thereof.

[0018] In some embodiments, the Queuine or the salt thereof, or the composition is administered at a dose of at least about 10 ng / kg, or the composition is administered at a dose in which the active ingredient comprises at least about 10 ng / kg of the Queuine or the salt thereof.

[0019] In some embodiments, the Queuine or the salt thereof, or the composition can be administered by enteral means, such as oral means, for example, in the form of tablets, emulsions, or capsules, or by parenteral means, such as in the form of injectable solutions or suspensions, by topical means, such as in the form of lotions, gels, ointments, or creams, or in the form of nasal agents or suppositories.

[0020] In some embodiments, Queuine or a pharmaceutically acceptable salt thereof, or a composition is used in combination with a second therapeutic agent in the manufacture of a medicament for delaying and / or ameliorating aging in a subject or for prolonging the life of a subject.

[0021] In some embodiments, the second therapeutic agent is selected from one or more of rapamycin, beta-nicotinamide mononucleotide (NMN), metformin, urolithin A, vitamin C, taurine, spermidine, quercetin, resveratrol, and other dietary supplements or drugs with anti-aging effects.

[0022] Queuine or a pharmaceutically acceptable salt thereof, or a composition can be administered in a conventional manner, in particular enterally, for example orally, for example in tablet, emulsion or capsule form, or parenterally, for example in the form of an injectable solution or suspension, topically, for example in the form of a lotion, gel, ointment or cream, or in the form of a nasal or suppository formulation, in the form of a pharmaceutical composition. In one aspect, when Queuine or a pharmaceutically acceptable salt thereof, or a composition is administered in combination (simultaneously or separately) with another therapeutic agent as described herein, both components can be administered by the same route (e.g. parenterally). Alternatively, the other therapeutic agent is administered by a different route relative to Queuine or a pharmaceutically acceptable salt thereof, or a composition. For example, Queuine or a pharmaceutically acceptable salt thereof, or a composition can be administered orally and the other therapeutic agent administered parenterally.

[0023] In some embodiments, the life of the subject is prolonged by at least about 5%.

[0024] In some embodiments, Queuine or a salt thereof is administered at a dose of at least about 10 ng / kg, or a composition is administered at a dose in which the active ingredient comprises at least about 10 ng / kg of the Queuine or salt thereof.

[0025] In some embodiments, Queuine or a salt thereof, or a composition can be administered enterally, for example orally, for example in tablet, emulsion or capsule form, or parenterally, for example in the form of an injectable solution or suspension, topically, for example in the form of a lotion, gel, ointment or cream, or in the form of a nasal or suppository formulation.

[0026] In some embodiments, Queuine or a pharmaceutically acceptable salt thereof, or a composition is administered in combination with a second therapeutic agent for delaying and / or ameliorating aging in a subject or for prolonging the life of a subject.

[0027] In some embodiments, Queuine or a salt thereof is administered at a dose of at least about 10 ng / kg daily, every two days, every three days, weekly, every two weeks, every three weeks, monthly, every two months, every three months, every six months, or every year, or a composition is administered at a dose in which the active ingredient comprises at least about 10 ng / kg of said Queuine or a salt thereof daily, every two days, every three days, weekly, every two weeks, every three weeks, monthly, every two months, every three months, every six months, or every year.

[0028] In another aspect, the present disclosure provides a method of treating paraquat poisoning comprising administering to a subject an effective amount of Queuine, or a pharmaceutically acceptable salt thereof, or a composition comprising the same or the same.

[0029] In another aspect, the present disclosure provides a use of Queuine, or a pharmaceutically acceptable salt thereof, or a composition comprising the same or the same in the manufacture of a detoxifying agent for treating paraquat poisoning.

[0030] In another aspect, the present disclosure provides a method of treating metabolic bone disease comprising administering to a subject an effective amount of Queuine, or a pharmaceutically acceptable salt thereof, or a composition comprising the same or the same.

[0031] In another aspect, the present disclosure provides a use of Queuine, or a pharmaceutically acceptable salt thereof, or a composition comprising the same or the same in the manufacture of a medicament for treating metabolic bone disease (e.g., osteoporosis).

[0032] The present disclosure first found that the content of manQ modification significantly decreased in old rats, mice, Drosophila aging models and the elderly, and the content of Queuine also decreased in the plasma of old rats. And the survival rate test of Drosophila and mouse aging models confirmed that Queuine has the effect of delaying aging and improving survival rate. The present disclosure can compensate for the decrease of Queuine-related modification in the aging process by exogenous Queuine supplement, and recover the biological function of manQ modification; through the behavioral indicators such as growth and development, motor ability, heat stress resistance, olfactory memory and antioxidant capacity of Drosophila parents and offspring, it is confirmed that Queuine has a significant anti-aging effect on natural aging mice. Comprehensive analysis shows that Queuine has good safety and can improve the behavior of elderly individuals and improve the health of the body, and can be used as an active ingredient of anti-aging drugs. The experiments of the present disclosure prove that Queuine has the activity of prolonging the lifespan of paraquat-induced mouse multi-organ failure aging model mice and improving the survival rate, and the activity is stronger with the increase of the dose. Queuine can be used as a detoxifying agent for paraquat poisoning. The experiments of the present disclosure also prove that the plasma of the Queuine intervention group mice significantly up-regulates the bone metabolism related substances, especially the content of Glucosamine and its derivative D-Glucosamine 1-phosphate which has the effect of anti-osteoporosis, suggesting that Queuine may delay aging-related bone degradation by enhancing bone synthetic metabolic pathways. Queuine can be used for the treatment of metabolic bone diseases (such as osteoporosis). BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 shows the qualitative and quantitative detection results of oligonucleotide fragments derived from tRNA in the kidneys of old (36 months old) and adult (6 months old) rats. Among them: (A) a heat map drawn with the content of oligonucleotide fragments in old and adult rats; (B) a volcano plot drawn with the content of oligonucleotide fragments in old and adult rats; (C) MS2 mapping qualitative example of tRNA oligonucleotide fragments (taking the fragment with the largest content difference as an example).

[0034] Figure 2 shows the quantitative detection results of oligonucleotide fragment CUC[manQ]UCA[m 5 C]G in rat kidneys. Among them: (A) total ion current (EIC) chromatogram of liquid chromatography-mass spectrometry detection of the oligonucleotide fragment in the kidneys of rats of different ages; (B) comparison of the content of the fragment in rats of different ages.

[0035] Figure 3 shows the relative content of oligonucleotide fragment CUC[manQ]UCA[m 5 C]G in multiple organs of old and adult rats.

[0036] Figure 4 shows the changes in the content of manQ in different month-old mice (A), different day-old fruit flies (B) and different age humans (C, D).

[0037] Figure 5 shows the changes in the content of Queuine in the plasma of different month-old rats (A) and different age humans (B).

[0038] Figure 6 shows the effects of adding Queuine on the lifespan (A) and survival rate (B) of fruit flies.

[0039] Figure 7 shows the effects of adding Queuine on manQ in fruit flies.

[0040] Figure 8 shows the effects of adding Queuine on the body weight of female fruit flies (A) and male fruit flies (B).

[0041] Figure 9 shows the effects of adding Queuine on the movement ability of fruit flies.

[0042] Figure 10 shows the effects of adding Queuine on the survival rate of fruit flies after high-temperature stimulation.

[0043] Figure 11 shows the effects of adding Queuine on the olfactory memory of fruit flies.

[0044] Figure 12 shows the effects of adding Queuine on the activities of antioxidant enzymes SOD (A) and GSH-Px (B) in fruit flies.

[0045] Figure 13 shows the EIC map (A) and relative quantitative results (B) of manQ content detected by liquid chromatography-mass spectrometry in paraquat-induced acute aging mice.

[0046] Figure 14 shows the effects of adding Queuine on the survival rate (A) and aging factors (B) of acute aging model mice.

[0047] Figure 15 shows the effects of adding Queuine on the content of free Queuine in the plasma of mice (A) and Queuosine in the kidneys of mice (B).

[0048] Figure 16 shows the effects of adding Queuine on the appearance of naturally aging mice.

[0049] Figure 17 shows the effects of adding Queuine on the blood routine of naturally aging mice, wherein: (A) leukocytes; (B) lymphocytes; (C) platelets; (D) red blood cells.

[0050] Figure 18 shows the effects of adding Queuine on the inflammatory factors of naturally aging mice.

[0051] Figure 19 shows the effect of adding Queuine on telomerase activity in naturally aging mice.

[0052] Figure 20 shows the effect of adding Queuine on the activity of antioxidant enzymes SOD (A) and GSH (B) in naturally aging mice.

[0053] Figure 21 shows the effect of adding Queuine on aging factors in naturally aging mice.

[0054] Figure 22 shows the effect of adding Queuine on open field score (A) and open field location (B) in naturally aging mice.

[0055] Figure 23 shows the effect of adding Queuine on the aging rate of naturally aging mice, where: (A) methylation age; (B) age acceleration analysis.

[0056] Figure 24 illustrates the multisystem rejuvenation of naturally aging mice induced by Queuine addition. (A) Cognitive function assessment: Novel Object Recognition Test (NORT, left) and Object Place Memory Test (OLT, right). (B) Exercise endurance performance. (C) Liver function indicators. (D) Blood glucose levels. (E) Blood lipid analysis. (F) Cardiac enzyme levels. (G) Renal function biomarkers.

[0057] Figure 25 shows tRNA in the kidneys of aged (36 months) and adult (6 months) rats. Asp(manQUC) Qualitative and quantitative detection results of monomers. (A) tRNA Asp(manQUC) (B) tRNA in rat kidney Asp(manQUC) The purification process. Right figure: urea-PAGE electrophoresis results (lane 1: low molecular weight labeling; lane 2: miRNA labeling; lanes 3–4: tRNA from 6-month-old and 36-month-old rats, respectively). Asp(manQUC) (C) TIC chromatograms of rat samples from 6-month-old and 36-month-old rats. (D) tRNA Asp (manQUC) unconvolution mass spectrum. (E) tRNA after RNase T1 digestion. Asp(manQUC) UHPLC-QTOF-MS chromatogram of the fragment. (F) Purified tRNA Asp(manQUC) UHPLC-QQQ-MS analysis results of manQ nucleoside.

[0058] Figure 26 shows Queuine alleviates paraquat-induced senescence in mice. (A) Lung histopathology: H&E staining. (B) Lung edema index: wet / dry weight ratio. (C) Lung and liver appearance comparison. (D) Hepatoprotective effect: serum liver function biomarker AST level. (E) Hepatoprotective effect: serum liver function biomarker ALP level. (F) Lung inflammation: cytokine level.

[0059] Figure 27 shows Queuine addition extends lifespan in naturally aging mice. (A) Dosing regimen: Queuine (10 pg / kg) was orally administered to 16-month-old C57BL / 6J male mice. (B) Lifespan extension effect: Kaplan-Meier survival curve of naturally aging mice (n = 30; log-rank test, p < 0.001).

[0060] Figure 28 shows the study of the relationship between synthetic enzyme GTDC1 and manQ modification and aging. (A) Schematic diagram of the biosynthetic pathway of manQ modification in tRNA in humans. (B) Expression changes of manQ-related enzymes in different age groups: the left panel shows the relative expression analysis in human peripheral leukocytes (young vs. old donors); the right panel shows the relative expression analysis in rat kidneys (6 months vs. 36 months). (C) Construct 2BS cell model to compare the expression changes of p16 / p21 in the 30th generation (young phenotype) and the 39th generation (senescent phenotype) cells. (D) Differences in manQ expression levels in the 30th and 39th generations of 2BS cells. (E) Changes in GTDC1 expression levels after knocking down GTDC1 in the 30th generation of 2BS cells using CRISPR / Cas9. (F) Changes in manQ expression levels after knocking down GTDC1. (G) Expression of senescence-related markers after knocking down GTDC1. (H) Results of β-galactosidase staining after knocking down GTDC1.

[0061] Figure 29 shows the study of the relationship between hydrolytic enzyme Man2c1 and manQ modification and aging. (A) Changes in Man2c1 expression levels after increasing Man2c1 in the 30th generation of 2BS cells using plasmid overexpression method. (B) Changes in manQ expression levels after overexpressing Man2c1. (C) Expression of senescence-related markers after overexpressing Man2c1. (D) Changes in manQ expression levels after knocking down Man2c1 expression levels in the 39th generation of 2BS cells using siRNA technology. (E) Expression of senescence-related markers after knocking down Man2c1.

[0062] Figure 30 shows the effect of Queuine addition on manQ level. Among them, (A) the effect of manQ promotion on 39th generation senescent 2BS cells; (B) the effect of manQ promotion on paraquat acute senescent mice; (C) the effect of manQ promotion on old fruit flies (30 days old).

[0063] Figure 31 shows the regulatory effect of Queuine addition on key metabolites related to aging. Among them: (A) the level of glucosamine (Glucosamine) and its derivative D- glucosamine 1-phosphate; (B) the level of anti-inflammatory metabolites and various specific pro-inflammatory resolution mediators; (C) the level of kynurenine; (D) the level of nicotinamide riboside.

[0064] Figure 32 shows (A) the appearance of Queuine-containing microemulsion; (B) Queuine- containing microemulsion appears Tyndall effect under laser pen irradiation.

[0065] Figure 33 shows the particle size and particle size distribution of Queuine-containing microemulsion.

[0066] Figure 34 shows (A) the appearance of Queuine-containing chitosan nanoparticles; (B) Queuine-containing chitosan nanoparticles appear Tyndall effect under laser pen irradiation.

[0067] Figure 35 shows the particle size and particle size distribution of Queuine-containing chitosan nanoparticles.

[0068] Figure 36 shows the average plasma concentration-time curve of Queuine in rats under different administration methods (n = 6; mean ± standard deviation). Among them: (A) intravenous injection of free Queuine low-dose group; (B) intravenous injection of free Queuine high-dose group; (C) oral administration of free Queuine low-dose group; (D) oral administration of free Queuine high-dose group; (E) oral administration of Queuine enteric-coated capsules low-dose group; (F) oral administration of Queuine enteric-coated capsules high-dose group; (G) oral administration of Queuine microemulsion low-dose group; (H) oral administration of Queuine microemulsion high-dose group; (I) oral administration of Queuine chitosan nanoparticles low-dose group; (J) oral administration of Queuine chitosan nanoparticles high-dose group. All low-dose groups are 0.5 mg / kg, and high-dose groups are 2 mg / kg.

[0069] Figure 37 shows the effect of Queuine hydrochloride intervention on the levels of 2BS cell senescence markers p16 and p21. DETAILED DESCRIPTION

[0070] In the present disclosure, the scientific and technical terms used herein have the meanings commonly understood by a person of ordinary skill in the art, unless otherwise indicated. Also, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology, and laboratory operation procedures used herein are those widely used in the corresponding fields and the conventional procedures. Meanwhile, for better understanding of the present disclosure, the definitions and explanations of the related terms are provided as follows.

[0071] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0072] As used herein, the terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to").

[0073] As used herein, the term "about" when used in reference to a numerical value or range allows for a certain degree of variation, for example, within 10% or within 5% of the stated value or the stated range.

[0074] Herein, "anti-aging" includes two aspects: one aspect is to delay and / or improve the aging of a subject; the other aspect is to prolong the life span of a subject.

[0075] As used herein, the term "aging" refers to the irreversible changes in the structure and function of an organism that gradually occur over time. Aging involves Cellular Senescence, Aging of organs, and Aging of the system.

[0076] As used herein, the term "cellular senescence" refers to a process in which cells irreversibly stop dividing and enter a permanent growth-arrested state without undergoing cell death. The term "cellular senescence" is selected from senescence characterized by currently recognized aging hallmarks, including: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient-sensing, mitochondrial dysfunction, stem cell exhaustion, and altered intercellular communication, disabled macroautophagy, chronic inflammation, and dysbiosis.

[0077] As used herein, the term "organ senescence" refers to the functional decline and degenerative phenomena of each organ in the body, such as the heart, liver, spleen, lung, kidney, brain, thymus, pancreas, skin, bone, etc. over time. Organ senescence not only includes the characteristics of cellular senescence, but also includes changes in the physiological structure and function of organs (Hainan Bao, et al. Biomarkers of aging. Science China. Life sciences. 2023 May, 66(5):893-1066).

[0078] As used herein, the term "physiological system senescence" refers to the phenomenon of reduced movement, cognition, metabolism, transport, respiration, absorption, excretion, and reproductive ability caused by the functional degradation of physiological systems of the body, including the motor system, nervous system, endocrine system, cardiovascular system, lymphatic system, respiratory system, digestive system, urinary system, reproductive system, immune system, skin system, and sensory system. System senescence encompasses the connections between organs and is a higher-dimensional manifestation.

[0079] As used herein, the term "life span" refers to the normal length of time of an organism's life.

[0080] As used herein, the term "natural aging" refers to the inevitable physiological and biochemical changes that occur in an organism as it ages. Unlike induced aging models, natural aging is a slow and continuous process that every organism goes through.

[0081] As used herein, the term "metabolic bone disease" refers to a disease that affects the metabolic processes of bone tissue. Metabolic bone diseases include pathologic bone demineralization, pathologic decalcification, lipoidosis, osteoporosis, pseudohypoparathyroidism, rickets, chronic kidney disease-mineral and bone disorder, osteomalacia, and hypophosphatemic rickets. Among them, osteoporosis includes postmenopausal osteoporosis.

[0082] As used herein, the term "delaying" refers to the slowing and / or stopping of the rate of progression of an individual from an existing younger or older age to a more aged state by an effective intervention. Such "delaying" can be at any stage of an individual's life and by affecting and modulating the biological mechanisms associated with aging, the stability of the individual's physiological functions and structures are prolonged, thereby slowing the progression of further decline.

[0083] As used herein, the term "improving" refers to the lessening of an individual's aged state by an effective intervention, or the restoration and / or reversal of an individual from a more severe aged state to a less severe aged state or a younger state. Such "improving" can be at any stage of an individual's life and by affecting and modulating the biological mechanisms associated with aging, the physiological and functional changes associated with aging are alleviated and / or reversed, thereby enhancing the overall health, quality of life, and physiological functions of the individual.

[0084] As used herein, the terms "patient," "individual," and "subject" are used interchangeably and refer to a human or animal patient, individual, and subject, and includes a living organism that is using a compound or pharmaceutical composition or undergoing delaying and / or improving aging or extending lifespan by a method as provided herein. Non-limiting examples include humans, other mammals, bovine, rats, mice, dogs, monkeys, goats, sheep, cows, deer, and other non-mammalian animals. However, it is to be understood that "patient" does not imply the presence of symptoms. In some embodiments, the patient is a human. In some embodiments, the subject is a human.

[0085] As used herein, the term "young adult" refers to a human who is older than 18 years of age and younger than 25 years of age.

[0086] As used herein, the term "middle-aged adult" refers to a human who is between 25 and 60 years of age.

[0087] As used herein, the term "elderly adult" refers to a human who is older than 60 years of age.

[0088] According to the methods provided herein, an effective amount of one or more of the agents provided herein (e.g., Queuine or a pharmaceutically acceptable salt thereof, or a composition comprising the same) is administered to the subject. An “effective amount” is an amount sufficient to achieve the stated purpose (e.g., to delay and / or ameliorate one or more symptoms of aging in a subject, or to extend the lifespan of a subject). An example of an “effective amount” is an amount sufficient to contribute to the delay and / or amelioration of one or more symptoms of aging in a subject, which can also be referred to as a “therapeutically effective amount.” For example, for a given parameter, a therapeutically effective amount will show at least about a 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100% increase or decrease. Therapeutic efficacy can also be expressed as a “fold” increase or decrease. For example, a therapeutically effective amount can have at least about a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect relative to a control.

[0089] As used herein, the term “administering” generally refers to the application of a compound or composition to a subject or system, to achieve delivery of the agent to the subject or system. Those of ordinary skill in the art will appreciate the various routes that can be used to administer to a subject (e.g., a human) as appropriate. For example, in some embodiments, the administration can be ocular, oral, parenteral, topical, etc. In some particular embodiments, the administration can be bronchial (e.g., by bronchial instillation), buccal, cutaneous (which can be or include, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteric, gastric, intramuscular, intranasal, intraperitoneal, within a particular organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), etc. In many embodiments provided by the present disclosure, the administration is oral administration. In some embodiments, the administration can involve only a single dose. In some embodiments, the administration can involve the application of a fixed number of doses. In some embodiments, the administration can involve intermittent (e.g., multiple doses separated in time) and / or periodic (e.g., individual doses separated by the same time period) dosing. In some embodiments, the administration can involve continuous dosing (e.g., perfusion) for at least a selected time period.

[0090] Herein, the rate of longevity is calculated as follows:

[0091] Rate of longevity = (average lifespan of dosed group - average lifespan of control group) / average lifespan of control group x 100%

[0092] In one aspect, the present disclosure provides the use of Queuine, or a pharmaceutically acceptable salt thereof, or a composition comprising the same or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for delaying and / or ameliorating aging in a subject or extending the lifespan of a subject.

[0093] In another aspect, the present disclosure provides a method of delaying and / or improving aging in a subject or prolonging lifespan in a subject, comprising administering to the subject an effective amount of Queuine, or a pharmaceutically acceptable salt thereof, or a composition comprising the same or the pharmaceutically acceptable salt thereof.

[0094] In some embodiments, the aging is selected from one or more of cellular aging, organ aging, and physiological system aging.

[0095] In some embodiments, the cellular aging is selected from one or more of epithelial cells, connective tissue cells, muscle cells, neural cells, blood cells, germ cells, stem cells, immune cells, sensory cells, and glandular cells.

[0096] In some embodiments, the organ aging is selected from one or more of heart, liver, spleen, lung, kidney, brain, thymus, pancreas, skin, bone, and the like.

[0097] In some embodiments, the Queuine level and / or the level of man-Queuosine modification in RNA of the subject is reduced or not reduced as compared to a control. The Queuine level and the level of man-Queuosine modification in RNA are obtained by detecting a biological sample. In some embodiments, the biological sample is selected from one or more of blood, heart, liver, spleen, lung, kidney, brain, and tissue.

[0098] In some embodiments, the Queuine level and / or the level of man-Queuosine modification in RNA of the subject is not reduced.

[0099] In some embodiments, the aging is an aging characterized by a decrease in the amount of manQ-modified tRNA in the subject; or the aging is an aging caused by a decrease in the amount of manQ-modified tRNA in the subject.

[0100] In some embodiments, the physiological system aging is selected from one or more of motor system, nervous system, endocrine system, cardiovascular system, lymphatic system, respiratory system, digestive system, urinary system, reproductive system, immune system, integumentary system, and sensory system.

[0101] In some embodiments, the subject is selected from a mammal and a non-mammal.

[0102] In some embodiments, the subject is a mammal.

[0103] In some embodiments, the subject is selected from a human, a cow, a dog, a monkey, a goat, a sheep, a dairy cow, a deer, a rat, a mouse, and other mammals.

[0104] In some embodiments, the subject is a human.

[0105] In some embodiments, the subject is at least about 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 years old.

[0106] In some embodiments, the subject is between about 16 and 95 years old, or between about 18 and 95 years old.

[0107] In some embodiments, the subject is between about 50 and 65 years old, or between about 60 and 75 years old, or between about 70 and 85 years old, or between about 80 and 95 years old.

[0108] In some embodiments, the subject is a middle-aged person between 25-60 years old.

[0109] In some embodiments, the subject is a young adult between 18 years old and below 25 years old.

[0110] In some embodiments, the subject is an elderly person over 60 years old.

[0111] In some embodiments, the subject is an elderly person over 65 years old.

[0112] In some embodiments, the medicament comprises at least about 300 ng of the Queuine or salt thereof.

[0113] In some embodiments, the medicament comprises at least about 500 ng, 750 ng, 1 pg, 2 pg, 3 pg, 4 pg, 5 pg, 6 pg, 9 pg, 15 pg, 20 pg, 25 pg, 30 pg, 35 pg, 40 pg, 45 pg, 50 pg, 100 pg, 150 pg, 250 pg, 500 pg, 750 pg, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.50 mg, 5 mg, 6 mg, 9 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 100 mg, 150 mg, 250 mg, or 500 mg of the Queuine or salt thereof.

[0114] In some embodiments, the Queuine or salt thereof is administered at a dose of at least about 10 ng / kg, or the composition is administered at a dose in which the active ingredient comprises at least about 10 ng / kg of the Queuine or salt thereof.

[0115] In some embodiments, Queuine or a salt thereof is administered, or a composition comprising at least about 10 ng to 10 mg / kg of said Queuine or a salt thereof as an active ingredient is administered, in a dose of at least about 10 ng to 10 mg / kg.

[0116] In some embodiments, Queuine or a salt thereof is administered at a dose of at least about 10 ng / kg, 15 ng / kg, 20 ng / kg, 25 ng / kg, 30 ng / kg, 35 ng / kg, 40 ng / kg, 45 ng / kg, 50 ng / kg, 55 ng / kg, 60 ng / kg, 65 ng / kg, 70 ng / kg, 75 ng / kg, 80 ng / kg, 85 ng / kg, 90 ng / kg, 95 ng / kg, 100 ng / kg, 120 ng / kg, 150 ng / kg, 180 ng / kg, 200 ng / kg, 250 ng / kg, 300 ng / kg, 350 ng / kg, 400 ng / kg, 450 ng / kg, 500 ng / kg, 550 ng / kg, 600 ng / kg, 650 ng / kg, 700 ng / kg, 750 ng / kg, 800 ng / kg, 850 ng / kg, 900 ng / kg, 950 ng / kg, 1 pg / kg, 2 pg / kg, 3 pg / kg, 5 pg / kg, 10 pg / kg, 15 pg / kg, 20 pg / kg, 25 pg / kg, 30 pg / kg, 35 pg / kg, 40 pg / kg, 45 pg / kg, 50 pg / kg, 55 pg / kg, 60 pg / kg, 65 pg / kg, 70 pg / kg, 75 pg / kg, 80 pg / kg, 85 pg / kg, 90 pg / kg, 95 pg / kg, 100 pg / kg, 120 pg / kg, 150 pg / kg, 180 pg / kg, 200 pg / kg, 250 pg / kg, 300 pg / kg, 350 pg / kg, 400 pg / kg, 450 pg / kg, 500 pg / kg, 550 pg / kg, 600 pg / kg, 650 pg / kg, 700 pg / kg, 750 pg / kg, 800 pg / kg, 850 pg / kg, 900 pg / kg, 950 pg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 55 mg / kg, 60 mg / kg, 65 mg / kg, 70 mg / kg, 75 mg / kg, 80 mg / kg, 85 mg / kg, 90 mg / kg, 95 mg / kg, 100 mg / kg, 120 mg / kg, 150 mg / kg, 180 mg / kg, 200 mg / kg, 250 mg / kg, 300 mg / kg, 350 mg / kg, 400 mg / kg, 450 mg / kg, 500 mg / kg, 550 mg / kg, 600 mg / kg, 650 mg / kg, 700 mg / kg, 750 mg / kg, 800 mg / kg, 850 mg / kg, 900 mg / kg, 950 mg / kg, 1 g / kg, 2 g / kg, 3 g / kg, 5 g / kg, 10 g / kg, 15 g / kg, 20 g / kg, 25 g / kg, 30 g / kg, 35 g / kg, 40 g / kg, 45 g / kg, 50 g / kg, 55 g / kg, 60 g / kg, 65 g / kg, 70 g / kg, 75 g / kg, 80 g / kg, 85 g / kg, 90 g / kg, 95 g / kg, 100 g / kg, 120 g / kg, 150 g / kg, 180 g / kg, 200 g / kg, 250 g / kg, 300 g / kg, 350 g / kg, 400 g / kg, 450 g / kg, 500 g / kg, 550 g / kg, 600 g / kg, 650 gor at least about 10 ng / kg, 15 ng / kg, 20 ng / kg, 25 ng / kg, 30 ng / kg, 35 ng / kg, 40 ng / kg, 45 ng / kg, 50 ng / kg, 55 ng / kg, 60 ng / kg, 65 ng / kg, 70 ng / kg, 75 ng / kg, 80 ng / kg, 85 ng / kg, 90 ng / kg, 95 ng / kg, 100 ng / kg, 120 ng / kg, 150 ng / kg, 180 ng / kg, 200 ng / kg, 250 ng / kg, 300 ng / kg, 350 ng / kg, 400 ng / kg, 450 ng / kg, 500 ng / kg, 550 ng / kg, 600 ng / kg, 650 ng / kg, 700 ng / kg, 750 ng / kg, 800 ng / kg, 850 ng / kg, 900 ng / kg, 950 ng / kg, 1 μg / kg, 2 μg / kg, 3 μg / kg, 5 μg / kg, 10 μg / kg, 15 μg / kg, 20 μg / kg, 25 μg / kg, 30 μg / kg, 35 μg / kg, 40 μg / kg, 45 μg / kg, 50 μg / kg, 55 μg / kg, 60 μg / kg, 65 μg / kg, 70 μg / kg, 75 μg / kg, 80 μg / kg, 85 μg / kg, 90 μg / kg, 95 μg / kg, 100 μg / kg, 120 μg / kg, 150 μg / kg, 180 μg / kg, 200 μg / kg, 250 μg / kg, 300 μg / kg, 350 μg / kg, 400 μg / kg, 450 μg / kg, 500 μg / kg, 550 μg / kg, 600 μg / kg, 650 μg / kg, 700 μg / kg, 750 μg / kg, 800 μg / kg, 850 μg / kg, 900 μg / kg, 950 μg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg of the active ingredient.

[0117] In some embodiments, the Queuine or salt thereof, or composition, can be administered by enteral means, e.g., orally, e.g., in tablet, emulsion, or capsule form, or by parenteral means, e.g., in injectable solution or suspension form, topically, e.g., in lotion, gel, ointment, or cream form, or in nasal or suppository form.

[0118] In some embodiments, the Queuine or salt thereof, or composition, is formulated for oral administration.

[0119] In some embodiments, the Queuine or salt thereof, or composition is a food, beverage, feed composition, or nutritional supplement.

[0120] In some embodiments, the Queuine or salt thereof, or composition is a liquid, syrup, emulsion, tablet, lozenge, gummy, capsule, powder, gel, or film.

[0121] In some embodiments, the Queuine or pharmaceutically acceptable salt thereof, or composition is used in combination with a second therapeutic agent in the manufacture of a medicament for delaying and / or improving aging in a subject or prolonging the life of a subject.

[0122] In some embodiments, the second therapeutic agent is selected from one or more of rapamycin, beta-nicotinamide mononucleotide (NMN), metformin, urolithin A, vitamin C, taurine, spermidine, quercetin, resveratrol, and other dietary supplements or drugs with anti-aging effects.

[0123] In some embodiments, the subject administered with the Queuine, or salt thereof, or composition lives at least over 10 days, 20 days, 1 month, 2 months, 3 months, half a year, 1 year, 2 years, 3 years, 5 years, 10 years, 20 years, or 30 years longer than a control not administered with the Queuine, or salt thereof, or composition.

[0124] In some embodiments, the subject administered with the Queuine, or salt thereof, or composition lives at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, or 90% or more longer than a control not administered with the Queuine, or salt thereof, or composition.

[0125] In some embodiments, the subject administered with the Queuine, or salt thereof, or composition experiences a delay or improvement in symptoms of aging of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 100%, 200%, or more compared to a control not administered with the Queuine, or salt thereof, or composition.

[0126] In some embodiments, the Queuine, or salt thereof, or composition can also be used in the manufacture of one or more of the following:

[0127] (1) improving learning ability in a subject;

[0128] (2) improving memory in a subject;

[0129] (3) improving cognitive ability in a subject;

[0130] (4) improving the exercise capacity of the subject;

[0131] (5) improving the antioxidant capacity of the subject;

[0132] (6) improving the anti-heat stress capacity of the subject.

[0133] In some embodiments, the improvement is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or more than the control level.

[0134] In another aspect, the present disclosure is a method of treating paraquat poisoning comprising administering to a subject an effective amount of Queuine, or a pharmaceutically acceptable salt thereof, or a composition comprising the same or the pharmaceutically acceptable salt thereof.

[0135] In another aspect, the present disclosure is a use of Queuine, or a pharmaceutically acceptable salt thereof, or a composition comprising the same or the pharmaceutically acceptable salt thereof, in the preparation of a detoxifying agent for treating paraquat poisoning.

[0136] In another aspect, the present disclosure provides a method of treating metabolic bone disease comprising administering to a subject an effective amount of Queuine, or a pharmaceutically acceptable salt thereof, or a composition comprising the same or the pharmaceutically acceptable salt thereof.

[0137] In another aspect, the present disclosure is a use of Queuine, or a pharmaceutically acceptable salt thereof, or a composition comprising the same or the pharmaceutically acceptable salt thereof, in the preparation of a drug for treating osteoporosis.

[0138] In some embodiments, the metabolic bone disease is osteoporosis.

[0139] In some embodiments, the Queuine or salt thereof is administered at a dose of at least about 10 ng / kg, or the composition is administered at a dose in which the active ingredient comprises at least about 10 ng / kg of the Queuine or salt thereof.

[0140] In some embodiments, the Queuine or salt thereof is administered at a dose of at least about 10 ng-10 mg / kg, or the composition is administered at a dose in which the active ingredient comprises at least about 10 ng-10 mg / kg of the Queuine or salt thereof.

[0141] In some embodiments, Queuine or a salt thereof is administered at a dose of at least about 10 ng / kg, 15 ng / kg, 20 ng / kg, 25 ng / kg, 30 ng / kg, 35 ng / kg, 40 ng / kg, 45 ng / kg, 50 ng / kg, 55 ng / kg, 60 ng / kg, 65 ng / kg, 70 ng / kg, 75 ng / kg, 80 ng / kg, 85 ng / kg, 90 ng / kg, 95 ng / kg, 100 ng / kg, 120 ng / kg, 150 ng / kg, 180 ng / kg, 200 ng / kg, 250 ng / kg, 300 ng / kg, 350 ng / kg, 400 ng / kg, 450 ng / kg, 500 ng / kg, 550 ng / kg, 600 ng / kg, 650 ng / kg, 700 ng / kg, 750 ng / kg, 800 ng / kg, 850 ng / kg, 900 ng / kg, 950 ng / kg, 1 pg / kg, 2 pg / kg, 3 pg / kg, 5 pg / kg, 10 pg / kg, 15 pg / kg, 20 pg / kg, 25 pg / kg, 30 pg / kg, 35 pg / kg, 40 pg / kg, 45 pg / kg, 50 pg / kg, 55 pg / kg, 60 pg / kg, 65 pg / kg, 70 pg / kg, 75 pg / kg, 80 pg / kg, 85 pg / kg, 90 pg / kg, 95 pg / kg, 100 pg / kg, 120 pg / kg, 150 pg / kg, 180 pg / kg, 200 pg / kg, 250 pg / kg, 300 pg / kg, 350 pg / kg, 400 pg / kg, 450 pg / kg, 500 pg / kg, 550 pg / kg, 600 pg / kg, 650 pg / kg, 700 pg / kg, 750 pg / kg, 800 pg / kg, 850 pg / kg, 900 pg / kg, 950 pg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 55 mg / kg, 60 mg / kg, 65 mg / kg, 70 mg / kg, 75 mg / kg, 80 mg / kg, 85 mg / kg, 90 mg / kg, 95 mg / kg, 100 mg / kg, 120 mg / kg, 150 mg / kg, 180 mg / kg, 200 mg / kg, 250 mg / kg, 300 mg / kg, 350 mg / kg, 400 mg / kg, 450 mg / kg, 500 mg / kg, 550 mg / kg, 600 mg / kg, 650 mg / kg, 700 mg / kg, 750 mg / kg, 800 mg / kg, 850 mg / kg, 900 mg / kg, 950 mg / kg, 1 g / kg, 2 g / kg, 3 g / kg, 5 g / kg, 10 g / kg, 15 g / kg, 20 g / kg, 25 g / kg, 30 g / kg, 35 g / kg, 40 g / kg, 45 g / kg, 50 g / kg, 55 g / kg, 60 g / kg, 65 g / kg, 70 g / kg, 75 g / kg, 80 g / kg, 85 g / kg, 90 g / kg, 95 g / kg, 100 g / kg, 120 g / kg, 150 g / kg, 180 g / kg, 200 g / kg, 250 g / kg, 300 g / kg, 350 g / kg, 400 g / kg, 450 g / kg, 500 g / kg, 550 g / kg, 600 g / kg, 650 gor at least about 10 ng / kg, 15 ng / kg, 20 ng / kg, 25 ng / kg, 30 ng / kg, 35 ng / kg, 40 ng / kg, 45 ng / kg, 50 ng / kg, 55 ng / kg, 60 ng / kg, 65 ng / kg, 70 ng / kg, 75 ng / kg, 80 ng / kg, 85 ng / kg, 90 ng / kg, 95 ng / kg, 100 ng / kg, 120 ng / kg, 150 ng / kg, 180 ng / kg, 200 ng / kg, 250 ng / kg, 300 ng / kg, 350 ng / kg, 400 ng / kg, 450 ng / kg, 500 ng / kg, 550 ng / kg, 600 ng / kg, 650 ng / kg, 700 ng / kg, 750 ng / kg, 800 ng / kg, 850 ng / kg, 900 ng / kg, 950 ng / kg, 1 μg / kg, 2 μg / kg, 3 μg / kg, 5 μg / kg, 10 μg / kg, 15 μg / kg, 20 μg / kg, 25 μg / kg, 30 μg / kg, 35 μg / kg, 40 μg / kg, 45 μg / kg, 50 μg / kg, 55 μg / kg, 60 μg / kg, 65 μg / kg, 70 μg / kg, 75 μg / kg, 80 μg / kg, 85 μg / kg, 90 μg / kg, 95 μg / kg, 100 μg / kg, 120 μg / kg, 150 μg / kg, 180 μg / kg, 200 μg / kg, 250 μg / kg, 300 μg / kg, 350 μg / kg, 400 μg / kg, 450 μg / kg, 500 μg / kg, 550 μg / kg, 600 μg / kg, 650 μg / kg, 700 μg / kg, 750 μg / kg, 800 μg / kg, 850 μg / kg, 900 μg / kg, 950 μg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg of the active ingredient.

[0142] In some embodiments, the Queuine or salt thereof, or composition, can be administered by enteral means, e.g., orally, e.g., in tablet, emulsion, or capsule form, or by parenteral means, e.g., in injectable solution or suspension form, topically, e.g., in lotion, gel, ointment, or cream form, or in nasal or suppository form.

[0143] In some embodiments, the Queuine or salt thereof, or composition, is formulated for oral administration.

[0144] In some embodiments, the Queuine or salt thereof, or composition is a food, beverage, feed composition, or nutritional supplement.

[0145] In some embodiments, the Queuine or salt thereof, or composition is a liquid, syrup, emulsion, tablet, lozenge, gummy, capsule, powder, gel, or film.

[0146] In some embodiments, the Queuine or pharmaceutically acceptable salt thereof, or composition is administered in combination with a second therapeutic agent for delaying and / or improving aging in a subject or extending the life of a subject. In some embodiments, the second therapeutic agent is selected from one or more of rapamycin, beta-nicotinamide mononucleotide (NMN), metformin, urolithin A, vitamin C, taurine, spermidine, quercetin, resveratrol, and other dietary supplements or drugs with anti-aging effects. The Queuine or pharmaceutically acceptable salt thereof, or composition is administered at the same time as, immediately before, or immediately after administration of the second therapeutic agent. The Queuine or pharmaceutically acceptable salt thereof, or composition can be administered to the patient separately or co-administered.

[0147] In some embodiments, the Queuine or salt thereof is administered or the composition is administered at least once, twice, or three times a day, every two days, every three days, every week, every two weeks, every three weeks, every month, every two months, every three months, every half year, or every year at a dose of at least about 10 ng / kg, or at a dose of active ingredient comprising at least about 10 ng / kg of the Queuine or salt thereof.

[0148] In some embodiments, the Queuine or salt thereof is administered or the composition is administered at least once, twice, or three times a day, every two days, every three days, every week, every two weeks, every three weeks, every month, every two months, every three months, every half year, or every year at a dose of at least about 10 ng / kg, or at a dose of active ingredient comprising at least about 10 ng / kg of the Queuine or salt thereof.

[0149] For purposes of clarity and brevity, features described as part of some embodiments herein are described as part of the same or separate embodiments, however, it is to be understood that the scope of the disclosure can include embodiments having combinations of all or some of the features described.

[0150] Hereinafter, the present disclosure will be described in greater detail with reference to specific examples, however, the examples are for illustrative purposes only and do not limit the present disclosure.

[0151] Examples

[0152] Main experimental materials and reagents:

[0153] Queuine (CAS: 72496-59-4, Catalog No. Q525000) was purchased from TRC Toronto Research Chemicals, Inc. Sucrose (CAS: 57-50-1, Catalog No. S8271), Yeast (ATCC 9763, Catalog No. LA9380) and Red Blood Cell Lysis Buffer (Catalog No. R1010) were purchased from Beijing Solabio Biotech Co., Ltd. Propionic acid (CAS: 79-09-4, Catalog No. 94425), Methylparaben (CAS: 99-76-3, Catalog No. 1432005), Ammonium acetate (CAS: 631-61-8, Catalog No. AX1222), Hexafluoroisopropanol (CAS: 920-66-1, Catalog No. 105228-500G), Triethylamine (CAS: 121-44-8, Catalog No. 471283-500ML), Methanol (CAS: 67-56-1, Catalog No. 1.06035, LC-MS grade) and Acetonitrile (CAS: 75-05-8, Catalog No. 1.00029, LC-MS grade) were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. Paraquat (CAS: 1910-42-5, Catalog No. M106760-100mg) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. TRIzol (Catalog No. 15596018), RevertAid™ First Strand cDNA Synthesis Kit (Catalog No. K16225), mirVana™ miRNA kit (Catalog No. AM1561), RNAse T1 (1000 U / μL, Catalog No. EN0541) and Bacterial Alkaline Phosphatase (2500 U, Catalog No. 18011015) were purchased from Shanghai Thermo Fisher Scientific Co., Ltd. RNAse P1 (10000 U, Catalog No. M0660S) was purchased from Beijing Anuoer Biological Technology Co., Ltd. Superoxide dismutase SOD kit (Catalog No. A001-3-2) and Glutathione Peroxidase GSH-PX kit (Catalog No. A005-1-2) were purchased from Nanjing Jiancheng Biological Engineering Institute. SYBR Green qPCR Mix (Catalog No. D7260-25ml) was purchased from Shanghai Biyun Tian Biotechnology Co., Ltd. Mouse Telomerase ELISA Kit (Catalog No. ml024416) and Mouse IL-6 ELISA Kit (Catalog No. ml001532) were purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd.

[0154] Table 1. Primer sequences

[0155] In the absence of specific sources of reagents in this document, such reagents can be obtained from any molecular biology reagent supplier, with quality / purity standards appropriate for molecular biology.

[0156] Changes of manQ-modified tRNA fragments in different stages of animal and human life cycle

[0157] 1.1 Collection of animal and clinical samples

[0158] (1) Collection of rat and mouse samples

[0159] Six 2-month-old, 6-month-old, 24-month-old and 36-month-old male SD rats of SPF level were purchased from Zhuhai Baiti Tong Biotechnology Co., Ltd. Fifteen 6-month-old and 20-month-old female Balb / c mice of SPF level were purchased from Bao Saigu Biotechnology Co., Ltd. The animals were raised in the clean animal room of the State Key Laboratory of Traditional Chinese Medicine Quality Research, University of Macau. The ambient temperature was maintained at 24-26°C, the humidity was 45-65%, and the light environment was 12h light and 12h darkness alternately. The animals could freely eat and drink water. After 3 days of adaptive feeding, the peripheral blood of SD rats and Balb / c mice was collected in a sodium citrate anticoagulant tube. The blood samples were centrifuged at 3000 r / min for 10 min to separate the plasma, which was stored in a -20°C refrigerator. After blood collection, the animals were sacrificed, and the organs such as heart, liver, spleen, lung, kidney and brain were collected, fully homogenized and ground, and then soaked in TRIzol and stored in a -80°C refrigerator.

[0160] (2) Collection of Drosophila melanogaster samples

[0161] Wild-type Drosophila melanogaster strain was purchased from Qidong Fangjing Biotechnology Co., Ltd. and cultured in standard yeast-cornmeal-sucrose medium (H2O 120 mL, cornmeal 12 g, sucrose 8 g, dry yeast 1.5 g, agar powder 1 g, propionic acid 0.5 mL and 10% nipagin methyl ester 0.5 mL). The incubator was set at a temperature of (25±1) °C and a humidity of (55±3)%, with a light / dark cycle of 12h / 12h. After brief CO2 anesthesia, Drosophila within 48h of new eclosion were collected and fed at a density of 20 per tube. During the experiment, they were transferred to fresh medium every 3 days. 120mg of Drosophila were collected on the 3rd, 10th and 30th days, fully homogenized and ground, and then soaked in TRIzol and stored in a -80°C refrigerator. In all experiments, the Drosophila were treated in accordance with the Guide for the Care and Use of Laboratory Animals: Eighth Edition (ISBN-8: 978-0-309-18663-6). The animal experiments in this work were based on all ethical regulations.

[0162] (3) Collection of clinical samples

[0163] Immediately after blood sample collection (EDTA anticoagulation), plasma and blood cells were separated by centrifugation at 3000 r / min for 10 min. The intermediate leukocyte layer was lysed with erythrocyte lysis buffer to remove excess erythrocytes, and then soaked in TRIzol and stored at -80℃.

[0164] 1.2 RNA extraction and sample pretreatment

[0165] After thawing the TRIzol-lysed tissue samples at room temperature, centrifuge at 12000g for 2 min to remove underlying tissue debris, and extract and separate small RNA using the mirVana™ miRNA kit.

[0166] RNA samples were hydrolyzed with RNase T1 enzyme (50 U / g RNA) in a 200 mM ammonium acetate buffer system for 2 h to obtain tRNA oligonucleotide fragments with guanine nucleotides at the 3' end. The RNA samples were then fully hydrolyzed with RNase P1 enzyme (100 U / g RNA) and bacterial alkaline phosphatase (10 U / g RNA) in reaction buffer for 8 h to obtain nucleoside samples.

[0167] 1.3 Chromatographic conditions and instrument parameters

[0168] (1) UHPLC-QTOF-MS

[0169] tRNA oligonucleotide fragment mass spectrometry analysis was performed using an Agilent UHPLC 1290 system and an Agilent 6545 time-of-flight mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). The oligonucleotide fragments were separated using UPLC-OST C. 18 column( The column was 2.1 × 100 mm, 1.7 μm, Waters, MA, USA. The mobile phase consisted of water (A) and 50% methanol (V / V) (B), with 100 mM hexafluoroisopropanol and 15 mM triethylamine (pH 8.5) added to the mobile phase. Elution was performed at a flow rate of 0.2 mL / min using a gradient elution program: 1.50 min for 1% B, 1.5–15 min for 1%–19% B, 15–18.5 min for 19–30% B, and 18.5–22 min for 30%. The column temperature was 60 °C, and the injection volume was 20 μL.

[0170] Qualitative and quantitative analysis was performed in negative mode using automatic MS / MS data acquisition mode. The MS1 scan range was m / z 600-3200 with a scan rate of 2 spectra / s, and the MS2 scan range was m / z 300-1800 with a scan rate of 3 spectra / s, and the collision energy was 10-65 eV. The instrument parameters were as follows: dry gas temperature 320 °C, flow rate 12 L / min, nebulizer pressure 35 psi, nitrogen sheath gas temperature 350 °C, flow rate 12 L / min, capillary voltage 3500 V, nozzle voltage 1700 V, fragmenter voltage 220 V, and the isolator voltage 65 V, and the ion funnel RF peak 750 V.

[0171] (2) UHPLC-QQQ-MS / MS

[0172] The mass spectrometric analysis of nucleosides and Queuine was performed using an Agilent UHPLC 1290 system and an Agilent 6490 triple quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). The separation of nucleosides was performed using an Agilent Poroshell 120 EC-C 18 column (2.7 μm, 4.6 x 100 mm) with a mobile phase consisting of 0.1% formic acid in water (V / V) (A) and 0.1% formic acid in acetonitrile (V / V) (B) at a flow rate of 0.4 mL / min, and a gradient elution program of 0.00-2.50 min, 1.5% B; 2.51-6.00 min, 1.5%-4% B; 6.01-12.00 min, 4%-15% B; 12.01-16.00 min, 15%-45% B; 16.01-20.00 min, 45% B. The column temperature was 30 °C, and the injection volume was 2 μL.

[0173] The 6490 ESI QQQ-MS was operated in positive ion mode with the following instrument parameters: dry gas temperature 200 °C, flow rate 14 L / min, nebulizer pressure 35 psi, sheath gas temperature 350 °C, sheath gas flow rate 11 L / min, capillary voltage 3000, and nozzle voltage 1500 V. The quantitative analysis of nucleosides and Queuine was performed in multiple reaction monitoring (MRM) mode: Man-Queuosine (m / z 572.1→163.1), Queuine (m / z 278.1→163.1).

[0174] 1.4 Experimental results

[0175] (1) The manQ-modified tRNA fragment was significantly down-regulated in aged rats

[0176] After the small RNA in each organ of different month-old SD rats was extracted and separated, the oligonucleotide fragments were obtained by RNase T1 enzyme digestion and analyzed by UHPLC-QTOF-MS. It was found that the oligonucleotide fragments of young mice (2 months old) and adult mice (6 months old) had a high degree of similarity, and the similarity of 24-month-old and 36-month-old old mice was high. Therefore, adult mice and 36-month-old old mice were selected for further study. By analyzing the oligonucleotide fragments in the kidney of adult mice and 36-month-old old mice, 52 tRNA fragments were located (Figure 1A), and the differential fragment CUC[manQ]UCA[m 5 C]G was significantly down-regulated in old mice (Figure 1B). The secondary mass spectrum could further determine the oligonucleotide sequence fragment (Figure 1C).

[0177] The differential fragment CUC[manQ]UCA[m 5 C]G (Figure 2A) in the kidney of rats of different months was quantitatively analyzed, and it was found that the fragment increased first and then decreased in the young (2 months old), adult (6 months old) and old (24 months old and 36 months old) stages (Figure 2B), and the content was very low in old individuals. Peter et al. reported that the content of manQ in multiple organs of adult individuals was significantly higher than that of young individuals (Thumbs P, et al. Synthesis of Galactosyl-Queuosine and Distribution of Hypermodified Q-Nucleosides in Mouse Tissues. Angew Chem Int Ed Engl. 2020, 59(30): 12352-12356), which is consistent with the results of the present embodiment. The inventors' research first found that manQ was significantly down-regulated in old mice.

[0178] The differential fragment CUC[manQ]UCA[m 5 C]G was detected in the heart, liver, spleen, lung, kidney and brain of adult rats (6 months old) and old rats (36 months old). The results showed that compared with adult rats, the differential fragment was significantly reduced in multiple organs of old rats (Figure 3).

[0179] (2) The manQ-modified tRNA fragment is also significantly down-regulated in other aging models

[0180] In order to systematically study the universality of manQ modification changes in other classic aging models, the inventors systematically collected the kidneys of aging mice, aging fruit flies and human peripheral blood leukocytes of different ages, extracted and separated small RNA, and analyzed the oligonucleotide fragments or mononucleotides by UHPLC-QQQ-MS / MS after cutting by restriction enzyme. The results are shown in Figure 4.

[0181] The manQ-modified fragment in the kidney of old mice (20 months old) was significantly lower than that in adult mice (6 months old) (Figure 4A); the change of manQ in the fruit fly model was basically similar to that in rats, the newly molted fruit fly (3 days old) had a low manQ content, the adult fruit fly (10 days old) had the highest manQ content, and the old fruit fly (30 days old) had a significantly reduced manQ (Figure 4B); the distribution of manQ in different age groups was roughly normally distributed, the manQ content in the young population (20-40 years old) was generally high, and there was a trend that women were higher than men, and the manQ content in people over 60 years old was significantly reduced (Figures 4C and 4D). It can be seen that the reduction of manQ-modified content generally occurs in old fruit flies, mice, rats and humans, and has a certain universality.

[0182] From the above experiments, it can be seen that the tRNA fragment containing manQ modification is significantly reduced in aging animals and old people.

[0183] Example 2: Change of Queuine in the plasma of aging animals

[0184] Take 200 μL of animal and human plasma samples prepared in Example 1, denature the protein therein by adding 800 μL of acetonitrile, and centrifuge at 4000 r / min for 10 min to remove the lower protein precipitate. Collect the supernatant, dry under nitrogen, and then re-dissolve in 50 μL of water, and then analyze Queuine. UHPLC-QQQ-MS was used to detect the Queuine content in the plasma of rats of different months (Figure 5A) and people of different ages (Figure 5B), and the results are shown in Figure 5. The UHPLC-QQQ-MS chromatography method and mass spectrometry parameters are the same as in Example 1. The highest Queuine content in rat plasma was 21.98 ng / mL (6 months old), and the lowest was 1.32 ng / mL (2 months old), and the Queuine content in rat plasma showed a trend of first increasing and then decreasing in the juvenile, adult and old stages. Among them, Annie et al. showed that the Queuine content in the liver of adult individuals was significantly higher than that of juvenile individuals (Costa A, et al. Determination of queuosine derivatives by reverse-phase liquid chromatography for the hypomodification study of Q-bearing tRNAs from various mammal liver cells. J Chromatogr B. 2004, 801(2): 237-247), which is consistent with the results of this example. The Queuine content in the plasma of people of different ages also showed a trend of significantly reduced Queuine content in old people.

[0185] The source of Queuine in animals and humans includes food and intestinal microorganisms, but the amount of Queuine ingested by different age rats through food is uniform, so the reason for the difference in the content of Queuine in rats of different ages may be: (1) The intestinal environment of young individuals is relatively immature, and the abundance of the intestinal flora is low, which leads to relatively less Queuine provided by the intestinal flora to the body; the intestinal function of old individuals is declining, and the diversity of the flora is disordered, which also leads to a decrease in the content of Queuine supplied by intestinal microorganisms to the body. (2) The functional development of young individuals is not perfect, and the utilization rate of exogenous Queuine is low, which leads to a limited amount of Queuine integrated into tRNA; during the aging process, inevitable organic changes occur, such as down-regulation of Qtrt1 enzyme expression, which leads to a decrease in the ability of the body to utilize Queuine, which also leads to a decrease in the content of Queuine in the body.

[0186] Example 3 Effect of Queuine intervention on the lifespan of Drosophila melanogaster

[0187] Wild-type strain Drosophila melanogaster was purchased from Qidong Fangjing Biotechnology Co., Ltd. To evaluate the effect of Queuine on the lifespan of Drosophila melanogaster, newly eclosed (48 h) Drosophila melanogaster was divided into a control group and a drug administration group (Q treatment group) after being anesthetized, with 200 flies in each group, half male and half female. The control group was fed with the standard yeast-cornmeal-sucrose medium in Example 1, and the drug administration group was fed with the medium with the addition of 50 ng of Queuine per tube based on the standard yeast-cornmeal-sucrose medium. Each tube was fed with 20 flies, and the medium was replaced every 3 days, and the other culture conditions were the same as in Example 1. The number of surviving and dead flies was recorded every day. The survival curve was analyzed using Graphpad Prism 7.0 software. The survival curve and survival rate results are shown in Figure 6.

[0188] The experimental results show that the average lifespan of the control group is 25.28 days, and the average lifespan of the drug administration group is 32.2 days (Figure 6A), and the lifespan extension rate of the addition of 50 ng of Queuine is 27.37%. The survival curve (Figure 6B) shows that the survival rate of the drug administration group is significantly higher than that of the control group at each sampling time point during the entire experimental period. Among them, the survival rate of the drug administration group at the 21st day sampling point increased by 20%. The experimental results of this example suggest that an appropriate concentration of Queuine can prolong the lifespan of Drosophila melanogaster and improve the survival rate of Drosophila melanogaster.

[0189] Example 4 Effect of Queuine intervention on the health of old Drosophila melanogaster

[0190] This example studies the effect of Queuine intervention on the health of old fruit flies. Among them, the wild type strain Drosophila melanogaster was purchased from Qidong Fangjing Biotechnology Co., Ltd. The control group used the standard yeast-cornmeal-sucrose medium in Example 1 to feed the fruit flies, and the drug group used the medium with 50 ng Queuine added to each tube based on the standard yeast-cornmeal-sucrose medium to feed the fruit flies. Each tube was fed at a density of 20, and the medium was replaced every 3 days. The other culture conditions were the same as in Example 1.

[0191] 4.1 Fruit fly behavior evaluation

[0192] The evaluation index of fruit fly health includes: growth and development of parents and offspring, motor ability, heat stress resistance, olfactory memory and antioxidant capacity.

[0193] (1) Detection of manQ modification level

[0194] Collect 3, 10 and 30-day-old fruit flies from the control group (C) and the drug group (T) respectively, 100 mg each, half male and half female, and do three repeats for each group. Extract and separate small RNA from fruit flies, and analyze the content of manQ modification by UHPLC-QQQ-MS method. The chromatographic method and mass spectrometry parameters are the same as in Example 1.

[0195] (2) Growth and development

[0196] Collect 10, 20 and 30-day-old fruit flies from the control group (C) and the drug group (T) respectively, 20 each, half male and half female, and accurately weigh the body weight of male and female fruit flies with an electronic balance. Each group was repeated three times.

[0197] (3) Offspring development

[0198] Transfer single female and male fruit flies from the control group (C) and the drug group (T) intervened for 30 days to fresh standard medium, collect eggs and place them in normal culture conditions to observe offspring development. Each group was repeated 3 times.

[0199] (4) Motor ability

[0200] Collect 10, 20 and 30-day-old fruit flies from the control group (C) and the drug group (T) for motor ability test. Take a long fruit fly tube and mark a line at a distance of 8 cm from the bottom of the tube. 20 fruit flies (10 males and 10 females) were transferred to the fruit fly tube and adapted for 10 min, then the test tube was knocked to make the fruit flies fall to the bottom of the test tube. Then start timing and record the number of fruit flies crossing the mark line within 15 s. Each group was set up 3 parallels.

[0201] (5) Heat stress resistance

[0202] Collect 30-day-old fruit flies in control group (C) and treatment group (T), 50 in each group, half male and half female. Put the fruit flies to be tested into empty tubes and place them in a 35℃ incubator for high-temperature treatment for 30 min. Record the number of dead fruit flies 24 h later. Repeat 3 times for each group.

[0203] (6) Olfactory memory

[0204] Prepare a 1000 mL beaker and place 2 50 mL centrifuge tubes with holes and containing bananas in it in advance. One of the centrifuge tubes is an unobstructed centrifuge tube, and the fruit flies can smell the odor and also enter the foraging through the hole. The other is an obstructed centrifuge tube, and the fruit flies can smell the odor but cannot enter the foraging. Collect 20 30-day-old fruit flies in control group (C) and treatment group (T), half male and half female. Put the fruit flies to be tested into empty tubes and starve them for 2 h, then place them in the beaker. Record the number of fruit flies entering the unobstructed centrifuge tube within 1 min. Repeat 3 times for each group.

[0205] (7) Antioxidant capacity

[0206] Collect 30-day-old fruit flies in control group (C) and treatment group (T), 30 in each group (half male and half female) and weigh them. Add pre-cooled physiological saline for grinding. The obtained homogenate is centrifuged at 12000 r / min for 15 min at -4℃ to remove tissue debris, and the supernatant is collected. Use the superoxide dismutase (SOD) kit and glutathione peroxidase (GSH-Px) kit to determine the activities of SOD and GSH-Px.

[0207] 4.2 Experimental results

[0208] (1) manQ modification level

[0209] Compared with the control group, the content of manQ in the 3, 10 and 30-day-old treatment groups was significantly increased (Figure 7). The results showed that the level of manQ modification in vivo could be improved by exogenous addition of Queuine, and there was no toxic side effect, so it was speculated that Queuine intervention could be applied to all stages of growth and development.

[0210] (2) Growth and development of parents and offspring

[0211] To evaluate the effects of Queuine on the development of Drosophila parents and offspring, the body weight of parent female and male Drosophila (10 per sample) was accurately weighed by an electronic balance to reflect the growth and development state, and whether the offspring could develop into healthy adults was observed. The results are shown in Figure 8. Compared with the control group, the exogenous addition of an appropriate amount of Queuine had no significant effect on the growth and development of female and male Drosophila (Figures 8A and 8B) (p>0.05), and the offspring of the administration group had the ability to develop into healthy adults. The results showed that the exogenously added Queuine had no toxic side effects, and it had no obvious adverse reactions on the growth and development of parents and offspring.

[0212] (3) Motor ability

[0213] With the increase of age, the motor ability of the body gradually decreases, and the strength of the motor ability can reflect the degree of aging, so the motor ability has always been one of the classic behavioral indicators for reflecting the aging of Drosophila. In this embodiment, the climbing experiment was used to reflect the motor ability of old Drosophila, and the results are shown in Figure 9. According to the number of Drosophila climbing to 8 cm within 10 s, there was no significant difference in the motor ability of 10-day-old Drosophila between the control group and the Queuine administration group (p>0.05); the motor ability of 20-day-old and 30-day-old Drosophila in the Queuine administration group was significantly higher than that in the control group (p<0.001). The results showed that Queuine could significantly improve the motor ability of old Drosophila.

[0214] (4) Heat stress resistance

[0215] After aging, the ability of the body to resist changes in the external environment also decreases. The heat stress test can be used to evaluate the tolerance of Drosophila to high temperature. As shown in Figure 10, the survival rate of 10-day-old, 20-day-old and 30-day-old Drosophila in the Queuine administration group after heat stimulation was significantly higher than that in the control group (p<0.001). The results showed that Queuine could significantly improve the heat tolerance of old Drosophila.

[0216] (5) Olfactory memory

[0217] During the aging process, the cognitive function of the brain decreases, and learning and memory are important components of cognitive function, so learning and memory are one of the important indicators for evaluating the aging of the body. Olfactory memory test couples banana odor and banana reward to induce Drosophila to form learning and memory, and further reflect its cognitive function. The results of olfactory memory are shown in Figure 11. Compared with 30-day-old control group of aging Drosophila, feeding Queuine could significantly increase the number of Drosophila entering the barrier-free centrifuge tube (p<0.001).

[0218] (6) Antioxidant ability

[0219] During the normal aging process of organisms, excessive reactive oxygen species (ROS) are produced, leading to oxidative stress, protein and DNA structure damage, and mitochondrial dysfunction, which in turn accelerates the aging and apoptosis of the body. SOD and GSH-Px are important antioxidant enzymes in the body, and their activities can reflect the antioxidant capacity of the body. In this experiment, the activities of SOD (Figure 12A) and GSH-Px (Figure 12B) in fruit flies were detected by kit, and the results are shown in Figure 12. Compared with 10-day-old young fruit flies, the activities of SOD and GSH-Px in 30-day-old old fruit flies were significantly reduced (p<0.001), indicating that the antioxidant capacity of old fruit flies decreased significantly. However, the activities of SOD and GSH-Px in 30-day-old old fruit flies fed with Queuine were significantly increased compared with those in the control group (p<0.001). The results showed that Queuine could significantly improve the antioxidant capacity of old fruit flies.

[0220] This example confirmed that Queuine has good safety and can improve the behavioral ability of old individuals and improve the health of the body through the evaluation of the growth and development, motor ability, heat stress resistance, olfactory memory, and antioxidant capacity of fruit fly parents and offspring. It can be used as an active ingredient of anti-aging drugs.

[0221] Example 5 Effect of Queuine intervention on the lifespan of acute aging model mice

[0222] Paraquat (PQ)-induced acute aging model in mice is one of the commonly used acute aging models for preclinical research, which can evaluate the anti-aging effect of drugs in a short period of time. Therefore, this example applies this model to evaluate the anti-aging effect of high and low doses of Queuine.

[0223] 5.1 Experimental method

[0224] Forty-eight healthy SPF male Balb / c mice (18-20 g) were purchased and raised in the clean level experimental animal room of the Guangdong Provincial Institute of Testing and Analysis (China Guangzhou Analysis and Testing Center). The animal room maintained an environmental temperature of 24-26°C, humidity of 45-65%, and a light environment of 12 h light and 12 h darkness alternately, and the mice could freely eat and drink water.

[0225] After one week of adaptive feeding, the mice were randomly divided into a control group and a model group, with 12 mice in each group. The blank control group was administered normal saline by gavage every day. After the model group was injected intraperitoneally with paraquat at a dose of 50 mg / kg, the mice were randomly divided into a model control group, a Queuine low-dose group (QL), and a Queuine high-dose group (QH). The model control group was administered normal saline by gavage every day. The Queuine low-dose group was administered 10 μg / kg by gavage every day. The Queuine high-dose group was administered 25 μg / kg by gavage every day. The number of dead mice and the time of death were recorded every day.

[0226] On the third day after modeling, 6 mice were randomly selected from each group, and the heart, liver, spleen, lung, kidney, and brain were removed after sacrifice. The organs were ground and soaked in TRIzol and stored in a -80°C refrigerator. After the TRIzol lysed tissue samples were thawed at room temperature, they were centrifuged at 12,000 g for 2 min to remove the bottom tissue debris. The mirVana™ miRNA kit was used to isolate small RNA.

[0227] Total RNA was extracted, and cDNA was synthesized using the RevertAid™ First-Strand cDNA Synthesis Kit. The relative expression of aging-related factors p16 and p21 was quantified by QPCR.

[0228] 5.2 Experimental results

[0229] (1) manQ modification content induced by paraquat

[0230] The small RNA was extracted and isolated from the kidneys of the paraquat-induced acute aging mouse model, and the small RNA was further hydrolyzed into nucleosides. The nucleosides were analyzed by UHPLC-QQQ-MS, and the method was the same as in Example 1. The results are shown in FIG. 13A. As shown in FIG. 13B, compared with the blank control group, the manQ content in the kidneys of the paraquat-induced acute aging mouse model was significantly reduced. This indicates that the same aging phenotype as the naturally aging mouse (20-month-old mouse in Example 1) exists, i.e., the reduction in the content of manQ modification.

[0231] (2) Effect of Queuine intervention on the survival rate and aging factors of mice

[0232] The results are shown in FIG. 14A. The average survival time of the PQ model mice was 78 h, the average survival time of the mice in the Queuine low-dose group was 102 h, the life extension rate was 30.76%, and the average survival time of the mice in the Queuine high-dose group was 132 h, the life extension rate was 69.23%. It was found that Queuine has the activity of prolonging the lifespan of the PQ-induced multi-organ failure aging model mice and improving the survival rate, and the activity is stronger as the dose increases.

[0233] In addition, as shown in FIG. 14B, in the PQ-induced acute aging model group of mice, the aging factors p16 and p21 of the model control group were significantly up-regulated, which was consistent with the natural aging model. The p16 and p21 of the Queuine low-dose and Queuine high-dose groups were significantly lower than those of the model control group.

[0234] The above experimental results show that both the survival rate experiment and the biological indicators suggest that Queuine has the effect of prolonging the life span of mice and improving the survival rate.

[0235] Example 6 Pharmacokinetic study

[0236] 6.1 Experimental method

[0237] Twenty-one healthy SPF male Balb / c mice (18-20 g) were purchased and raised in the clean level experimental animal room of the Guangdong Provincial Institute of Testing and Analysis (China Guangzhou Analysis and Testing Center). The animal room maintained an environmental temperature of 24-26°C, a humidity of 45-65%, and a light environment of 12 h light and 12 h darkness alternately, and the mice could freely eat and drink water.

[0238] After the mice were adaptively fed for 3 days, Queuine was orally administered at a dose of 10 μg / kg, and the mouse plasma and kidney tissue were collected at 0, 0.5, 1, 2, 4, 8, and 24 h after administration, respectively. Three animal samples were collected at each time point.

[0239] The pretreatment of plasma and the analysis method of Queuine are referred to Example 2. After the tissue was collected, it was ground thoroughly and soaked in TRIzol and stored in a -80°C refrigerator. Small RNA was extracted and separated by the method of Example 1, hydrolyzed into oligonucleotides, and then analyzed by UHPLC-QQQ-MS. Using Graphpad Prism 7.0 software, the pharmacokinetic curve was plotted with time as the abscissa and the content of free Queuine in plasma and bound Queuosine in tissue as the ordinate.

[0240] 6.2 Experimental results

[0241] The pharmacokinetic results of Queuine are shown in FIG. 15. At 24 h, the free Queuine in serum was about 2 times that at 0 h (FIG. 15A), and the bound Q-tRNA / manQ-tRNA in tissue was also significantly higher than that at 0 h (FIG. 15B). According to the dose conversion relationship between mice and humans, it is recommended that the Queuine dose for humans is 10 ng-10 mg / kg, and the administration method is oral.

[0242] Example 7 Effect of Queuine intervention on the health of naturally aging mice

[0243] Natural aging animals are the best model to reflect the normal aging process of the organism, which is convenient for observing and analyzing the biological mechanism of aging, and is often used to evaluate the effectiveness and safety of anti-aging drugs. Therefore, this example further applies this model to evaluate the anti-natural aging effect of Queuine.

[0244] 7.1 Experimental method

[0245] 16-month-old healthy SPF male C57 mice were purchased from Zhisan Beijing Health Medical Research Institute, 2-month-old healthy SPF male C57 mice were purchased from Zhuhai Baisitong Biotechnology Co., Ltd., and were raised in the clean level experimental animal room of the Guangdong Academy of Sciences Test Analysis Institute (China Guangzhou Analysis Test Center). The animal room maintains an ambient temperature of 24-26°C, humidity of 45-65%, and a light environment of 12h light and 12h darkness alternately, and the mice can freely eat and drink water.

[0246] The 2-month-old young mice were used as the control group (Control) without any treatment. After 3 days of adaptive feeding, the 16-month-old old mice were randomly divided into a blank group (Blank) and a Queuine administration group, each group of 6. The blank group was administered with normal saline, and the Queuine administration group was administered with 10 μg / kg, with a dosing frequency of 1 time / 3 days. After 4 months of administration, the mice peripheral blood was collected from the inner canthus, and the plasma and white blood cells were separated according to the method of Example 1. The plasma was used for biochemical index detection, and the white blood cells were used for RNA extraction.

[0247] (1) Blood routine

[0248] 20 μL of peripheral blood of the control group (6-month-old), blank group (20-month-old old mice), and Queuine administration group (20-month-old old mice) was collected in an anticoagulant tube, and blood routine analysis was performed on the Meiyade animal full-automatic blood cell analyzer.

[0249] (2) Inflammatory factors

[0250] The serum of the control group (6-month-old), blank group (20-month-old old mice), and Queuine administration group (20-month-old old mice) was collected in an anticoagulant tube, and the IL-6 content in the serum was detected using a mouse IL-6 ELISA kit.

[0251] (3) Telomerase activity

[0252] The serum of the control group (6-month-old), blank group (20-month-old old mice), and Queuine administration group (20-month-old old mice) was collected in an anticoagulant tube, and the telomerase level in the serum was detected using a mouse telomerase ELISA kit.

[0253] (4) Antioxidant capacity

[0254] Reference Example 4. SOD and GSH-Px activities in mouse serum were measured using a Superoxide dismutase (SOD) kit and a Glutathione peroxidase (GSH-Px) kit, respectively.

[0255] (5) Senescence factors

[0256] Reference Example 5. Total RNA was extracted from leukocytes, reverse-transcribed into cDNA, and the relative expression of senescence factors p16 and p21 was measured using QPCR.

[0257] (6) Behavioral evaluation

[0258] The control group (6 months old), blank group (20-month-old old mice), and Queuine administration group (20-month-old old mice) were placed one by one into an open field box (40 cm x 40 cm x 40 cm) and allowed to move freely for 7 min. Tracker software was used to count the movement trajectory of the mice in the open field, the open field score (the number of times crossing each square), and the time spent in the central area (20 cm x 20 cm) and the four corners.

[0259] (7) Methylation clock sequencing

[0260] DNA was extracted from leukocytes, and Illumina base calling software was used to identify the sequencing sequences, which were aligned with the mouse reference genome using BSMAP software (an alignment software for bisulfite sequence data and methylation sequencing data). The methylation level of cytosine (CpG site) in each sample was estimated as the number of reads supporting C divided by the total number of reads supporting C or T. According to the proprietary DNA methylation age prediction model (Stubbs, T. M., et al. (2017). Multi-tissue DNA methylation age predictor in mouse. Genome biology, 18(1), 68. https: / / doi.org / 10.1186 / s13059-017-1203-5), the DNA methylation age was evaluated using the DNA methylation value calculated from the sequence data.

[0261] (8) Memory and recognition ability evaluation

[0262] The spatial memory or recognition ability was evaluated using the Object Location Test (OLT) and the Novel Object Recognition Test (NORT).

[0263] (9) Motor coordination and fatigue resistance evaluation

[0264] Motor coordination and fatigue resistance test was performed using a Rotarod. The time (in seconds) that each mouse persisted on the Rotarod was recorded. This time reflects its neuromuscular coordination and fatigue resistance capacity. All tests were performed under the same environmental conditions and the operator remained blind to avoid experimental bias.

[0265] (10) Organ function and blood biochemical evaluation

[0266] Peripheral blood (20 μL) was collected from the tail vein of mice using an anticoagulant tube, and immediately mixed to prevent coagulation. Subsequently, the plasma was separated from the whole blood using centrifugation. The separated plasma was used for the detection of biochemical indicators. The plasma samples were analyzed using an automatic biochemical analyzer (Chemray 800), and the detection items included liver function, kidney function, myocardial enzyme spectrum, blood glucose level, and blood lipid indicators. All sample detections were completed under the automatic operation conditions of the equipment to ensure the accuracy and consistency of the results.

[0267] 7.2 Experimental results

[0268] (1) Appearance changes

[0269] Compared with the young mice in the control group, the natural aging mice in the blank group had lighter, sparser hair, and decreased gloss (see Figure 16). The above appearance was improved to some extent in the Queuine administration group compared with the blank group.

[0270] (2) Changes in blood routine

[0271] The results of peripheral blood routine analysis of the control group (6-month-old), blank group (20-month-old old mice), and Queuine administration group (20-month-old old mice) are shown in Figure 17. Compared with the young control group, the red blood cells in the blood cells of the natural aging mice in the blank group were significantly reduced, and the white blood cells, lymphocytes, and platelets were significantly increased, indicating that chronic inflammation and infection occurred in the old mice. Compared with the blank group, the red blood cells in the peripheral blood of the Queuine administration group were significantly increased, and the white blood cells, lymphocytes, and platelets were significantly reduced, indicating that the Queuine intervention mice were significantly younger in terms of blood routine indicators.

[0272] (3) Changes in inflammatory factors

[0273] The IL-6 detection results in the plasma of the control group (6-month-old), blank group (20-month-old old mice) and Queuine administration group (20-month-old old mice) are shown in FIG. 18. The IL-6 in the body of the old mice in the blank group was significantly higher than that of the 6-month-old adult mice; the IL-6 level in the body of the Queuine administration group was significantly lower than that of the blank group, indicating that Queuine effectively alleviated the inflammation in the body of the old mice.

[0274] (4) Changes in telomerase activity

[0275] Telomerase can synthesize new telomere DNA sequences, thereby extending the length of the telomeres of chromosomes and preventing cell aging and death. In other words, the higher the level of telomerase in the body, the lower the degree of telomere loss. The telomerase detection results in the plasma of the control group (6-month-old), blank group (20-month-old old mice) and Queuine administration group (20-month-old old mice) are shown in FIG. 19. As shown in FIG. 19, compared with the young control group, the telomerase in the plasma of the old mice in the blank group was significantly reduced; but Queuine intervention significantly improved the level of telomerase in the body of the old mice.

[0276] (5) Changes in antioxidant capacity

[0277] SOD and GSH are important antioxidant enzymes in the body. The SOD and GSH detection results in the plasma of the control group (6-month-old), blank group (20-month-old old mice) and Queuine administration group (20-month-old old mice) are shown in FIG. 20. As shown in FIG. 20, compared with the young control group, the SOD (FIG. 20A) and GSH (FIG. 20B) in the plasma of the old mice in the blank group were significantly reduced; but the results of the Queuine administration group showed that Queuine intervention significantly improved the levels of SOD and GSH in the body of the old mice.

[0278] (6) Changes in aging factors

[0279] The aging factor p16 and p21 results in the peripheral blood leukocytes of the control group, blank group (naturally aged mice) and Queuine intervention mice are shown in FIG. 21. As shown in FIG. 21, compared with the young mice, the aging-related phenotypes p16 and p21 in the leukocytes of the old mice in the blank group were significantly increased. Compared with the naturally aged mice in the blank group, the p16 and p21 in the leukocytes of the Queuine intervention mice were significantly reduced, indicating that Queuine had obvious anti-aging effect.

[0280] (7) Behavioral evaluation

[0281] The motion trajectory of the control group (6 months old), the blank group (20-month-old old mice), and the Queuine administration group (20-month-old old mice) was studied. The statistical results of the open field scores (A) and the four-week and central area residence time (B) of the mice in the three groups are shown in FIG. 22. As shown in FIG. 22, the open field score of the blank group was significantly lower than that of the young mice, and the Queuine administration group significantly improved the exercise capacity of the old mice. The statistical results of the motion trajectory and residence time analysis showed that, compared with the control group, the residence time of the mice in the blank group in the four-week period was significantly prolonged, and the Queuine administration group significantly improved the autonomous and exploratory behavior of the old mice.

[0282] (8) DNA methylation age

[0283] DNA methylation age (DNAm age) is one of the most promising indicators of physiological age that has attracted much attention in recent years. This indicator not only accurately identifies physiological age, but also has good performance in predicting health outcomes such as death and cardiovascular disease. The use of gene methylation maps to determine physiological age has a high accuracy of 98%.

[0284] As shown in FIG. 23, the average methylation age of the blank group of naturally aging mice was 23.97 months, and the methylation age of the Queuine administration group of old mice was 17.27 months (see FIG. 23A). The methylation age of the administration group was significantly lower than that of the blank group. Through age acceleration analysis (see FIG. 23B), the average reduction in methylation age of the Queuine administration group was 4.72 months. According to the mouse and human age comparison table (Table 2), if a human being starts to regularly supplement Queuine (1 μg / kg) from the age of 44, their physiological age will be about 28 years younger than their actual age at the age of 76.

[0285] Table 2. Mouse age and human age comparison table

[0286] (9) Memory and recognition ability evaluation

[0287] The OLT and NORT behavioral test results showed that Queuine intervention significantly improved the spatial memory and recognition ability of mice. Compared with the blank control group, the proportion of exploration time of mice in the Queuine treatment group in the new location (OLT) and the new object (NORT) increased significantly, showing stronger spatial location recognition ability and novelty object recognition preference (FIG. 24A). This result suggests that Queuine can effectively improve the function of the central nervous system in information memory and processing, and improve the performance of cognitive-related behaviors.

[0288] (10) Exercise coordination and fatigue resistance evaluation

[0289] The results of the rotarod test showed that Queuine intervention significantly improved the mice's residence time under high-speed rotating conditions. Compared with the control group, the Queuine-treated mice showed longer sustained running ability on the rotarod (Figure 24B), indicating that their neuromuscular coordination and anti-fatigue ability were significantly enhanced. This result suggests that Queuine may play a positive regulatory role in motor function by improving neuromotor control or enhancing muscle endurance.

[0290] (11) Organ function and blood biochemical evaluation

[0291] The results of blood biochemical tests showed that Queuine intervention exhibited significant regulatory effects on multiple physiological function indicators. Specifically, the liver function (Figure 24C), blood glucose level (Figure 24D), blood lipid level (Figure 24E), and myocardial enzyme spectrum (Figure 24F), kidney function (Figure 24G), and other key biochemical indicators of Queuine-treated mice were significantly improved compared to the blank old group. The above indicators tended to approach the normal level of young mice after Queuine intervention, suggesting that it has good multi-system regulation potential and can effectively alleviate aging-related metabolic disorders and organ function decline.

[0292] Example 8 Changes in manQ-modified tRNA monomers at different stages of the rat's life cycle

[0293] According to the rat tRNA Asp(manQUC) Sequence (SEQ ID NO. 7) (Figure 25A), the tRNA monomer purification probe was designed, and a biotin-labeled probe was synthesized. Different age rats were taken, and small RNA was prepared according to the method of Example 1. The tRNA Asp(manQUC) monomer was purified by probe-magnetic bead capture method, and the purity and content of the purified sample were evaluated by nanodrop, and the integrity of the purified sample was evaluated by polyacrylamide gel method. As shown in Figure 25B, the purified tRNA monomer had good purity and integrity.

[0294] The purified tRNA monomer was analyzed by UHPLC-QTOF-MS as described in Example 1. The total ion current chromatogram (TIC) (Figure 25C) showed that both 6-month-old and 36-month-old rats contained tRNA Asp(GUC) without manQ modification at the anticodon site. However, only 6-month-old rats contained tRNA Asp(manQUC) with manQ modification (chromatographic peak retention time 10.90-11.30 min) (Figure 25D), while tRNA Asp(manQUC) with manQ modification completely disappeared in 36-month-old rats. The mass spectrometry total ion current chromatogram (EIC) also showed that only 6-month-old rats contained tRNA Asp(manQUC)(molecular weight 24,544.25), while the mass spectrum signal of tRNA Asp(manQUC) in 36-month-old rats completely disappeared.

[0295] The purified tRNA Asp(manQUC) monomer was fragmented and each fragment was quantitatively analyzed by UHPLC-QTOF-MS. As shown in Fig. 25E, among the fragments produced by tRNA Asp(manQUC) , the fragment containing manQ modification (CUC[manQ]UCA[m 5 C]G) only existed in 6-month-old rats. While in 36-month-old rats, the fragment containing manQ modification completely disappeared, and the rest of the fragments were not different from those in 6-month-old rats, indicating that the change in manQ modification specifically caused the change in tRNA monomer level.

[0296] The purified tRNA Asp(manQUC) monomer was completely hydrolyzed by RNase P1 according to the method described in Example 1, and all the types of nucleosides contained in tRNA Asp(manQUC) were obtained and quantitatively analyzed by UHPLC-QTOF-MS. As shown in Fig. 25F, the content of manQ nucleosides contained in tRNA Asp(manQUC) monomer in 36-month-old rats was much lower than that in 6-month-old rats.

[0297] From the above experiments, it can be seen that the expression level of tRNA monomer containing manQ modification is significantly down-regulated in aged rats.

[0298] Example 9 Effect of Queuine intervention on the health of acute aging model mice

[0299] This example studies the effect of Queuine intervention on the health of acute aging model mice. The acute aging model of mice induced by paraquat (PQ) was constructed according to the method described in Example 5. After modeling, the mice were randomly divided into different groups for Queuine intervention: high-dose group (QH, 200 μg / kg), low-dose group (QL, 25 μg / kg), model control group (Model, without intervention) and normal control group (Ctrl, without treatment). Queuine was administered orally once a day for 5 consecutive days. At the end of the intervention, the mice were euthanized and their lung, liver and other organs and blood samples were collected. Changes in typical pathological phenotypes such as lung and liver injury were observed.

[0300] (1) Lung histopathology evaluation: Hematoxylin-Eosin (H&E) staining was used to observe the structural changes of lung tissue, especially the inflammatory cell infiltration; the wet / dry weight ratio (W / D ratio) was used to assess the degree of pulmonary edema. (2) Liver function evaluation: The levels of aspartate aminotransferase (AST) and alkaline phosphatase (ALP) in serum and liver tissue were measured to assess the degree of liver function damage. (3) Inflammatory factor analysis: The levels of IL-6, TNF-a and other pro-inflammatory factors in lung tissue were detected by ELISA to evaluate the inhibitory effect of Queuine on inflammatory response.

[0301] The results of this study showed that Queuine had obvious protective effect in the acute aging mouse model, especially in the repair of lung and liver damage in a dose-dependent manner. (1) Lung protection: In the H&E staining results of lung tissue, the alveolar structure of QH group mice was significantly improved, and the infiltration of inflammatory cells was significantly reduced (Figure 26A). In addition, the W / D ratio of pulmonary edema was also significantly reduced, from 5.8±0.3 in the model group to 3.7±0.3 in the QH group (p<0.01) (Figure 26B). (2) Liver protection: In the biochemical detection of liver function, the serum AST and ALP levels of QH group mice were significantly lower than those of the model group, decreased by 34.7% (AST) and 26.4% (ALP) respectively (p<0.01) (Figures 26C, D and E), showing that Queuine had significant protective effect on PQ-induced liver damage. (3) Inhibition of inflammatory response: Analysis of inflammatory factors showed that Queuine significantly inhibited PQ-induced inflammatory response. In QH group mice, the levels of inflammatory factors such as IL-6, IL-1β and TNF-a were significantly lower than those in the model group (Figure 26F), suggesting that Queuine may regulate immune response, reduce oxidative stress and inflammatory response, and further reduce tissue damage.

[0302] Example 10 Effect of Queuine intervention on the lifespan of naturally aging mice

[0303] In this example, 60 male C57BL / 6 mice aged 16 months (equivalent to 50 years old in humans) were selected as experimental objects and randomly divided into two groups. The experimental group was given Queuine orally once every 3 days (dose of 10 μg / kg, Figure 27A), and the control group was given the same volume of normal saline. All mice were raised under the same conditions, and their survival time was continuously monitored until natural death.

[0304] The results showed that Queuine significantly prolonged the lifespan of aged mice (Figure 27B). The average lifespan of the Queuine-treated group was extended from 747.13 days in the control group to 861.13 days, an increase of about 15.3% (p < 0.001). The median lifespan was increased from 738 days to 864 days, with an extension of 17.1%. In addition, the maximum lifespan was extended from 813 days to 984 days, an increase of 141 days. The hazard ratio was 0.19 (95% confidence interval: 0.09-0.38), indicating that Queuine intervention significantly reduced the risk of death. This result fully confirmed the potential of Queuine in delaying the aging process and improving overall lifespan, laying an experimental foundation for its application as an anti-aging intervention agent.

[0305] Example 11 Mechanism of Queuine intervention in regulating aging and health

[0306] The synthesis process of manQ modification of biological tRNA mainly involves four key enzymes, Qtrt1, Qtrt2, Man2c1, and GTDC1 (Figure 28A). The expression levels of these key synthesis enzymes in human white blood cells and mouse kidneys were quantified by PCR, and the results showed that Qtrt1 and Qtrt2 were significantly up-regulated in aged individuals, while GTDC1 expression was significantly down-regulated (Figure 28B). This suggests that the down-regulation of GTDC1 expression in aged individuals may have caused a decline in the function of tRNA manQ synthesis, ultimately leading to the aging phenotype.

[0307] The present embodiment takes 2BS cells (human embryonic lung diploid fibroblasts, purchased from China Typical Culture Collection Center) as a model to verify the role of GTDC1 in the regulation of aging. This model is an in vitro cell model widely used in anti-aging research. Cells at 30 generations exhibit a young phenotype, and gradually age as the number of passages increases, with cells at 39 generations exhibiting a clear aging phenotype. As shown in FIG. 28C, the aging markers p16 and p21 of 2BS cells at 39 generations were significantly higher than those at 30 generations. The results of SA-β-gal staining also showed that the degree of aging of 39 generations was significantly higher than that of 30 generations. In addition, the level of manQ in 39-generation cells was also significantly lower than that in 30-generation young cells (FIG. 28D). The GTDC1 in 30-generation young cells was knocked down using the CRISPR / Cas9 method (FIG. 28E). In the figure, A and B represent two knockout schemes. The results showed that the level of manQ modification in 2BS cells with knocked-out GTDC1 was significantly reduced (FIG. 28F). At the same time, the aging markers p16 and p21 were significantly up-regulated in young cells after knockout (FIG. 28G). The results of SA-β-gal staining also showed that the degree of aging of cells after GTDC1 knockout was significantly higher than that of natural 30-generation 2BS cells (FIG. 28H). The above research results show that the decrease in the expression of GTDC1 leads to a decrease in the level of manQ, which is an important mechanism of aging.

[0308] Example 12 Mechanism of Queuine Intervention in Regulating Aging and Health

[0309] The decomposition process of manQ modification of biological tRNA is mainly the key enzyme Man2c1 (FIG. 28A). The expression level of this key synthetic enzyme in human white blood cells and mouse kidneys was quantified by PCR, and the results showed that Man2c1 was significantly up-regulated in old individuals (FIG. 28B). It is possible that the increased expression of Man2c1 in old individuals accelerates the decomposition of tRNA manQ, ultimately leading to an aging phenotype.

[0310] To verify the role of Man2c1 in the regulation of aging, we used 2BS cells (human embryonic lung diploid fibroblasts) as a model. We used plasmid overexpression to increase the expression level of Man2c1 in 30-year-old young cells (Figure 29A). The results showed that the manQ modification level was significantly reduced in 2BS cells overexpressing Man2c1 (Figure 29B). At the same time, the expression of the aging markers p16 and p21 was significantly upregulated in young cells overexpressing Man2c1 (Figure 29C). Conversely, when we used siRNA technology to knock down the expression level of Man2c1 in 39-year-old 2BS cells, the manQ modification level was significantly increased (Figure 29D). At the same time, the expression of the aging markers p16 and p21 was significantly reduced in aging cells with knocked down Man2c1 (Figure 29E). The above research results show that the increase in the expression of Man2c1 causes a decrease in the level of manQ, which is an important mechanism of aging. The research in this embodiment from the perspective of accelerated decomposition of manQ in aging cells first proves the role of the key hydrolytic enzyme Man2c1 in aging.

[0311] Example 13 Mechanism of Queuine intervention in the regulation of aging and health

[0312] Based on the results of Example 11 and Example 12, it can be reasonably assumed that in aging individuals, the key enzyme GTDC1 responsible for manQ synthesis is down-regulated, while the key enzyme Man2c1 responsible for the decomposition of manQ is up-regulated. These two factors together or independently cause the down-regulation of the manQ modification level of tRNA in aging individuals, thereby ultimately causing the aging phenotype. The mechanism of Queuine intervention in the regulation of aging may be to supplement the substrate necessary for the synthesis of manQ in the body, thereby promoting the recovery of the body's manQ level, thereby achieving the purpose of anti-aging and regulating health.

[0313] To verify this hypothesis, this embodiment used Queuine to intervene in 39-year-old 2BS cells. The results showed that the expression of manQ in 39-year-old cells supplemented with Queuine (10 ng / mL) was significantly increased, and its level could be restored to that of 30-year-old young cells (Figure 30A).

[0314] The manQ level in the paraquat acute aging model mice in Example 5 was detected. The results showed that after Queuine (200 μg / kg) intervention, the manQ level of the acute aging mice was significantly improved compared with the model control group (without treatment), and its level could be restored to that of normal mice (without paraquat) (Figure 30B).

[0315] The manQ level in the fruit fly aging model in Example 4 was detected. The results showed that after Queuine (50 ng / bottle) intervention, the manQ level of 30-day-old fruit flies was significantly improved compared with the model group (untreated) (Figure 30C).

[0316] The above results show that Queuine supplementation can significantly improve the expression level of manQ in aging individuals, thereby improving the aging phenotype.

[0317] Example 14 Mechanism of Queuine intervention in regulating aging and health

[0318] This example aims to study the regulatory effect of Queuine on the metabolic function of naturally aging animals and clarify its potential value in improving metabolic disorders in aging individuals. The naturally aging mice and their Queuine intervention group grown to 22 months of age in Example 7 were selected as research objects, and their tail vein plasma samples were collected. After protein precipitation and impurity removal with methanol / acetonitrile, metabolite extraction was performed, followed by quantitative analysis of key metabolites using an ultra-high performance liquid chromatography-tandem mass spectrometry system (UHPLC-QTRAP / MS, 6500+, AB Sciex) to reveal the effects of Queuine on aging-related metabolic pathways.

[0319] The results of metabolomics detection showed that the bone metabolism-related substances in the plasma of Queuine intervention group mice were significantly up-regulated, especially the content of Glucosamine and its derivative D-Glucosamine 1-phosphate, which have anti-osteoporosis effect (Figure 31A), suggesting that Queuine may delay aging-related bone degradation by enhancing bone synthetic metabolic pathways. In addition, Queuine intervention significantly increased the expression of various anti-inflammatory metabolites, including prostaglandin derivatives such as 6-keto-PGF1α and various specific pro-inflammatory resolution mediators (SPMs) such as 11-HEPE (11-hydroxy-5Z,8Z,12E,14Z,17Z-eicosapentaenoic acid) and 5-HEPE (5-Hydroxy-6,8,11,14,17-eicosapentaenoic acid) (Figure 31B), indicating that it may play an anti-aging role by activating inflammation resolution mechanisms.

[0320] Notably, Queuine effectively inhibited the generation of kynurenine, a neurotoxic metabolite in the tryptophan metabolic pathway, suggesting that it may slow the development of neurodegenerative diseases by blocking the Kynurenine pathway (Figure 31C).

[0321] In addition, the level of nicotinamide riboside, a longevity-related molecule in the plasma of the Queuine intervention group, was significantly increased (Figure 31D). As a precursor of NAD+, the increase of nicotinamide riboside can promote cellular energy metabolism and DNA repair, thereby delaying the aging process of multiple systems.

[0322] In summary, Queuine exhibits significant regulatory capacity in improving bone metabolism, inflammation regulation, neuroprotection, and NAD+ synthesis in naturally aging mice through multi-target and multi-pathway metabolic remodeling. The synergistic changes in these metabolic characteristics reveal the potential of Queuine as an anti-aging metabolic regulator and provide an experimental basis for its application in delaying aging-related metabolic disorders.

[0323] Example 15 Preparation of Queuine formulation

[0324] This example studies the formulation suitable for Queuine to verify whether the bioavailability of Queuine can be further improved by optimizing the dosage form, thereby improving the anti-aging effect.

[0325] 15.1 Experimental methods

[0326] 15.1.1 Enteric capsule formulation

[0327] (1) Preparation of enteric capsules

[0328] Queuine is packaged with common enteric capsules. Queuine is mixed with microcrystalline cellulose at a weight ratio of 1:10 and then filled into capsules to ensure uniformity and sealing.

[0329] (2) Characterization and evaluation of enteric capsules

[0330] ①Uniformity test: randomly select 10 capsules, and detect the content of Queuine in each capsule by the UHPLC-QQQ-MS / MS method described in Example 1. The relative standard deviation should be less than 6%. ②Disintegration test: place the capsules in artificial gastric juice (pH 1.2) at 37°C for 2 hours. The capsules should remain intact without breakage. Then transfer the capsules to artificial intestinal juice (pH 6.8). The capsules should completely disintegrate within 45 minutes. ③Dissolution test: perform the test in simulated gastric juice and intestinal juice, respectively, and draw the cumulative release curve. The amount of Queuine released in the gastric juice within 2 hours should be less than 10%, and the amount released in the intestinal juice within 45 minutes should be more than 80%.

[0331] 15.1.2 Microemulsion

[0332] (1) Preparation of microemulsion

[0333] Isopropyl myristate was selected as the oil phase, and a mixed surfactant was prepared by mixing soybean phospholipid and anhydrous ethanol at a mass ratio of 1:1. The preparation process was as follows: Queuine was dissolved in 0.83 mL of aqueous phase at low dose (1.2 mg) and high dose (4.8 mg), respectively. 1.25 mL of oil phase and 1.92 mL of mixed surfactant were mixed uniformly, and then the aqueous phase containing Queuine was slowly added. The mixture was stirred at a constant speed of 1200 revolutions per minute on a magnetic stirrer for 30 minutes to form a stable water-in-oil microemulsion. The final concentration of Queuine in the microemulsion prepared by this process was 0.3 mg / mL (low dose group) and 1.2 mg / mL (high dose group), respectively.

[0334] (2) Characterization and evaluation of microemulsion

[0335] ① Appearance: observe the appearance of the Queuine-containing microemulsion, record its transparency, color, homogeneity, and flowability, and observe whether the Tyndall effect appears under laser pen illumination. ② Physicochemical property test: at room temperature, use an Abbe refractometer to measure the refractive index of the Queuine-containing microemulsion, and use an acidity meter to measure the pH value of the Queuine-containing microemulsion. ③ Particle size and size distribution: take an appropriate amount of Queuine-containing microemulsion, place it in a particle size determination cup, and measure the particle size and size distribution of the microemulsion at room temperature for 120 seconds using a Zetasizer Nano ZS90 analyzer.

[0336] 15.1.3 Chitosan nanoparticles

[0337] (1) Preparation of chitosan nanoparticles

[0338] The Queuine-loaded chitosan nanoparticles were prepared by ionic gelation technique. First, low molecular weight chitosan was weighed and dissolved in 0.1% acetic acid solution (pH 5.0) at room temperature with sufficient stirring until completely dissolved to obtain a clear and transparent chitosan solution (1 mg / mL). An appropriate amount of tripolyphosphate (TPP) was dissolved in pure water to prepare a TPP solution with a concentration of 2 mg / mL. The nanoparticles were prepared as follows: 50 mL of the previously prepared chitosan solution (1 mg / mL) was taken, 10 mg of Queuine was added to the solution, and the solution was stirred at a constant speed of 1200 rpm on a magnetic stirrer for 30 minutes to ensure that the drug was well dispersed. Then, under continuous stirring, a total of 10 mL of the TPP solution (2 mg / mL) was added dropwise at a constant flow rate using a microsyringe, and the stirring speed was maintained at 1200 rpm for 12 hours, finally forming a suspension with a Queuine concentration of 0.167 mg / mL.

[0339] (2) Characterization and evaluation of chitosan nanoparticles

[0340] The method is the same as (2) in 15.1.2.

[0341] 15.2 Experimental results

[0342] Characterization and evaluation of enteric capsules: The enteric capsules used in this study were commercialized enteric capsules (product number: YG00-2) from Yurui Instrument Co., Ltd., which met the requirements of the study in terms of enteric delivery characteristics.

[0343] Characterization and evaluation of microemulsions: As shown in Figure 32A, the Queuine-containing microemulsion appeared as a light yellow transparent homogeneous liquid, which did not separate upon standing, and had good homogeneity and flowability. As shown in Figure 32B, the Dindal effect appeared when irradiated with a laser pen. The pH value of the Queuine-containing microemulsion was 6.33 ± 0.01, and the refractive index was 1.4150 ± 0.0005. The particle size and size distribution of the Queuine-containing microemulsion are shown in Figure 33. The results showed that the average particle size of the Queuine-containing microemulsion was 76.58 ± 0.07 nm, and the polydispersity index (PDI) value was 0.186 ± 0.020. This indicates that the Queuine microemulsion has small particle size and narrow distribution range, and is a uniform and stable dispersion system.

[0344] Characterization and evaluation of chitosan nanoparticles: As shown in Figure 34A, Queuine-containing chitosan nanoparticles appeared as a milky white suspension, which did not precipitate upon standing, and had good homogeneity and fluidity. As shown in Figure 34B, the Tyndall effect appeared upon irradiation with a laser pointer. The pH value of Queuine-containing chitosan nanoparticles was 4.30 ± 0.02. The particle size and size distribution of Queuine-containing chitosan nanoparticles are shown in Figure 35. The results showed that the average particle size of Queuine-containing chitosan nanoparticles was 480.40 ± 3.84 nm, and the polydispersity index (PDI) value was 0.122 ± 0.003. This indicates that the Queuine-containing chitosan nanoparticles have a large particle size but a narrow distribution range, and are a uniform and stable dispersion system.

[0345] Example 16 Comparative study of Queuine pharmacokinetics (PK) of different dosage forms

[0346] 16.1 Experimental methods

[0347] 16.1.1 Experimental animals and grouping

[0348] 60 healthy male Sprague-Dawley (SD) rats (weight range 250-300 g, purchased from Zhuhai Baisitong Biotechnology Co., Ltd.) were randomly divided into 10 groups, 6 rats in each group. They were divided into (1) intravenous injection of free Queuine (Q) low-dose group; (2) intravenous injection of free Queuine high-dose group; (3) oral administration of free Queuine low-dose group; (4) oral administration of free Queuine high-dose group; (5) oral administration of Queuine enteric-coated capsules low-dose group; (6) oral administration of Queuine enteric-coated capsules high-dose group; (7) oral administration of Queuine microemulsion low-dose group; (8) oral administration of Queuine microemulsion high-dose group; (9) oral administration of Queuine chitosan nanoparticles low-dose group; (10) oral administration of Queuine chitosan nanoparticles high-dose group.

[0349] 16.1.2 Dosing and sampling

[0350] The doses of the low-dose and high-dose groups were 0.5 mg / kg and 2 mg / kg, respectively. The dosing methods were as follows: free Queuine was dissolved in physiological saline and injected through the tail vein; free Queuine was dissolved in physiological saline and administered orally; enteric-coated capsules were used to directly deliver the intact capsules into the stomach using a rat oral administration device; microemulsions and chitosan nanoparticles were administered orally.

[0351] The intravenous injection group, at 0, 0.033, 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 36 and 48 hours after administration; the oral free group, at 0, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, 24, 36 and 48 hours after administration; the enteric capsule group, at 0, 1, 2, 4, 6, 8, 10, 12, 24, 36 and 48 hours after administration; the microemulsion and chitosan nanoparticle groups, at 0, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24 and 48 hours after administration, all about 200 μL of blood was collected from the tail vein into a heparinized centrifuge tube.

[0352] 16.1.3 Plasma sample processing

[0353] The blood sample was immediately centrifuged at 3500 rpm for 10 minutes, the plasma was separated and stored in a -80°C refrigerator until sample processing and analysis. When processing the sample, 50 μL of the plasma sample was taken and 150 μL of acetonitrile / methanol (2:1) was added for protein precipitation. After centrifugation at 14000 rpm for 10 minutes at 4°C, the supernatant was transferred to a new tube and dried under a nitrogen stream. The dried sample was redissolved with 50 μL of pure water containing an internal standard, vortexed for 30 seconds, and then centrifuged again (14000 rpm, 5 minutes, 4°C), and the supernatant was taken for UHPLC-MRM-MS analysis.

[0354] 16.1.4 UHPLC-MRM-MS analysis conditions

[0355] Chromatographic separation was performed using an Agilent 1290 Infinity UHPLC system. The chromatographic conditions were as follows: the chromatographic column was an Agilent InfinityLab Poroshell 120Aq-C 18Column (2.1 x 150 mm, 2.7 pm); flow rate: 0.4 mL / min; column temperature: 40 °C; mobile phase: purified water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B), gradient: 0-12 min, 0-5% B; 12-14 min, 5-80% B; 14-15 min, 80-80% B; 15-16 min, 80-0% B; 16-19 min, 0-0% B. Mass spectrometry detection was performed on an Agilent 6490 triple quadrupole (QQQ) mass spectrometer in positive ion mode using multiple reaction monitoring (MRM). The mass spectrometer parameters were set as follows: drying gas temperature 250 °C, drying gas flow rate 16 L / min, nebulizer pressure 35 psi, sheath gas temperature 400 °C, sheath gas flow rate 11 L / min, capillary voltage 4000 V. The MRM parameters for Queuine were: precursor ion m / z 278.1 → product ion m / z 136.1, collision energy 18 eV. The MRM parameters for internal standard thymine-d4 were: precursor ion m / z 131.1 → product ion m / z 88.0, collision energy 18 eV.

[0356] 16.1.5 Pharmacokinetic parameter calculation

[0357] The following parameters were calculated using Phoenix WinNonlin 8.1 software and Linear Up Log Down method: maximum plasma concentration (C max ), time to reach maximum plasma concentration (T max ), elimination half-life (t 1 / 2 ), area under the plasma concentration-time curve (AUC 0-t and AUC0-∞). Oral bioavailability (F) was calculated according to the following formula: F (%) = (AUC 口服 / AUC 静脉 ) x 100%. The relative bioavailability of each oral formulation (enteric capsule, microemulsion and chitosan nanoparticle) relative to free Queuine was calculated according to the following formula: relative F (%) = (AUC 制剂 / AUC 游离型 ) x 100%.

[0358] 16.2 Experimental results

[0359] Based on the plasma concentration data determined at each sampling time point, the plasma concentration-time curves of each dosage form under high and low dose conditions were plotted (as shown in Figures 36A, B, C, D, E, F, G, H, I, J). Further, the pharmacokinetic parameters of each formulation under high and low dose levels were calculated by non-compartment analysis method, including C max , Tmax , t 1 / 2 , AUC 0-t , AUC0-∞and F, the results are shown in Table 3.

[0360] Intravenous administration, as the reference dosage form, exhibited a maximum plasma concentration of 2035.76 ± 1145.42 ng / mL at low dose, while it increased to 7015.39 ± 1780.66 ng / mL at high dose, about 3.4-fold increase. The T max max was 0.03 hour at both doses, consistent with the pharmacokinetic profile of intravenous injection. As a reference, the bioavailability of intravenous administration was set to 100%. max

[0361] Oral free Queuine exhibited a C max max of 41.54 ± 13.52 ng / mL at low dose, while it increased to 50.70 ± 8.29 ng / mL at high dose. The T max max was 3.17 ± 1.83 hours at low dose, while it shortened to 1.75 ± 0.42 hours at high dose. The half-life of the drug remained relatively stable, 28.87 ± 45.69 hours at low dose and 32.83 ± 18.96 hours at high dose. The AUC 0-t values significantly increased from 463.93 ± 84.40 h*ng / mL at low dose to 1020.86 ± 280.47 h*ng / mL at high dose, about 2.2-fold increase. The oral bioavailability was 25.10 ± 4.57% at low dose, while it significantly increased to 40.65 ± 11.17% at high dose, suggesting that the increased dose might improve the absorption or reduce the first-pass effect. The relative bioavailability of oral formulation was 100%, as a reference to other oral dosage forms.

[0362] The significant feature of enteric-coated capsules was the longer T max max, 10.33 ± 1.97 hours at low dose and 9.00 ± 2.76 hours at high dose, reflecting its delayed release property. The C max max was 33.20 ± 14.89 ng / mL at low dose, while it increased to 44.01 ± 12.55 ng / mL at high dose. The relative bioavailability was as high as 145.35 ± 96.62% at low dose, while it was 91.33 ± 24.82% at high dose, suggesting that the absorption efficiency of enteric-coated capsules was superior to that of ordinary oral formulations at low dose, but this advantage was weakened at high dose.

[0363] Microemulsion exhibited a significantly prolonged half-life (94.50 ± 38.70 hours) at low dose, much longer than other dosage forms, but it decreased to 42.95 ± 9.26 hours at high dose. The T max ​The duration of action was 7.83 ± 3.82 hours at low doses and 4.17 ± 2.04 hours at high doses, which was also longer than that of conventional oral formulations. max The value was 38.49±5.47 ng / mL at low doses and increased slightly to 42.62±3.30 ng / mL at high doses. The relative bioavailability of the microemulsion reached 278.12±43.33% at low doses, the highest among all dosage forms, indicating that it significantly improved drug absorption; while at high doses it decreased to 117.03±16.54%, still higher than that of conventional oral formulations.

[0364] The most prominent feature of chitosan nanoparticles is their extremely long half-life, reaching 257.16±302.27 hours at low doses, but with a large standard deviation, indicating significant individual variability; at high doses, it decreases to 35.45±13.71 hours. max The relative bioavailability was relatively low across all dosage forms, at 29.31±2.90 ng / mL at low doses and 39.22±7.35 ng / mL at high doses. The relative bioavailability was 112.60±15.32% at low doses, but decreased to 45.96±6.55% at high doses, lower than that of standard oral formulations.

[0365] Overall, in terms of absorption rate and mode, intravenous administration provided immediate systemic exposure, oral formulations and chitosan nanoparticles provided relatively rapid absorption, and microemulsions exhibited moderately delayed absorption (T0.05). max Approximately 4-8 hours), while enteric-coated capsules exhibit a significantly delayed absorption pattern (T0). max (Approximately 9-10 hours). In terms of relative bioavailability, microemulsions (278.12%) offered the best absorption efficiency at low doses, significantly superior to other dosage forms. Enteric-coated capsules (145.35%) and chitosan nanoparticles (112.60%) also outperformed standard oral formulations. However, this advantage significantly diminished at higher doses. Regarding the extended half-life effect, microemulsions exhibited a significantly prolonged half-life at low doses. However, this effect was strongly dose-dependent, with its effectiveness significantly reduced at higher doses.

[0366] Table 3. Comparison of pharmacokinetic parameters of different dosage forms under high and low dose conditions (n=6; mean ± standard deviation) Note: C max Peak plasma concentration; T max Peak time; t 1 / 2 Half-life; AUC 0-t Area under the blood drug concentration-time curve from 0 to the last sampling time point; AUC 0-∞ Area under the plasma concentration-time curve from 0 to infinity; F: bioavailability. The F value for intravenous injection is set to 100% as a reference.

[0367] Example 17 Effect of Queuine hydrochloride intervention on a model of senescent cells

[0368] This example uses 2BS cells (human embryonic lung diploid fibroblasts) as a cell senescence model to evaluate the role of Queuine hydrochloride intervention in senescence regulation. This model is an in vitro cell model widely used in anti-aging research. Cells at 30 generations exhibit a young phenotype, and gradually senesce as the number of passages increases, with cells at 39 generations exhibiting a clear senescent phenotype. As shown in Figure 28C, the senescence markers p16 and p21 in 39-generation 2BS cells were significantly higher than in 30-generation cells, indicating that 39-generation 2BS cells have senescence characteristics. In this example, 2BS cells were intervened with 10 ng / mL and 50 ng / mL of Queuine hydrochloride, and the expression levels of the senescence markers p16 and p21 after intervention were determined by PCR to evaluate the anti-aging effect of Queuine hydrochloride.

[0369] It was found that Queuine hydrochloride intervention significantly reduced the levels of p16 and p21 in senescent cells (39 generations), and had a dose-dependent effect, indicating that Queuine hydrochloride has good anti-aging effects (Figure 37).

Claims

1. Use of Queuine, or a pharmaceutically acceptable salt thereof, or a composition comprising the same or the pharmaceutically acceptable salt thereof, in the manufacture of a medicament for delaying and / or improving aging in a subject or prolonging lifespan in a subject.

2. Use according to claim 1, wherein, The aging is selected from one or more of cellular aging, organ aging, and system aging.

3. Use according to claim 2, wherein, The cellular aging is selected from one or more of epithelial cells, connective tissue cells, muscle cells, neural cells, blood cells, germ cells, stem cells, immune cells, sensory cells, and gland cells.

4. The use according to claim 2, wherein, The organ aging is selected from one or more of heart, liver, spleen, lung, kidney, brain, thymus, pancreas, skin, bone, and the like.

5. The use according to claim 2, wherein, The system aging is selected from one or more of motor system, nervous system, endocrine system, cardiovascular system, lymphatic system, respiratory system, digestive system, urinary system, reproductive system, immune system, skin system, and sensory system.

6. The use according to any one of claims 1 to 5, wherein, The subject is selected from a mammal and a non-mammal.

7. The use according to any one of claims 1 to 6, wherein, The subject is a mammal.

8. The use according to any one of claims 1 to 7, wherein, The subject is selected from a human, a cow, a dog, a monkey, a goat, a sheep, a dairy cow, a deer, a rat, a mouse, and other mammals.

9. The use according to any one of claims 1 to 8, wherein, The subject is a human.

10. The use according to any one of claims 1 to 9, wherein, The subject is at least about 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 years of age.

11. Use according to any one of claims 1 to 10, wherein, The subject is between about 16 and 95 years of age, or between about 18 and 95 years of age.

12. The use according to any one of claims 1 to 11, wherein, The subject is between about 50 and 65 years of age, or between about 60 and 75 years of age, or between about 70 and 85 years of age, or between about 80 and 95 years of age.

13. The use according to any one of claims 1 to 12, wherein, The subject is an adult between 25 and 60 years of age.

14. The use according to any one of claims 1 to 13, wherein, The subject is a young adult between 18 years of age and below 25 years of age.

15. The use according to any one of claims 1 to 14, wherein, The subject is an elderly person above 60 years of age.

16. The use according to any one of claims 1 to 15, wherein, The subject is an elderly person above 65 years of age.

17. The use according to any one of claims 1 to 16, wherein, The medicament comprises at least about 300 ng of the Queuine or the salt thereof.

18. The use according to claim 17, wherein, The medicament comprises at least about 500 ng, 750 ng, 1 µg, 2 µg, 3 µg, 4 µg, 5 µg, 6 µg, 9 µg, 15 µg, 20 µg, 25 µg, 30 µg, 35 µg, 40 µg, 45 µg, 50 µg, 100 µg, 150 µg, 250 µg, 500 µg, 750 µg, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 6 mg, 9 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 100 mg, 150 mg, 250 mg, or 500 mg of the Queuine or the salt thereof.

19. The use according to any one of claims 1-18, wherein, The Queuine or the salt thereof is administered at a dose of at least about 10 ng / kg, or the composition is administered at a dose in which the active ingredient comprises at least about 10 ng / kg of the Queuine or the salt thereof.

20. The use according to any one of claims 1-19, wherein, The Queuine or salt thereof, or the composition is administered at a dose of at least about 10 ng to 10 mg / kg, or the composition is administered at a dose of active ingredient comprising at least about 10 ng to 10 mg / kg of the Queuine or salt thereof.

21. The use according to any one of claims 1-20, wherein, at least about 10 ng / kg, 15 ng / kg, 20 ng / kg, 25 ng / kg, 30 ng / kg, 35 ng / kg, 40 ng / kg, 45 ng / kg, 50 ng / kg, 55 ng / kg, 60 ng / kg, 65 ng / kg, 70 ng / kg, 75 ng / kg, 80 ng / kg, 85 ng / kg, 90 ng / kg, 95 ng / kg, 100 ng / kg, 120 ng / kg, 150 ng / kg, 180 ng / kg, 200 ng / kg, 250 ng / kg, 300 ng / kg, 350 ng / kg, 400 ng / kg, 450 ng / kg, 500 ng / kg, 550 ng / kg, 600 ng / kg, 650 ng / kg, 700 ng / kg, 750 ng / kg, 800 ng / kg, 850 ng / kg, 900 ng / kg, 950 ng / kg, 1 μg / kg, 2 μg / kg, 3 μg / kg, 5 μg / kg, 10 μg / kg, 15 μg / kg, 20 μg / kg, 25 μg / kg, 30 μg / kg, 35 μg / kg, 40 μg / kg, 45 μg / kg, 50 μg / kg, 55 μg / kg, 60 μg / kg, 65 μg / kg, 70 μg / kg, 75 μg / kg, 80 μg / kg, 85 μg / kg, 90 μg / kg, 95 μg / kg, 100 μg / kg, 120 μg / kg, 150 μg / kg, 180 μg / kg, 200 μg / kg, 250 μg / kg, 300 μg / kg, 350 μg / kg, 400 μg / kg, 450 μg / kg, 500 μg / kg, 550 μg / kg, 600 μg / kg, 650 μg / kg, 700 μg / kg, 750 μg / kg, 800 μg / kg, 850 μg / kg, 900 μg / kg, 950 μg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 55 mg / kg, 60 mg / kg, 65 mg / kg, 70 mg / kg, 75 mg / kg, 80 mg / kg, 85 mg / kg, 90 mg / kg, 95 mg / kg, 100 mg / kg, 120 mg / kg, 150 mg / kg, 180 mg / kg, 200 mg / kg, 250 mg / kg, 300 mg / kg, 350 mg / kg, 400 mg / kg, 450 mg / kg, 500 mg / kg, 550 mg / kg, 600 mg / kg, 650 mg / kg, 700 mg / kg, 750 mg / kg, 800 mg / kg, 850 mg / kg, 900 mg / kg, 950 mg / kg, 1 g / kg, 2 g / kg, 3 g / kg, 5 g / kg, 10 g / kg, 15 g / kg, 20 g / kg, 25 g / kg, 30 g / kg, 35 g / kg, 40 g / kg, 45 g / kg, 50 g / kg, 55 g / kg, 60 g / kg, 65 g / kg, 70 g / kg, 75 g / kg, 80 g / kg, 85 g / kg, 90 g / kg, 95 g / kg, 100 g / kg, 120 g / kg, 150 g / kg, 180 g / kg, 200 g / kg, 250 g / kg, 300 g / kg, 350or at least about 10 ng / kg, 15 ng / kg, 20 ng / kg, 25 ng / kg, 30 ng / kg, 35 ng / kg, 40 ng / kg, 45 ng / kg, 50 ng / kg, 55 ng / kg, 60 ng / kg, 65 ng / kg, 70 ng / kg, 75 ng / kg, 80 ng / kg, 85 ng / kg, 90 ng / kg, 95 ng / kg, 100 ng / kg, 120 ng / kg, 150 ng / kg, 180 ng / kg, 200 ng / kg, 250 ng / kg, 300 ng / kg, 350 ng / kg, 400 ng / kg, 450 ng / kg, 500 ng / kg, 550 ng / kg, 600 ng / kg, 650 ng / kg, 700 ng / kg, 750 ng / kg, 800 ng / kg, 850 ng / kg, 900 ng / kg, 950 ng / kg, 1 μg / kg, 2 μg / kg, 3 μg / kg, 5 μg / kg, 10 μg / kg, 15 μg / kg, 20 μg / kg, 25 μg / kg, 30 μg / kg, 35 μg / kg, 40 μg / kg, 45 μg / kg, 50 μg / kg, 55 μg / kg, 60 μg / kg, 65 μg / kg, 70 μg / kg, 75 μg / kg, 80 μg / kg, 85 μg / kg, 90 μg / kg, 95 μg / kg, 100 μg / kg, 120 μg / kg, 150 μg / kg, 180 μg / kg, 200 μg / kg, 250 μg / kg, 300 μg / kg, 350 μg / kg, 400 μg / kg, 450 μg / kg, 500 μg / kg, 550 μg / kg, 600 μg / kg, 650 μg / kg, 700 μg / kg, 750 μg / kg, 800 μg / kg, 850 μg / kg, 900 μg / kg, 950 μg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg of the active ingredient.

22. The use according to any one of claims 1-21, wherein, The Queuine or salt thereof, or the composition can be administered enterally, e.g., orally, e.g., in tablet, emulsion, or capsule form, or parenterally, e.g., in injectable solution or suspension form, topically, e.g., in lotion, gel, ointment, or cream form, or in nasal or suppository form.

23. The use according to any one of claims 1-22, wherein, The Queuine or salt thereof, or the composition is formulated for oral administration.

24. The use according to any one of claims 1 to 23, wherein, The Queuine or salt thereof, or the composition is a food, beverage, feed composition, or nutritional supplement.

25. The use according to any one of claims 1-24, wherein, The Queuine or salt thereof, or the composition is a liquid, syrup, emulsion, tablet, lozenge, gummy, capsule, powder, gel, or film.

26. The use according to any one of claims 1 to 25, wherein, The use of the Queuine or pharmaceutically acceptable salt thereof, or the composition in combination with a second therapeutic agent in the manufacture of a medicament for delaying and / or improving aging in a subject or prolonging the life of a subject.

27. The use according to any one of claims 1 to 26, wherein, The second therapeutic agent is selected from one or more of rapamycin, beta-nicotinamide mononucleotide (NMN), metformin, urolithin A, vitamin C, taurine, spermidine, quercetin, resveratrol, and other dietary supplements or drugs with anti-aging effects.

28. The use according to any one of claims 1 to 27, wherein, The subject administered with the Queuine or salt thereof, or the composition lives at least more than 10 days, 20 days, 1 month, 2 months, 3 months, half a year, 1 year, 2 years, 3 years, 5 years, 10 years, 20 years, or 30 years longer than a control not administered with the Queuine or salt thereof, or the composition.

29. The use according to any one of claims 1 to 28, wherein, The subject administered with the Queuine or salt thereof, or the composition lives at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, or 90% or more longer than a control not administered with the Queuine or salt thereof, or the composition.

30. The use according to any one of claims 1 to 29, wherein, The subject administered with the Queuine or salt thereof, or the composition experiences a delay or improvement in symptoms of aging of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 100%, 200%, or more than a control not administered with the Queuine or salt thereof, or the composition.

31. The use according to any one of claims 1 to 30, wherein, The Queuine or salt thereof, or the composition can also be used in the manufacture of one or more of the following: (1) improving learning ability in a subject; (2) improving memory in a subject; (3) improving cognitive ability in a subject; (4) improving motor ability in a subject; (5) improving antioxidant ability in a subject; (6) improving heat stress resistance in a subject.

32. The use of claim 31, wherein, The increase is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more over the control level.

33. A method of delaying and / or improving aging in a subject or prolonging lifespan in a subject comprising administering to the subject an effective amount of Queuine, or a pharmaceutically acceptable salt thereof, or a composition comprising the same or a pharmaceutically acceptable salt thereof.

34. The method of claim 33, wherein, The Queuine or salt thereof is administered at a dose of at least about 10 ng / kg daily, every two days, every three days, weekly, every two weeks, every three weeks, monthly, every two months, every three months, every six months, or every year, or the composition is administered at a dose in which the active ingredient comprises at least about 10 ng / kg of the Queuine or salt thereof daily, every two days, every three days, weekly, every two weeks, every three weeks, monthly, every two months, every three months, every six months, or every year.

35. The method of claim 33, wherein, The Queuine or salt thereof is administered at a dose of at least about 10 ng / kg daily, every two days, every three days, weekly, every two weeks, every three weeks, monthly, every two months, every three months, every six months, or every year, or the composition is administered at a dose in which the active ingredient comprises at least about 10 ng / kg of the Queuine or salt thereof daily, every two days, every three days, weekly, every two weeks, every three weeks, monthly, every two months, every three months, every six months, or every year.

36. A method of treating paraquat poisoning comprising administering to the subject an effective amount of Queuine, or a pharmaceutically acceptable salt thereof, or a composition comprising the same or a pharmaceutically acceptable salt thereof.

37. A method of treating metabolic bone disease comprising administering to the subject an effective amount of Queuine, or a pharmaceutically acceptable salt thereof, or a composition comprising the same or a pharmaceutically acceptable salt thereof. Preferably, the metabolic bone disease is osteoporosis.

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