Pharmaceutical use of reduced β-nicotinamide mononucleotide

Abstract

Provided is pharmaceutical use of reduced β-nicotinamide mononucleotide (NMNH). Specifically, the present invention relates to use of reduced β-nicotinamide mononucleotide (NMNH) and / or a corresponding salt thereof in the preparation of a drug or health-care product for relieving fatigue, improving sleep, promoting heart health, and improving cognition and / or combating aging.

Description

Pharmaceutical use of reduced beta-nicotinamide mononucleotide

[0001] Cross-reference to related applications

[0002] This application claims priority to the Chinese patent application No. 202410466701.8, filed on April 17, 2024, entitled “Pharmaceutical use of reduced beta-nicotinamide mononucleotide”, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present application belongs to the technical field of pharmaceutical preparations, and specifically relates to a pharmaceutical use of reduced beta-nicotinamide mononucleotide, and more specifically relates to a use of reduced beta-nicotinamide mononucleotide (NMNH) for preparing a drug for relieving fatigue, improving sleep, promoting heart health, improving cognition, and / or anti-aging. BACKGROUND

[0004] Aging is a progressive, irreversible pathophysiological process, mainly manifested as a decline in tissue and cellular function, and a significantly increased risk of various aging-related diseases, including neurodegenerative diseases, cardiovascular diseases, metabolic diseases, musculoskeletal diseases, and immune system diseases. Although the development of modern medicine has promoted human health and greatly extended the life expectancy of humans, with the aging of society, various chronic diseases have gradually become the most important cause of disability and death in the elderly. Currently, research on aging mainly focuses on elucidating how various endogenous and exogenous stresses participate in the regulation of aging, such as genomic instability, telomere dysfunction, epigenetic changes, loss of protein homeostasis, impaired autophagy, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, altered intercellular communication, and impaired nutrient sensing. In addition, the field is eagerly awaiting in-depth research on the pathogenesis of aging to determine interventions (such as caloric restriction, microbial transplantation, and nutritional interventions) that promote health and longevity and clinical treatment methods (such as senolytic therapy, stem cell therapy, antioxidant, and anti-inflammatory therapy) for aging-related diseases.

[0005] At present, as one of the most popular molecules in the field of anti-aging, nicotinamide adenine dinucleotide (NAD) has become the center of anti-aging substances without exception. NAD exists in two forms, namely oxidized form (NAD+) and reduced form (NADH), wherein the oxidized form NAD+ accepts a hydrogen ion to become the reduced form NADH, and this conversion process is crucial to central carbon metabolism. As a coenzyme of redox reaction, NAD+ is an important part of energy metabolism; NAD+ is also an essential cofactor for non-oxidation-reduction NAD+-dependent enzymes, including sirtuins, CD38 and poly (adp-ribose) polymerase. NAD+ can directly or indirectly affect many key cellular functions, including metabolic pathways, DNA repair, chromatin remodeling, cell aging and immune cell function. These cellular processes and functions are crucial for maintaining tissue and metabolic homeostasis and healthy aging. Notably, aging is often accompanied by a gradual decline in tissue and cellular NAD+ levels in a variety of model organisms, including rodents and humans. The decline in NAD+ levels is causally related to many age-related diseases, including cognitive decline, cancer, metabolic disease, muscle loss and weakness. Many age-related diseases can be slowed down or even reversed by restoring NAD+ levels. Therefore, targeting NAD+ metabolism has become a potential therapeutic method to improve age-related diseases and prolong human healthy life.

[0006] NAD+ can be synthesized in a de novo biosynthesis pathway using tryptophan, or in a preiss-handler pathway using nicotinic acid (NA), and in a salvage pathway using nicotinamide (NAM), nicotinamide riboside (NR), nicotinamide mononucleotide (NMN), reduced nicotinamide riboside (NRH) or reduced nicotinamide mononucleotide (NMNH). In particular, as a key intermediate of NAD+, NAM, NR, NRH and NMN have been widely studied for their potential therapeutic effects in many mouse disease models, among which NMN is considered to be the most suitable NAD+ precursor at present, and NMN has been hot-selling in the global market and favored by consumers. Therefore, it is of positive significance to develop more new drugs based on the improvement and therapeutic effect of NAD+ activity. SUMMARY

[0007] To this end, the technical problem to be solved by the present application is to provide a new use of reduced β-nicotinamide mononucleotide (NMNH) as a drug, more specifically to a use of reduced β-nicotinamide mononucleotide (NMNH) for preparing a drug for relieving fatigue, improving sleep, promoting heart health, improving cognition and / or anti-aging;

[0008] A second technical problem to be solved by the present application is to provide a medicament having the effects of relieving fatigue, improving sleep, promoting heart health, improving cognition, and / or anti-aging.

[0009] To solve the above technical problems, the present application provides the use of reduced nicotinamide mononucleotide (NMNH) and / or a salt thereof for preparing a preparation having at least one of the following effects (1)-(5):

[0010] (1) relieving fatigue;

[0011] (2) improving sleep;

[0012] (3) promoting heart health;

[0013] (4) improving cognition;

[0014] (5) anti-aging.

[0015] Specifically, the daily dosage of the reduced nicotinamide mononucleotide (NMNH) and / or a salt thereof is 1-1000 mg / kg body weight.

[0016] Specifically, the salt of the reduced nicotinamide mononucleotide (NMNH) includes one or a mixture of several of sodium salt, calcium salt, magnesium salt, potassium salt, iron salt, zinc salt, or manganese salt.

[0017] Specifically, the preparation includes food, medicine, and / or health products.

[0018] Specifically, the preparation includes at least one of oral preparations, injection dosage forms, respiratory administration dosage forms, skin administration dosage forms, mucosal administration dosage forms, or cavity administration dosage forms.

[0019] Specifically, the preparation includes one or more of the forms of tablets, capsules, granules, aqueous preparations, enteric preparations, injections, serums, emulsions, creams, foams, sprays, ointments, gels, lotions, pads, roll-on preparations, caplets, lozenges, troches, chewable tablets, gums, jellies, syrups, liquid solutions, suspensions, buccal films, sublingual films, oral adhesive films, powders, solid crystals, orally disintegrating tablets, or pastes.

[0020] Specifically, the preparation further includes food, pharmaceutical, and / or health product acceptable adjuvants or carriers.

[0021] The present application also discloses a composition, wherein the active ingredients of the composition include reduced nicotinamide mononucleotide (NMNH) and / or a salt thereof.

[0022] The application also discloses a food, medicine and / or health product with effects of relieving fatigue, improving sleep, promoting heart health, improving cognition and / or anti-aging, which comprises reduced nicotinamide mononucleotide (NMNH) and / or a salt thereof.

[0023] Specifically, the food, medicine and / or health product with effects of relieving fatigue, improving sleep, promoting heart health, improving cognition and / or anti-aging, the content of the reduced nicotinamide mononucleotide (NMNH) and / or a salt thereof is 0.01-100 wt%.

[0024] The application also discloses a method for relieving fatigue, improving sleep, promoting heart health, improving cognition and / or anti-aging, which comprises administering reduced nicotinamide mononucleotide (NMNH) and / or a salt thereof to a subject in need.

[0025] Specifically, the daily dose of the reduced nicotinamide mononucleotide (NMNH) and / or a salt thereof is 1-1000 mg / kg body weight.

[0026] Specifically, the salt of the reduced nicotinamide mononucleotide (NMNH) comprises one or a mixture of several of sodium salt, calcium salt, magnesium salt, potassium salt, iron salt, zinc salt or manganese salt.

[0027] The application uses reduced nicotinamide mononucleotide (NMNH) or a corresponding salt thereof as an active ingredient, and applies it in the preparation of a medicine or health product for relieving fatigue, improving sleep, promoting heart health, improving cognition and anti-aging, effectively solving the problems of poor efficacy, high cost and toxic side effects of existing medicines or health products.

[0028] The application uses reduced nicotinamide mononucleotide (NMNH) or a corresponding salt thereof as an active ingredient, which not only proves that it has effects of relieving fatigue, improving sleep, promoting heart health, improving cognition and anti-aging, but also can more effectively improve the NAD+ level in blood, brain, liver, kidney, heart, gastrocnemius muscle and the like compared with NMN of the same dose, which makes NMNH a more potential NAD+ enhancer and has potential application prospects in the anti-aging field, effectively expanding the health application field of NMNH. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to make the content of the application more easily understood, the application will be further described in detail below according to specific embodiments of the application and in combination with the drawings, in which,

[0030] Figure 1 is the staining result of myocardial cells under fluorescence microscope, wherein 1-5 are myocardial cell apoptosis of blank control group, positive control group-coenzyme Q10, NMNH-100mg / kg group, NMNH-200mg / kg group and NMNH-300mg / kg group in turn. Blue is the staining negative cell, i.e. the surviving myocardial cell, and green is the staining positive cell, i.e. the apoptotic myocardial cell. DETAILED DESCRIPTION

[0031] In the following examples of the present application, the test substance is NMNH, and the preparation method is pure water. The blank control is ultrapure water. Both are prepared on the same day and cannot be prepared overnight.

[0032] In the following examples of the present application, the experimental mice are administered by gavage or intraperitoneal injection. Oral gavage is a method of administering drug solution or suspension directly into the stomach of animals using a gavage needle. It is the most commonly used method of drug administration in animal experiments, especially in modeling of experimental animals in physiology, pharmacology, immunology and other disciplines, and in research on drug toxicity, efficacy and dose determination. Both gavage solution and suspension can be used, as long as the drug is uniform and stable, and can be repeatedly administered. The selection of gavage tools is generally based on the age of the mice. For example, naked mice or mice under 5 weeks of age can use a 6-gauge gavage needle, mice weighing less than 30g can use an 8-gauge or 9-gauge gavage needle, and mice weighing more than 30g can use a 12-gauge gavage needle.

[0033] In the following examples of the present application, the specific gavage experimental steps are as follows:

[0034] 1. Preparation: The mice should be fasted for 4-8 hours before gavage to avoid too much stomach contents hindering gavage injection and affecting drug absorption rate;

[0035] 2. Drug preparation: The drug is prepared according to the concentration marked on each group, and the dosage is calculated according to the body weight of each mouse;

[0036] 3. Gavage preparation: The mice are weighed, the gavage needle and syringe are installed, the dosage is drawn according to the body weight, and the drug is placed aside for standby;

[0037] 4. Mouse catching and fixing: The mouse is caught by grabbing the tail, and the animal is stimulated as little as possible. The tail part should not be too close to the tail end. The mouse is placed on a rough surface, the left hand (here the operator is right-handed) thumb and index finger grab the mouse ear and head and neck skin, the left hand palm and little finger hold the tail, the head, neck and body (oral cavity and esophagus) are in a straight line, the abdomen is upward, and the mouse is fixed firmly without moving. The skin of the neck should not be pulled too tightly. If the mouse is not caught properly, it should be released and caught again to avoid problems in subsequent operations;

[0038] 5. Intragastric administration: The right hand holds the syringe and inserts the needle into the oral cavity from the corner of the mouth, avoiding the teeth, and slowly inserts the needle along the tongue into the upper palate, avoiding damage to the esophagus. If resistance is encountered, the needle can be slightly moved up and down. When the mouse swallows, the cardiac muscle relaxes, and the feeling of resistance disappears. Slowly push the needle in. Generally, when the needle is inserted into the mouse 3-4 cm, i.e., the intragastric needle is 3 / 4, the syringe is backflushed without air backflow, indicating that the needle has entered the stomach, and the injection can be performed. If the animal struggles strongly, has difficulty breathing, or encounters significant resistance during needle insertion, stop the needle insertion immediately and remove the needle. After the animal has recovered, try again. After the injection is complete, release the mouse and observe if there is any abnormal breathing. If not, the intragastric administration is considered successful.

[0039] In the following examples of the present application, the dissection tools, including forceps, scalpels, and scissors, were disinfected with 75% alcohol wipes and thoroughly rinsed with double distilled water (ddH2O); the area was disinfected with 70% ethanol spray; and PBS for washing the tissue was prepared in a sterile petri dish.

[0040] Example 1: Sleep improvement function experiment

[0041] The experiments of this example are based on the "Health Food Function Test and Evaluation Method (2022 Edition)", "Health Food Function Test and Evaluation Technical Guidelines (2022 Edition)".

[0042] Experimental principle: Sodium pentobarbital hinders the transmission of excitation impulses to the cerebral cortex, thereby exerting an inhibitory effect on the central nervous system. In small doses, the experimental animal exhibits a hypnotic effect. On the basis of sodium pentobarbital-induced sleep, it is observed whether the test drug can prolong sleep time.

[0043] This example involves instruments and reagents, including mouse cages and sodium pentobarbital.

[0044] Experimental protocol

[0045] The experimental protocol of this example is designed as shown in Table 1.

[0046] Table 1: Sleep improvement experimental protocol design

[0047] Experimental procedure

[0048] 1. Pre-experiment

[0049] A pre-experiment was conducted before the animal experiment to determine the dose of sodium pentobarbital that would induce 100% sleep in mice without causing excessive sleep time.

[0050] 2. Formal experiment

[0051] C57 mice were orally gavaged for 20 consecutive days, and after the last administration, each group of mice was intraperitoneally injected with sodium pentobarbital at a dose of 50 mg / kg. After placing the animals, the loss of the mouse righting reflex was used as an indicator to record the sleep time of each mouse. The loss of the righting reflex refers to the back remaining for more than 30 s and not appearing again within 15 s.

[0052] In this example, the statistical results of each group of experiments are as follows in Table 2.

[0053] Table 2: Results of each experimental group

[0054] In this example, the p-value results of the statistical results of each group of experiments are shown in Table 3 below.

[0055] Table 3: Statistical data p results

[0056] It can be seen that under the experimental conditions, compared with the control group, the positive control group's melatonin and different concentrations of reduced nicotinamide mononucleotide (NMNH) can effectively improve the sleep of mice, and the effect of the test substance NMNH in improving sleep shows a certain dose-dependent effect.

[0057] Example 2: Fatigue relief experiment

[0058] The test basis of this example is "Health Food Function Test and Evaluation Method (2022 Edition)", "Health Food Function Test and Evaluation Technical Guidelines (2022 Edition)". Somatic fatigue is caused by a series of biochemical changes in the body caused by exercise, leading to a decrease in muscle strength. Therefore, there are mainly two methods for evaluating fatigue: endurance test and biochemical change detection.

[0059] In this example, the experimental design is as follows in Table 4.

[0060] Table 4: Fatigue relief experiment design

[0061] Weighted swimming experiment

[0062] Test principle: The most important manifestation of fatigue is the decline in exercise tolerance, and exercise tolerance is the most direct and objective indicator of body fatigue. The length of swimming time can reflect the degree of animal exercise fatigue.

[0063] Test steps: C57 mice were intraperitoneally injected every other day for 15 days, and 30 min after the last administration, the mice were weighed by 5% lead at the root of the tail, placed in a swimming box with water temperature of 25±1℃ and water depth of 40 cm, and the time from the start of swimming to death was recorded with a stopwatch, i.e. the weighted swimming time of mice.

[0064] In this embodiment, the results of the weight-loaded swimming experiment are shown in Table 5 below.

[0065] Table 5 Weight-loaded swimming experiment results

[0066] Blood lactic acid determination

[0067] The detection steps are as follows:

[0068] 1) Standard sample addition: Set standard sample holes and sample holes, and add different concentrations of standard samples 50 μL to each standard sample hole;

[0069] 2) Sample addition: Set blank holes (blank control holes do not add samples and enzyme-labeled reagents, and the rest of the steps are the same) and sample holes. Add sample diluent 40 μl to the sample hole on the enzyme-labeled coating plate, and then add the sample to be tested 10 μl (the final dilution of the sample is 5 times). Add the sample to the bottom of the enzyme-labeled plate hole, try not to touch the hole wall, and gently shake to mix;

[0070] 3) Add enzyme: Add enzyme-labeled reagent 100 μl to each hole, except for the blank hole;

[0071] 4) Incubation: After sealing the plate with a sealing film, incubate at 37°C for 60 minutes;

[0072] 5) Liquid preparation: Dilute the 20-fold concentrated washing solution with distilled water 20 times for standby;

[0073] 6) Washing: Carefully remove the sealing film, discard the liquid, and shake dry. Add washing solution to each hole, stand for 30 seconds, then discard. Repeat this process 5 times, and tap dry;

[0074] 7) Color development: Add color developing agent A 50 μl to each hole, then add color developing agent B 50 μl, shake gently to mix, and develop color at 37°C for 15 minutes in the dark;

[0075] 8) Termination: Add termination solution 50 μl to each hole to terminate the reaction (at this time, the blue color turns yellow);

[0076] 9) Measurement: Zero the blank hole, measure the absorbance (OD value) of each hole at 450 nm, and the measurement should be completed within 15 minutes after adding the termination solution.

[0077] In this embodiment, the blood lactic acid experiment data after 15 days of administration are shown in Table 6 below.

[0078] Table 6 Blood lactic acid determination experiment results after 15 days of administration

[0079] In this embodiment, the blood lactic acid experiment data p value results after 15 days of administration are shown in Table 7.

[0080] Table 7 Blood lactic acid data P value

[0081] It can be seen that under the experimental conditions, compared with the control group, taurine and different concentrations of reduced nicotinamide mononucleotide (NMNH) in the positive control group can effectively relieve the fatigue of mice, prolong the weight-bearing swimming time and reduce the blood lactate content. The fatigue-relieving effect of the test substance NMNH is dose-dependent.

[0082] Example 3 Experiment on promoting heart health function

[0083] The inspection basis of this embodiment includes: "Health Food Function Inspection and Evaluation Method (2022 Edition)" and "Health Food Function Inspection and Evaluation Technical Guidelines (2022 Edition)".

[0084] Principle of the test: Cell apoptosis detection is an excellent solution for detecting cell health and function, in which the breakage of chromosomal DNA is a gradual and staged process. Chromosomal DNA is first degraded into large fragments of 50-300kb by the action of endogenous nucleases, and then about 30% of the chromosomal DNA is broken down by Ca 2+ and Mg 2+ Under the action of a template-dependent endonuclease, DNA is randomly cleaved between nucleosome units, forming 180-200 bp nucleosomal DNA polymers. Therefore, in the late stages of apoptosis, DNA is degraded into 180-200 bp fragments, exposing numerous 3'-OH termini on the fragmented genomic DNA. Terminal deoxynucleotidyl transferase (TdT) is a template-independent DNA polymerase that catalyzes the incorporation of deoxynucleotides into the 3'-OH termini of broken DNA molecules. Therefore, the TUNEL (TdT-mediated dUTP Nick End Labeling) apoptosis assay can be used to detect nuclear DNA fragmentation in tissue cells during the late stages of apoptosis. The principle is that, under the action of TdT enzyme, fluorescein-labeled dUTP (FITC-12-dUTP) is incorporated into the 3'-OH termini exposed by genomic DNA fragmentation, allowing for detection using a fluorescence microscope or flow cytometer (FITC excitation 450-500 nm, emission 515-565 nm).

[0085] Experimental plan

[0086] The experimental plan for this embodiment is shown in Table 8 below.

[0087] Table 8 Experimental design

[0088] The experimental steps are as follows:

[0089] 1. C57 mice are orally gavaged with different concentrations of the sample to be tested, a positive control, or ultrapure water for 20 consecutive days, and are euthanized with saturated carbon dioxide, and heart tissue is taken, deparaffinized in xylene for 5-10 minutes; fresh xylene is used, and deparaffinization is performed again for 5-10 minutes; anhydrous ethanol for 5 minutes, 90% ethanol for 2 minutes, 70% ethanol for 2 minutes, and distilled water for 2 minutes;

[0090] 2. 20 μg / ml of proteinase K (recommended use: Biyun Tian ST532 / ST533 proteinase K (20 mg / ml), diluted 1000 times with P0106 immunostaining washing solution or 10 mM Tris-HCl pH 7.4-7.8 to obtain 20 μg / ml of proteinase K without DNase) is added dropwise, and 20-37°C is allowed to act for 15-30 minutes (the optimal temperature and time for different tissues need to be explored by oneself);

[0091] 3. PBS or HBSS is used for washing 3 times, with the note that this step must wash the proteinase K clean, otherwise it will seriously interfere with the subsequent labeling reaction;

[0092] 4. TUNEL detection solution is prepared: refer to the following to prepare an appropriate amount of TUNEL detection solution, and mix well; note that the prepared TUNEL detection solution must be used up at one time, and cannot be frozen;

[0093] 1 sample: TdT enzyme 5 μl, fluorescent labeling solution 45 μl, TUNEL detection solution 50 μl;

[0094] 5 samples: TdT enzyme 25 μl, fluorescent labeling solution 225 μl, TUNEL detection solution 250 μl;

[0095] 10 samples: TdT enzyme 50 μl, fluorescent labeling solution 450 μl, TUNEL detection solution 500 μl;

[0096] 5. 50 μl of TUNEL detection solution is added to the sample, and incubation is performed at 37°C in the dark for 60 minutes;

[0097] 6. PBS or HBSS is used for washing 3 times;

[0098] 7. After sealing with an anti-fluorescence quenching mounting medium, observation is performed under a fluorescence microscope, and the excitation wavelength range that can be used is 450-500 nm, and the emission wavelength range is 515-565 nm (green fluorescence).

[0099] In this embodiment, the myocardial cell staining result under the fluorescence microscope is shown in FIG. 1, the apoptotic myocardial cells are dyed green, and the surviving myocardial cells are dyed blue, and the proportion of green cells is further calculated to evaluate the apoptosis of the myocardial cells.

[0100] In this embodiment, the statistical results are shown in Table 9 below.

[0101] Table 9 Experimental statistical results

[0102] In this embodiment, the p-value results of each experimental group are shown in Table 10 below.

[0103] Table 10 P-value results

[0104] It can be seen that under the experimental conditions, compared with the blank control group, coenzyme Q10 and different concentrations of reduced nicotinamide mononucleotide (NMNH) in the positive control group can effectively reduce the apoptosis rate of cardiomyocytes, have the effect of protecting the heart, and the heart protection effect of NMNH is better than that of coenzyme Q10, showing a certain dose-dependent effect.

[0105] Example 4: Cognitive improvement experiment

[0106] In this embodiment, the test basis includes: Health Food Function Test and Evaluation Method (2022 Edition) and Health Food Function Test and Evaluation Technical Guidelines (2022 Edition).

[0107] Acetylcholine, as an important neurotransmitter in the brain, is the biochemical basis of memory and learning. Studies have shown that acetylcholinesterase (AchE) can decompose acetylcholine between synapses and affect individual learning and memory activities. Patients with cognitive impairment have elevated serum AchE levels. Under normal circumstances, SOD and MDA are in a balanced state, but in patients with cognitive impairment, MDA is abnormally elevated. SOD is an important oxygen free radical metabolite in the body that can effectively scavenge oxygen free radicals and prevent cellular damage from oxygen free radicals. Abnormally high MDA exceeds the range of SOD clearance, which can cause an increase in oxidative stress levels, increase the release of oxygen free radicals, damage brain cells and brain neurons, and cause cognitive-related regions of the brain to have impaired cholinergic pathways, abnormal cholinergic function, and decreased levels of cholinergic neurotransmitters. By detecting the levels of acetylcholinesterase (AchE) and MDA, the impact on cognition can be reflected.

[0108] Malondialdehyde (MDA) in lipid peroxidation product can condense with thio-barbituric acid (TBA) to form a red product with maximum absorption at 532 nm. This method is called TBA method because the substrate is thio-barbituric acid (TBA). Acetylcholinesterase (AChE) hydrolyzes acetylcholine to produce choline and acetic acid. Choline can react with sulfhydryl color reagent to form a TNB (sym-trinitrobenzene) yellow compound. The amount of hydrolysis product choline can reflect the activity of cholinesterase according to the color depth.

[0109] The instruments used in this example include an enzyme label instrument (Tecan; Sunrise); reagent consumables include a malondialdehyde (MDA) detection kit, a 96-well enzyme label plate; an acetylcholinesterase (AChE) detection kit, a 96-well enzyme label plate.

[0110] Experimental design

[0111] The experimental design of this example is shown in Table 11 below.

[0112] Table 11 Experimental design

[0113] The specific experimental steps of this example include:

[0114] 1. Sample pretreatment:

[0115] C57 mice were orally gavaged for 20 consecutive days, and serum / plasma was directly measured. If the measured range is exceeded, physiological saline can be used for dilution before sampling;

[0116] 2. Experimental steps:

[0117] According to the sample addition table shown in Tables 12-13 below, blank tubes, standard tubes, test tubes, and control tubes are set up, and then the corresponding reagents are added to the enzyme label plate according to the operation table. Cover the centrifuge tube with a cover, prick a small hole in the cover with a needle, mix with a vortex mixer, 95°C water bath (or boil with a kettle with the cover open) for 40 minutes, then cool with running water, then centrifuge at 3500-4000 rpm for 10 minutes. (The centrifugation time needs to be extended below 3000 rpm to completely precipitate). Take the supernatant, 532 nm, 1 cm light path, distilled water zero, measure the absorbance value of each tube;

[0118] Table 12 Malondialdehyde (MDA) sample addition table

[0119] [Note]: a*: indicates the amount of sample, standard, ethanol, and reagent I, which are equal. (a* is generally 0.1-0.2 mL) For example, if the sample is 0.1 mL, the standard, ethanol, and reagent I are also 0.1 mL. If the sample is 0.2 mL, the standard, ethanol, and reagent I are also 0.2 mL. Because the absorbance is proportional to the sample volume, the results are not affected. Generally, only 1-2 standard tubes, blank tubes, and control tubes are needed per batch. If there is no hemolysis or lipemia in the sample, the control tube can not be measured and the blank tube can be used instead of the control tube. When the supernatant is colorimetrically measured, it is best to use a pipette to suck the supernatant into the cuvette to avoid pouring to prevent the precipitate from entering the cuvette and affecting the absorbance.

[0120] Calculation formula:

[0121] Serum (plasma) MDA content = (measured OD value - control OD value) / (standard OD value - blank OD value) * standard concentration * dilution factor.

[0122] Table 13 Acetylcholinesterase (AChE) sample addition table

[0123] [Note]: 1. The control tube must be done for each sample because the absorbance difference of each sample control tube is large. 2. The test tube is placed at room temperature for 15 minutes before colorimetry. If the room temperature is too low, the precipitate or turbidity may appear. At this time, please place the test tube in a 37°C water bath for a few minutes to clarify. After clarification, the experiment can be colorimetrically measured and the results will not be affected. 3. Because the reaction time is short, the number of samples measured per batch should not be too large. The reaction time should be accurately controlled, otherwise the experimental accuracy will be affected. 4. a* indicates the amount of sample, standard, and distilled water, which are equal. ① The serum (plasma) is diluted 10 times with normal saline before testing. The reference sample volume is 30-50 μL. ② The reference sample volume of 10% brain homogenate is 30-50 μL. ③ The diluted whole blood diluent is 0.1 mL. Shake well before sampling.

[0124] Calculation formula:

[0125] (1) Definition: 1 μmol of substrate in the hydrolysis reaction system per mL of serum sample at 37°C for 6 minutes is 1 unit of activity.

[0126] (2) Calculation formula:

[0127] Serum (plasma) AChE = (A measured - A control) / (A standard - A blank) * C * N activity (U / mL);

[0128] C standard: standard concentration, 1 μmol / mL;

[0129] N: dilution fold before sample testing.

[0130] In this embodiment, the results of the cognitive improvement experiment are shown in Table 14 below.

[0131] Table 14 Cognitive Improvement Experiment Results

[0132] In this embodiment, the statistical results of the p value of the experiment are shown in Table 15 below.

[0133] Table 15 p value results

[0134] It can be seen that under the experimental conditions, compared with the control group, pyrroloquinoline quinone (PQQ) and low-dose reduced nicotinamide mononucleotide (NMNH) in the positive control group can effectively reduce the level of acetylcholinesterase (AchE), and have the potential to improve cognition. Although the positive control can reduce the level of malondialdehyde (MDA), there is no statistically significant difference, and the medium and high doses of NMNH do not show better ability to reduce the levels of MDA and AchE, which are positively correlated with cognitive impairment. The reason is not clear at present, and further research is needed to explore its molecular mechanism.

[0135] Example 5 Anti-aging experiment

[0136] The test specifications in this embodiment include: Health Food Function Test and Evaluation Methods (2022 Edition) and Health Food Function Test and Evaluation Technical Guidelines (2022 Edition).

[0137] Test principle: The growth, aging and apoptosis of cells are closely related to telomeres. Telomeres gradually shorten with cell division, and when they shorten to a certain extent, cell division is blocked, replication ability is lost, and cells undergo apoptosis. Telomerase can catalyze the synthesis of telomere DNA, offset or delay telomere shortening. By measuring the length and wear rate of telomeres, the degree of cell aging can be detected. The fluorescence signal of the telomere product amplified by the telomere (T) primer is compared with the single copy gene product amplified by the single copy gene (S) primer. Amplify T and S with two reaction tubes containing equal amounts of samples at the same time, detect the fluorescence signal of the product, and use specific analysis software to obtain the relative telomere length.

[0138] This embodiment relates to instruments and reagents including: quantitative PCR instrument (ABI; Quantstudio7), ultramicro spectrophotometer (Thermo; Nanodrop One), centrifuge (Eppendorf; 5425), vortex oscillator (Beijing Dalong; MX-F); related reagent consumables include mouse telomere Qpcr detection kit, fluorescence quantitative PCR plate and sealing film (ABI).

[0139] The experimental design of this embodiment is shown in Table 16 below.

[0140] Table 16 Experimental design

[0141] The specific experimental steps of this embodiment include:

[0142] 1. C57 mice were orally gavaged for 20 consecutive days, and blood was taken from the eye vein for testing;

[0143] 2. Blood DNA extraction and quality control: blood DNA extraction was performed according to the instructions of the Tian Gen kit (DP348), and the extracted DNA was quality controlled using Nanodrop One. DNA with a purity of A260 / A280 ≈ 1.8 was qualified, the DNA content was calculated, and the TE was dissolved and stored in an ultra-low temperature refrigerator for standby;

[0144] 3. Fluorescent quantitative PCR detection, the reaction system is shown in Table 17 below, and the reaction procedure includes: 95℃ for 3min, 95℃ for 30sec, 50℃ for 1min, 72℃ for 30sec, a total of 40 cycles;

[0145] Table 17 PCR reaction system

[0146] Note: The primer group detects telomere genes and internal reference genes in one reaction, and the standard is a specific length telomere repeat DNA template.

[0147] It should be noted that the telomere length calculation method (T / S): first calculate the △CT of the standard and the sample respectively, CT(telomere)-CT(internal reference), and then calculate T / S = 2-^(△CT(sample)-△CT(standard));

[0148] Note: T is the sample to be tested, S is the standard, and T / S is the relative length ratio of the telomere of the sample to be tested and the standard.

[0149] In this embodiment, the relative telomere length of the experimental sample and the statistical analysis results are shown in Table 18.

[0150] Table 18 Relative length of telomere

[0151] In this embodiment, the p-value statistical results of each experimental group are shown in Table 19 below.

[0152] Table 19 Statistical analysis results

[0153] It can be seen that, under the experimental conditions, α-lipoic acid with strong free radical scavenging ability cannot significantly prolong the relative length of telomeres after oral administration for 20 days, and the same result occurs in the low-dose group of reduced nicotinamide mononucleotide (NMNH). The medium and high-dose NMNH groups show a significant ability to prolong the relative length of telomeres, and have the potential to delay aging or alleviate aging-related diseases.

[0154] Example 6

[0155] The food, drug and / or health product with the effects of relieving fatigue, improving sleep, promoting heart health, improving cognition and / or anti-aging in the present application takes reduced nicotinamide mononucleotide (NMNH) and / or a salt thereof as the active ingredient, and the salt of reduced nicotinamide mononucleotide (NMNH) includes one or a mixture of several of sodium salt, calcium salt, magnesium salt, potassium salt, iron salt, zinc salt or manganese salt. Optionally, the content of reduced nicotinamide mononucleotide (NMNH) and / or a salt thereof is 0.01-100wt%.

[0156] The food, drug and / or health product with the effects of relieving fatigue, improving sleep, promoting heart health, improving cognition and / or anti-aging in the present application can be prepared into conventional dosage forms known in the art, including oral preparations, injection dosage forms, respiratory administration dosage forms, skin administration dosage forms, mucosal administration dosage forms or cavity administration dosage forms, etc. The specific dosage forms are in the form of one or more of tablets, capsules, granules, aqueous preparations, enteric preparations, injections, serums, emulsions, creams, foams, sprays, ointments, gels, lotions, pads, roll-on preparations, caplets, lozenges, troches, chewable tablets, gums, jellies, syrups, liquid solutions, suspensions, buccal films, sublingual films, oral adhesive films, powders, solid crystals, oral disintegrating tablets or pastes.

[0157] In the present embodiment, the food, drug and / or health product with the effects of relieving fatigue, improving sleep, promoting heart health, improving cognition and / or anti-aging is preferably an enteric capsule or enteric tablet, i.e. an enteric preparation, and the excipients can be selected from the raw materials known in the art.

[0158] Obviously, the above examples are only examples for clearly illustrating, but not limitation of the embodiments. Based on the above description, other different forms of changes or variations can also be made by those skilled in the art. Here, it is not necessary and also impossible to enumerate all the embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. Use of reduced nicotinamide mononucleotide (NMNH) and / or a salt thereof for the preparation of a preparation having at least one of the following effects (1)-(5): (1) alleviating fatigue; (2) improving sleep; (3) promoting heart health; (4) improving cognition; (5) anti-aging. The daily dosage of the reduced nicotinamide mononucleotide (NMNH) and / or a salt thereof is 1-1000 mg / kg body weight. The salt of the reduced nicotinamide mononucleotide (NMNH) includes one or a mixture of several of sodium salt, calcium salt, magnesium salt, potassium salt, iron salt, zinc salt, or manganese salt. The preparation includes a food, a drug, and / or a health product. The preparation includes at least one of an oral preparation, an injection dosage form, a respiratory administration dosage form, a skin administration dosage form, a mucosal administration dosage form, or a cavity administration dosage form. The preparation includes one or more of a tablet, a capsule, a granule, an aqueous preparation, an enteric preparation, an injection, a syrup, an emulsion, a cream, a foam, a spray, an ointment, a gel, a lotion, a pad, a roll-on preparation, a caplet, a lozenge, a troche, a chewable tablet, a gum, a gummy, a syrup, a liquid solution, a suspension, a buccal film, a sublingual film, an oral adhesive film, a powder, a solid crystal, an orally disintegrating tablet, or a paste.

2. Use according to claim 1, characterized in that, The preparation further includes a food, a pharmaceutical, and / or a health product acceptable excipient or carrier.

3. Use according to claim 1 or 2, characterized in that, The active ingredient of the composition includes reduced nicotinamide mononucleotide (NMNH) and / or a salt thereof.

4. Use according to any one of claims 1 to 3, characterized in that, The composition including reduced nicotinamide mononucleotide (NMNH) and / or a salt thereof, or the composition of claim 8.

5. Use according to any one of claims 1 to 4, characterized in that, The content of the reduced nicotinamide mononucleotide (NMNH) and / or a salt thereof is 0.01-100 wt%.

6. Use according to claim 5, characterized in that, 11. A method of alleviating fatigue, improving sleep, promoting heart health, improving cognition, and / or anti-aging, comprising administering reduced nicotinamide mononucleotide (NMNH) and / or a salt thereof to a subject in need thereof.

7. Use according to any one of claims 1 to 6, characterized in that, 12. The method of claim 11, wherein the daily dosage of the reduced nicotinamide mononucleotide (NMNH) and / or a salt thereof is 1-1000 mg / kg body weight.

8. A composition characterized in that, 13. The method of claim 11 or 12, wherein the salt of the reduced nicotinamide mononucleotide (NMNH) includes one or a mixture of several of sodium salt, calcium salt, magnesium salt, potassium salt, iron salt, zinc salt, or manganese salt.

9. A food, drug and / or health product having an effect of alleviating fatigue, improving sleep, promoting heart health, improving cognition and / or anti-aging, characterized by, ​ 10. The food, drug and / or health product having an effect of alleviating fatigue, improving sleep, promoting heart health, improving cognition and / or anti-aging effect according to claim 9, characterized by, ​ ​ ​ ​

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