JP2023058828A - HUMAN eNAMPT-EVs PURIFICATION METHOD, COMPOSITION FOR INCREASING BIOSYNTHESIS OF NMN AND/OR NAD COMPRISING HUMAN eNAMPT-EVs, ANTI-AGING COMPOSITION, AND METHOD FOR MEASURING NAD SYNTHETIC ACTIVITY OF TEST SUBSTANCE COMPRISING eNAMPT-EVs - Google Patents

Abstract

To provide a purification method that can yield human eNAMPT-EVs with few impurities in simple and efficient manner, a composition for increasing biosynthesis of NMN and/or NAD comprising human eNAMPT-EVs yielded by the purification method, and an anti-aging composition, and to establish a novel method for measuring NAD synthetic activity of eNAMPT-EVs in a biological sample, making it possible to determine the degree of aging of the subject.SOLUTION: A human eNAMPT-EVs purification method includes the step of subjecting (A) a solution containing a human biological sample to ultracentrifugation under conditions of 180,000×g-250,000×g, 30-80 minutes, and 4°C-37°C. The present invention also encompasses a composition for increasing biosynthesis of NMN and/or NAD comprising human eNAMPT-EVs yielded by the purification method, and an anti-aging composition.SELECTED DRAWING: None

Description

The present invention provides a method for purifying human eNAMPT-EVs, a composition for increasing the biosynthesis of NMN and/or NAD containing human eNAMPT-EVs, an anti-aging composition, and a test substance containing eNAMPT-EVs. It relates to a method for measuring NAD synthesis activity.

Aging is a significant risk factor for tissue dysfunction and chronic disease. In recent years, NAD+ metabolism has become a central theme in the field of aging and longevity, as nicotinamide adenine dinucleotide (NAD+) levels decline throughout the body with aging. It has also been demonstrated that age-related decline in NAD+ triggers various pathophysiological changes (see Non-Patent Documents 1-4).

In mammals, age-related decline in NAD+ is thought to be caused by two major events: decreased NAD+ biosynthesis and increased NAD+ consumption (5-6). The former may be caused by chronic inflammation with increased oxidative stress and/or increased pro-inflammatory cytokines, the latter by increased DNA damage. As a result, with aging, the amount of NAD+ decreases in multiple tissues such as adipose tissue, skeletal muscle, liver, pancreas, skin, neurosensory retina, and brain (see Non-Patent Documents 1-4 and 7). Since it has become clear that such a decrease in systemic NAD+ is one of the causes of age-related pathophysiology, nicotinamide mononucleotide (NMN) and nicotinamide, which are major intermediates of NAD+, are currently being investigated. Developments are underway to use riboside (NR) and the like for anti-aging (see Non-Patent Documents 2 and 4). In fact, the efficacy of NMN and NR has already been proven by many studies, and has been found to alleviate age-related functional decline and treat age-related disease states (Non-Patent Document 2, 4).

In mammals, nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in the major NAD+ biosynthetic pathway, converting nicotinamide and 5′ phosphoribosyl-pyrophosphate (PRPP) to NMN (8, 9). . There are two different forms of NAMPT in mammals: intracellular NAMPT and extracellular NAMPT (iNAMPT and eNAMPT, respectively) (see Non-Patent Document 10). Although iNAMPT's function as a NAD+ biosynthetic enzyme is well known, the physiological function of eNAMPT has long been unclear. The present inventors have elucidated the physiological functions of eNAMPT in vivo by deleting or introducing the Nampt gene specifically into adipose tissue (see Non-Patent Documents 11 and 12). In addition, in adipose tissue-specific Nampt knockout mice (ANKO), eNAMPT levels circulating in the body are significantly reduced, and NAD + levels are also significantly reduced in tissues other than adipose tissue (Non-Patent Document 11). reference).

Subsequent further research also revealed that eNAMPT has a new function of enhancing NAD+. It was also revealed that eNAMPT enhances SIRT1 activity and activates nerves in the hypothalamus. These findings suggest the existence of a new inter-tissue communication system mediated by eNAMPT between adipose tissue and hypothalamus (see Non-Patent Document 5). However, it remains to be clarified how eNAMPT specifically regulates NAD+ levels in the hypothalamus.

In addition, the present inventors recently found that blood levels of eNAMPT in mice and humans significantly decrease with aging (see Non-Patent Document 12). They also found that by overexpressing NAMPT specifically in adipose tissue of aged mice, the amount of NAD+ in multiple tissues increases, the function of the tissues improves, and the healthy lifespan of the mice can be extended. Interestingly, eNAMPT is carried throughout the body of mice and humans in the form of extracellular vesicles (EVs), is taken up into cells, and promotes NAD+ biosynthesis. Supplementation with EVs (extracellular vesicles) containing eNAMPT isolated from young mice (eNAMPT-EVs) was found to significantly improve motor function and extend lifespan in aged mice. This reveals that eNAMPT-EVs promote systemic NAD+ biosynthesis and suppress aging (see Non-Patent Document 12).

Under these circumstances, there is a movement to utilize eNAMPT, especially eNAMPT-EVs in the form of microparticles, for anti-aging pharmaceuticals, functional foods, and the like. However, since anti-aging functional foods, pharmaceuticals, etc. are generally taken by humans over a long period of time, it is important that they have excellent anti-aging effects and high safety. Therefore, it is considered desirable that the content of impurities is as low as possible, and the same is required for eNAMPT-EVs. In addition, it is also important to be able to supply a sufficient amount as a product, so there is a demand for a manufacturing method that can not only obtain eNAMPT-EVs with few impurities, but also can supply a sufficient amount simply and efficiently.

Canto et al., Cell Metabolism, vol.22, Issue 1, Pages 31-53, 2015 Rajman, L., Chwalek, K., and Sinclair, D.A. (2018). Therapeutic potential of NAD-boosting molecules: the in vivo evidence. Cell Metab. 27, 529-547. Verdin, E. (2015). NAD+ in aging, metabolism, and neurodegeneration. Science 350, 1208-1213. Yoshino, J., Baur, J.A., and Imai, S.I. (2018). NAD+ intermediates: the biology and therapeutic potential of NMN and NR. Cell Metab. 27, 513-528. Imai, S.I. (2016). The NAD World 2.0: the importance of the inter-tissue communication mediated by NAMPT/NAD+/SIRT1 in mammalian aging and longevity control. NPJ Syst. Biol. Appl. 2, 16018. Imai, S., and Guarente, L. (2014). NAD+ and sirtuins in aging and disease. Trends Cell Biol. 24, 464-471. Lin, J.B., Kubota, S., Mostoslavsky, R., and Apte, R.S. (2018). Role of sirtuins in retinal function under basal conditions. Adv. Exp. Med. Biol. 1074, 561-567. Garten, A., Schuster, S., Penke, M., Gorski, T., de Giorgis, T., and Kiess, W. (2015). Physiological and pathophysiological roles of NAMPT and NAD metabolism. Nat. Rev. Endocrinol 11, 535-546. Imai, S. (2009). Nicotinamide phosphoribosyltransferase (Nampt): a link between NAD biology, metabolism, and diseases. Curr. Pharm. Des. 15, 20-28. Revollo, J.R., Ko¨rner, A., Mills, K.F., Satoh, A., Wang, T., Garten, A., Dasgupta, B., Sasaki, Y., Wolberger, C., Townsend, R.R., et al. (2007). Nampt/PBEF/visfatins regulates insulin secretion in β cells as a systemic NAD biosynthetic enzyme. Cell Metab. 6, 363-375. Yoon, M.J., Yoshida, M., Johnson, S., Takikawa, A., Usui, I., Tobe, K., Nakagawa, T., Yoshino, J., and Imai, S. (2015). SIRT1- mediated eNAMPT secretion from adipose tissue regulates hypothalamic NAD+ and function in mice. Cell Metab. 21, 706-717. Yoshida, M., Satoh, A., Lin, J.B., Mills, K.F., Sasaki, Y., Rensing, N., Wong, M., Apte, R.S., and Imai, S. (2019) Extracellular vesicle contained eNAMPT delays aging and extends lifespan in mice. Cell Metab. 30, 329-342.

The present invention has been made in view of such circumstances, and an object of the present invention is to provide a simple, efficient, and sufficient amount of human eNAMPT-EVs with few impurities. Furthermore, another object of the present invention is to provide a composition for increasing the biosynthesis of NMN and/or NAD, and an anti-aging composition, containing human eNAMPT-EVs obtained by the purification method. Another object is to establish a novel method for measuring the NAD synthesis activity of eNAMPT-EVs in a biological sample, and to make it possible to determine the degree of aging of a subject.

The present inventors attempted to isolate eNAMPT-EVs from human plasma. When human plasma was used as a sample for direct Western blotting, eNAMPT-EVs could not be detected with an anti-NAMPT antibody, unlike mouse. Although the reason for this is not clear, it is possible that there are many substances such as lipids that inhibit the detection of eNAMPT in human plasma. Therefore, when a purification method using the size exclusion method was tested on human plasma, a clear eNAMPT band was detected at around 70 kDa, indicating that highly pure eNAMPT-EVs could be obtained. However, the size-exclusion method has a limit to the amount that can be processed at one time, and the yield is extremely low. Therefore, we attempted to purify eNAMPT-EVs by ultracentrifugation, but under generally known conditions such as experimental conditions for purifying eNAMPT-EVs from mouse plasma, eNAMPT-EVs with high purity cannot be separated. It was impossible. As a result of various examinations of ultracentrifugation conditions, it became clear that human eNAMPT-EVs with high purity can be obtained only when a specific combination of ultracentrifugal force and centrifugation time is used, and finally the human eNAMPT-EV of the present invention. A method for purifying EVs was completed. Furthermore, it was also confirmed that the human eNAMPT-EVs obtained by this method have excellent NAD synthesis activity and the like. In addition, a new method for measuring the NAD synthesis activity of eNAMPT-EVs in biological samples has been established, making it possible to determine the degree of aging using biological samples from subjects. The gist of the present invention is as follows.

[1] (A) A method for purifying human eNAMPT-EVs, comprising the step of ultracentrifuging a solution containing a human biological sample at 180,000×g to 250,000×g for 30 to 80 minutes.
[2] The purification method of [1], wherein the biological sample is serum or plasma.
[3] The purification method according to [1] or [2], wherein the solution containing the biological sample is a solution obtained by diluting a human biological sample with a buffer solution.
[4] The purification method according to any one of [1] to [3], wherein the ultracentrifugation conditions in step (A) are 190,000×g to 220,000×g for 40 minutes to 75 minutes. .
[5] The purification method according to any one of [1] to [4], wherein the ultracentrifugation conditions in step (A) are 210,000 xg for 50 minutes.
[6] A composition for increasing NMN and/or NAD biosynthesis, comprising human eNAMPT-EVs obtained by the purification method according to any one of [1] to [5].
[7] The composition of [6], further comprising a carrier and/or excipient.
[8] An anti-aging composition containing human eNAMPT-EVs obtained by the purification method according to any one of [1] to [5].
[9] The anti-aging composition of [8], further comprising a carrier and/or excipient.
[10] (a) a step of culturing human cultured cells in a bovine serum-containing medium for a certain period of time;
(b) changing the medium of the human cultured cells obtained in step (a) to a serum-free medium and culturing for a certain period of time;
(c) adding a test substance or negative control substance containing eNAMPT-EVs to the human cultured cells obtained in step (b) and culturing for a certain period of time; and (d) obtained in step (c), A test containing eNAMPT-EVs, including the step of measuring and comparing the amount of NAD per certain number of cells of human cultured cells added with a test substance containing eNAMPT-EVs and human cultured cells with a negative control substance added A method for measuring the NAD synthesis activity of a substance.
[11] The method for measuring NAD synthesis activity according to [10], wherein the test substance containing eNAMPT-EVs is a biological sample from a subject.
[12] The test substance containing the eNAMPT-EVs is plasma of the subject or eNAMPT-EVs purified from the plasma of the subject by the method according to any one of [1] to [5], [10] or The method for measuring NAD synthesis activity according to [11].
[13] Using the method according to any one of [10] to [12], the plasma of the subject, or the eNAMPT-EVs purified from the plasma of the subject, aging of the subject including the step of measuring the NAD synthesis activity method of determining the degree of

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a simple and efficient purification method for obtaining human eNAMPT-EVs with few impurities. In addition, human eNAMPT-EVs obtained by this purification method have excellent NAD synthesis activity, etc., and are suitable for use in compositions for increasing NMN and/or NAD biosynthesis and anti-aging compositions. can do. Furthermore, according to the novel method for measuring the NAD synthesis activity of eNAMPT-EVs in biological samples, it is possible to determine the degree of aging of subjects using biological samples.

FIG. 1 shows the results of Western blotting detection of eNAMPT-EVs in human plasma. FIG. 2 shows the results of examination of the conditions for purification of mouse eNAMPT-EVs by ultracentrifugation. FIG. 3 shows the results of purification of human eNAMPT-EVs by size exclusion method. FIG. 4 shows the results of examination of conditions for a method for purifying human eNAMPT-EVs by ultracentrifugation. FIG. 5 is a diagram showing the results of confirmation of purification conditions for human eNAMPT-EVs by ultracentrifugation. FIG. 6 shows a method for measuring the NAD synthesis activity of eNAMPT-EVs using cultured human HEK293 cells and the results thereof. FIG. 7 shows the results of measuring the NAD synthesis activity of human eNAMPT-EVs using cultured human HEK293 cells. FIG. 8 is a diagram showing results showing individual differences in NAD synthesis activity of human eNAMPT-EVs using cultured human HEK293 cells and specificity.

The present invention will be described in detail below. In addition, the terms used in this specification are interpreted as meanings commonly used in the technical field unless otherwise specified.

<Purification method of human eNAMPT-EVs>
The method for purifying human eNAMPT-EVs of the present invention includes (A) a step of ultracentrifuging a solution containing a human biological sample under conditions of 180,000 × g to 250,000 × g for 30 minutes to 80 minutes. characterized by According to the purification method of the present invention, human eNAMPT-EVs with few impurities can be obtained simply and efficiently. In addition, human eNAMPT-EVs obtained by this purification method have excellent NAD synthesis activity, etc., and are suitable for use in compositions for increasing NMN and/or NAD biosynthesis and anti-aging compositions. can do.

(human eNAMPT-EVs)
NAMPT (nicotinamide phosphoribosyltransferase) is the rate-limiting enzyme of the major NAD+ (nicotinamide adenine dinucleotide) biosynthetic pathway and converts nicotinamide and 5′ phosphoribosyl-pyrophosphate (PRPP) into NMN. (nicotinamide mononucleotide; nicotinamide mononucleotide) (see Non-Patent Documents 8 and 9). There are two different forms of NAMPT in mammals, intracellular NAMPT and extracellular NAMPT (iNAMPT and eNAMPT, respectively) (see Non-Patent Document 10), where "eNAMPT" is extracellular NAMPT. eNAMPT is contained in extracellular vesicles (EVs), is transported throughout the body of mice and humans, is taken up into cells, and promotes NAD+ biosynthesis. "eNAMPT-EVs" refers to the form of eNAMPT contained in extracellular vesicles (EVs).

(biological sample)
In the method for purifying human eNAMPT-EVs of the present invention, the biological sample is not particularly limited as long as it is a biological sample containing eNAMPT-EVs, but from the viewpoint of human eNAMPT-EVs content and ease of purification, Human serum or plasma is preferred, and plasma is more preferred.

Human serum is a supernatant obtained by collecting blood with a blood collection tube that does not contain an anticoagulant such as heparin and leaving it for a certain period of time. Serum contains little or no clotting factors. On the other hand, human plasma is a supernatant obtained by sedimentation of blood cell components when blood is collected with a blood collection tube containing an anticoagulant such as heparin and left to stand or centrifuged. Plasma contains clotting factors.

Plasma components consist of water (90%), protein (about 7-8%), minerals such as cations and anions (0.9%), carbohydrates, lipids, urea, uric acid, amino acids (2%). and accounts for 55% of the total blood volume. Proteins in plasma include albumin, globulin (α-globulin, β-globulin, Φ-fibrinogen, γ-globulin), and human eNAMPT-EVs. Serum components are plasma components minus clotting factors, and include human eNAMPT-EVs as well as plasma.

The biological sample can be stored frozen at -80°C. When subjecting a biological sample after frozen storage to ultracentrifugation, after thawing by heating at a temperature of about 37° C., it is centrifuged at 2,000×g for about 15 minutes to precipitate clots and the like. , the supernatant can be collected and used. Furthermore, immediately before ultracentrifugation, clots, etc. are removed through a filter (pore size 0.22 μm, etc.) blocked with a buffer such as PBS containing 5% BSA (fatty acid-free), etc., and placed in a similarly blocked centrifuge tube. .

In the method for purifying human eNAMPT-EVs of the present invention, the biological sample to be ultracentrifuged is preferably a solution obtained by diluting the biological sample with a buffer or the like. The buffer at that time is not particularly limited as long as it has a buffering action. Examples include caprylic acid, deoxycholic acid, salicylic acid, triethanolamine, fumaric acid, other organic acids, carbonate buffer, Tris buffer, histidine buffer, imidazole buffer, and the like. Among these, phosphate buffer and Tris buffer are preferred, and phosphate buffer saline (PBS), phosphate buffer (PB), Tris buffer saline (TBS) and Tris buffer (TB) are more preferred. Phosphate-buffered saline (PBS) and Tris-buffered saline (TBS) are more preferred, and phosphate-buffered saline (PBS) is particularly preferred.

The concentration of the buffer is generally 1-500 mM, preferably 5-100 mM, more preferably 10-20 mM. When subjected to ultracentrifugation, the biological sample is usually diluted 2-fold to 20-fold with these buffers, preferably 3-fold to 15-fold, and preferably 5-fold to 10-fold. More preferably, it is even more preferably diluted about 8 times. By diluting the biological sample by such a factor, the efficiency of separation by ultracentrifugation is improved, and contamination by impurities can be reduced.

(ultracentrifuge)
In the method for purifying human eNAMPT-EVs of the present invention, the conditions for ultracentrifugation for a solution containing a biological sample are 180,000 × g to 250,000 × g for ultracentrifugal force, and 190,000 × g to It is preferably 220,000 x g, more preferably 195,000 x g to 215,000 x g, even more preferably around 210,000 x g, and 210,000 x g. is particularly preferred. Centrifugation time is 30 minutes to 80 minutes, preferably 40 minutes to 75 minutes, more preferably 45 minutes to 60 minutes, and around 50 minutes in the case of the above ultracentrifugal force. is more preferred, and 50 minutes is particularly preferred. In addition, the temperature for ultracentrifugation is in the range of 4° C. to 37° C., and is preferably adjusted appropriately so that the activity of human eNAMPT-EVs is maintained and the contamination of impurities is suppressed.

In the method for purifying human eNAMPT-EVs of the present invention, the conditions for ultracentrifugation for a solution containing a biological sample are 180,000×g to 250,000×g, 30 minutes to 80 minutes, 4° C. to 37° C., 190,000×g to 220,000×g, 40 to 75 minutes, preferably 4° C. to 37° C. 195,000×g to 215,000×g, 40 to 75 minutes, 4° C. to 37°C is more preferable, 210,000 × g, 40 to 75 minutes, 4°C to 37°C is even more preferable, 210,000 × g, 45 to 60 minutes, 4°C to 37°C is particularly preferred, and 210,000×g for 50 minutes at 4° C. to 37° C. is even more preferred.

(Embodiment of the method for purifying human eNAMPT-EVs of the present invention)
In an embodiment of the method for purifying human eNAMPT-EVs of the present invention, the human plasma collected by heparinization is subjected to an operation to remove clots and the like, and a buffer solution such as PBS is added 2-fold to 20-fold, preferably Diluted 3 to 15 times, more preferably 5 to 10 times, more preferably about 8 times, 180,000 × g to 250,000 × g, 30 minutes to 80 minutes, 4 ° C. to 37 ° C., preferably is 190,000 × g to 220,000 × g, 40 minutes to 75 minutes, 4 ° C. to 37 ° C., more preferably 195,000 × g to 215,000 × g, 40 minutes to 75 minutes, 4 ° C. to 37 ° C., more preferably 210,000 x g, 40 min to 75 min, 4 ° C to 37 ° C, particularly preferably more than 210,000 x g, 45 min to 60 min, 4 ° C to 37 ° C, more particularly preferably 210 ,000×g for 50 minutes at 4° C. to 37° C. to obtain a pellet of human eNAMPT-EVs.

In the purification method of the present invention, it is possible to suppress the contamination of impurities and purify a sufficient amount of human eNAMPT-EVs by setting the conditions as described above. Purification of eNAMPT-EVs from human biological samples presents challenges that are different from those of other animal species such as mice. That is, when purifying eNAMPT-EVs from mouse plasma, eNAMPT-EVs detection was not inhibited by extending the ultracentrifugation time, and the amount detected increased as the ultracentrifugation time increased. bottom. On the other hand, in the case of human plasma, the extension of the ultracentrifugation time tends to inhibit the detection of eNAMPT-EVs, and it is expected that the yield of eNAMPT-EVs will increase as the ultracentrifugation time increases. At the same time, it is thought that a large amount of impurities that hinder detection are precipitated. When considering the use of eNAMPT-EVs in medicine, such as readministration of eNAMPT-EVs to living bodies, contamination with such impurities is undesirable. Therefore, as a method for purifying eNAMPT-EVs from human plasma, the purification method of the present invention, which is fixed under the conditions described above, is desirable.

Human eNAMPT-EVs obtained by the purification method described above have excellent NAD synthesis activity and the like, and have anti-aging effects. A composition for increasing NMN and / or NAD biosynthesis, It can be suitably used in anti-aging compositions. These compositions are described below.

<Composition for increasing NMN and/or NAD biosynthesis>
The composition for increasing NMN and/or NAD biosynthesis of the present invention is characterized by containing human eNAMPT-EVs obtained by the purification method of the present invention described above. According to the purification method of the present invention, human eNAMPT-EVs with little contamination of impurities can be obtained simply and efficiently. Human eNAMPT-EVs thus obtained function as an enzyme in the biosynthetic pathway of NMN and/or NAD in vivo or in various cells. Cellular NMN and/or NAD biosynthesis can be increased. Such various cells are not particularly limited, but for example, reproductive cells (eggs, egg cells, sperm, sperm cells, fertilized eggs, unfertilized eggs, etc.), stem cells (embryonic stem cells, somatic stem cells, iPS cells, etc.), differentiated cells, and the like. Moreover, when using the composition of the present invention for such cells, it is conceivable to add the composition of the present invention to the culture medium of these cells. For the purification method of the present invention, the contents described in the section on the purification method of human eNAMPT-EVs can be applied as they are.

The composition for increasing NMN and/or NAD biosynthesis of the present invention can be used for pharmaceuticals, quasi-drugs, foods, functional foods, and the like. Such a composition of the present invention, in addition to the human eNAMPT-EVs obtained by the purification method of the present invention described above, may contain other components depending on the application and form within a range that does not impair the effects of the present invention. may contain.

Examples of the above-mentioned other components include pharmaceutically acceptable carriers and additives. Examples of such carriers and additives include water, tonicity agents, thickeners, sugars, sugar alcohols, preservatives (preservatives), bactericides or antibacterial agents, pH adjusters, stabilizers, Chelating agents, oily bases, gel bases, surfactants, suspending agents, binders, excipients, lubricants, disintegrants, foaming agents, fluidizing agents, dispersing agents, emulsifiers, buffers, dissolving agents Adjuvants, antioxidants, sweeteners, acidulants, coloring agents, flavoring agents, flavoring agents or cooling agents, etc., may include, but are not limited to, these.

The amount of human eNAMPT-EVs included in the composition for increasing NMN and/or NAD biosynthesis of the present invention ranges from 1 wt% to 80 wt%, from 1 wt% to 50 wt%, or from 1 wt% to 20 wt%. % by weight.

The vesicle size of human eNAMPT-EVs contained in the composition for increasing NMN and/or NAD biosynthesis of the present invention has an average particle size of 10 nm to 200 nm, 10 nm to 100 nm, or 20 nm to 100 nm.

The compositions for increasing NMN and/or NAD biosynthesis of the present invention can be administered orally or parenterally, systemically or locally. Examples of administration routes include intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracerebroventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, rectal, and the like. be done. Among these, oral, intranasal, intraperitoneal, intravenous, intramuscular, rectal, and transdermal are preferred routes of administration.

The composition for increasing NMN and/or NAD biosynthesis of the present invention comprises human eNAMPT-EVs obtained by the purification method of the present invention, a pharmaceutically acceptable carrier and/or excipient, etc. It can be prepared and formulated into a formulation by mixing or the like by a conventional method.

When the composition for increasing the biosynthesis of NMN and/or NAD of the present invention is provided as a pharmaceutical or quasi-drug, dosage forms include tablets (including sugar-coated tablets), capsules, granules, powders, Oral preparations such as lozenges, chewables, pills, liquids for internal use, emulsions, syrups, suspensions and elixirs; external preparations such as ointments, gels, creams and patches; subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections), parenteral agents such as infusions and suppositories. In addition, when the composition for increasing the biosynthesis of NMN and/or NAD of the present invention is provided as a food or functional food, it may be in the form of ordinary food, capsules, granules, powders, troches. It may also be used as supplements in the form of tablets, chewables, pills, liquids for internal use, emulsions, syrups, suspensions, elixirs and the like.

The composition for increasing the biosynthesis of NMN and/or NAD of the present invention is effective for decreased physical activity, decreased sleep quality, decreased cognitive function, decreased glucose metabolism, decreased visual acuity, type II diabetes, and obesity. , age-related obesity, age-related increase in blood lipid levels, age-related decrease in insulin sensitivity, age-related loss or decrease in memory function, age-related loss or decrease in eye function, aging Age-related physiological decline, impaired glucose-stimulated insulin secretion, type I diabetes, improved mitochondrial function, neuronal death, Alzheimer's disease, cardiac ischemia, reperfusion injury, maintenance of neural stem cell/neural progenitor cell populations, post-injury Recovery of mitochondrial function and arterial function of skeletal muscle, fungal diseases (diseases associated with muscle weakness, muscle atrophy, muscle weakness, specifically sarcopenia, dynapenia, cachexia, muscular dystrophy, muscle stiffness disorder, spinal cord It can be suitably used for muscular atrophy, myopathy, etc.

The dosage of the composition for increasing NMN and/or NAD biosynthesis of the present invention should be determined according to the conditions of the patient in need of treatment and the subject in need of improvement. And the administration frequency can be adjusted so that the effect of human eNAMPT-EVs, which is an active ingredient, is sufficiently exhibited. In embodiments of the invention, compositions of the invention are administered at doses of 1 mg to 500 mg, 10 mg to 500 mg, or 50 mg to 500 mg of human eNAMPT-EVs per day.

The subjects of administration of the composition for increasing NMN and/or NAD biosynthesis of the present invention are mammals, preferably humans, horses, cows, dogs, cats, etc., more preferably humans.

<Method for increasing NMN and/or NAD biosynthesis>
The present invention provides a method for increasing NMN and/or NAD biosynthesis in a subject or various cells, wherein human eNAMPT-EVs obtained by the purification method of the present invention or a composition containing the same is administered to the subject. Alternatively, it also includes a method comprising adding to various cells of a subject. Such various cells are not particularly limited, but for example, reproductive cells (eggs, egg cells, sperm, sperm cells, fertilized eggs, unfertilized eggs, etc.), stem cells (embryonic stem cells, somatic stem cells, iPS cells, etc.), differentiated cells, and the like. Moreover, when using the composition of the present invention for such cells, it is conceivable to add the composition of the present invention to the culture medium of these cells. The purification method of the present invention, the human eNAMPT-EVs obtained by the purification method of the present invention, the composition containing the same, and the administration method are described in the section on the purification method of human eNAMPT-EVs, the generation of NMN and / or NAD. See discussion under Compositions for Increased Synthesis.

<Anti-aging composition>
The anti-aging composition of the present invention is characterized by containing human eNAMPT-EVs obtained by the above purification method of the present invention. According to the purification method of the present invention, human eNAMPT-EVs with little contamination of impurities can be obtained simply and efficiently. Human eNAMPT-EVs thus obtained function as an enzyme in the biosynthetic pathway of NMN and/or NAD in vivo or in various cells. It can increase cellular NMN and/or NAD biosynthesis, resulting in an anti-aging effect. Such various cells are not particularly limited, but for example, reproductive cells (eggs, egg cells, sperm, sperm cells, fertilized eggs, unfertilized eggs, etc.), stem cells (embryonic stem cells, somatic stem cells, iPS cells, etc.), differentiated cells, and the like. Moreover, when using the composition of the present invention for such cells, it is conceivable to add the composition of the present invention to the culture medium of these cells. For the purification method of the present invention, the contents described in the section [Purification method of human eNAMPT-EVs] can be applied as they are.

Since the anti-aging composition of the present invention is substantially the same as the composition for increasing NMN and/or NAD biosynthesis described above, the specific description is The description in the section on compositions for increasing biosynthesis can be applied by substituting compositions for increasing NMN and/or NAD biosynthesis with anti-aging compositions.

<Method for exhibiting anti-aging effect>
The present invention is a method for exerting an anti-aging effect on a subject or various cells, wherein human eNAMPT-EVs obtained by the purification method of the present invention or a composition containing the same is administered to a subject, or various Also included are methods comprising adding to a cell. Such various cells are not particularly limited, but for example, reproductive cells (eggs, egg cells, sperm, sperm cells, fertilized eggs, unfertilized eggs, etc.), stem cells (embryonic stem cells, somatic stem cells, iPS cells, etc.), differentiated cells, and the like. Moreover, when using the composition of the present invention for such cells, it is conceivable to add the composition of the present invention to the culture medium of these cells. The purification method of the present invention, the human eNAMPT-EVs obtained by the purification method of the present invention, the composition containing the same, and the administration method are described in the section of the purification method of human eNAMPT-EVs and the section of the anti-aging composition. See the explanation in

<Method for measuring NAD synthesis activity of test substance containing eNAMPT-EVs>
A method for measuring the NAD synthesis activity of a test substance containing eNAMPT-EVs of the present invention comprises:
(a) culturing human cultured cells in bovine serum-containing medium for a certain period of time;
(b) changing the medium of the human cultured cells obtained in step (a) to a serum-free medium and culturing for a certain period of time;
(c) adding a test substance or negative control substance containing eNAMPT-EVs to the human cultured cells obtained in step (b) and culturing for a certain period of time; and (d) obtained in step (c), It is characterized by including a step of measuring and comparing the amount of NAD per certain number of cells in human cultured cells to which a test substance containing eNAMPT-EVs has been added and human cultured cells to which a negative control substance has been added. Each step will be described below.

(Step (a))
This step is a step of culturing human cultured cells in a bovine serum-containing medium for a certain period of time. Human cultured cells are not particularly limited as long as they are adherent cells, and examples thereof include HEK293 cells, OP9 cells, C2C12 cells and the like. Among these, HEK293 cells are preferable from the viewpoint of excellent sensitivity in measuring NAD synthesis activity. In this step, human cultured cells that have been grown in a growth medium (10% FBS-containing DMEM or the like) in advance are treated with a cell detachment agent such as 0.05% trypsin/EDTA and peeled off from the plate. is added and suspended, washed by centrifugation or the like, and suspended again in the growth medium. The cells are passed through a cell strainer with a pore size of about 100 μm and seeded on a Type I collagen-coated 24-well plate or the like. A guideline for the number of cells to be seeded when using a 24-well plate is 5×10 ⁴ cells/well.

(Step (b))
This step is a step of changing the culture medium of the human cultured cells obtained in step (a) to a serum-free medium and culturing the cells for a certain period of time. This medium exchange is performed 18 to 30 hours, 20 to 28 hours, 22 to 26 hours, or 24 hours after seeding the cells in step (a). The serum-free medium is not particularly limited, but αMEM, DMEM, etc., can be used, for example. As a positive control, the growth medium is used without exchanging the medium with serum-free medium. The certain period of time is 0 to 10 hours, preferably 0 to 5 hours, more preferably 0 to 2 hours, even more preferably 0 to 30 minutes. Particularly preferred is between hours and 10 minutes.

(Step (c))
This step is a step of adding a test substance containing eNAMPT-EVs or a negative control substance to the human cultured cells obtained in step (b) and culturing for a certain period of time. Where the medium was changed to serum-free medium in step (b), the test substance or negative control substance was added, 15 minutes to 2 hours, 20 minutes to 1.5 hours, 30 minutes to 1.2 hours, or 1 Incubate for hours. After that, the cells are sufficiently washed with a buffer solution such as PBS, the liquid is completely removed, and an appropriate amount of H ₂ Cl ₄ is added. The standard amount to be added when using a 24-well plate is 300 μL/well. If it is not possible to proceed to step (d) immediately, it can be stored frozen at -80°C. As the test substance, a biological sample from a subject can be used. For example, the subject's plasma or eNAMPT-EVs purified from the subject's plasma by the method of the present invention can be used as the test substance.

(Step (d))
In this step, the human cultured cells to which the test substance containing eNAMPT-EVs was added and the human cultured cells to which the negative control substance was added, obtained in the step (c), were measured. This is the step of comparison. Although the method for measuring the amount of NAD per certain number of cells is not particularly limited, measurement by HPLC is preferable from the viewpoint of excellent accuracy and simplicity of measurement. The cells obtained in step (c) are disrupted by sonication, scraped from the bottom surface with a cell scraper or the like, and transferred to a tube. Centrifuge at 20,000×g for 5 minutes to collect the supernatant. Add about 83 μL of 3M K ₂ CO ₃ per 250 μL, stir with a vortex or the like, and incubate on ice for about 15 minutes. Centrifuge at 20,000×g for 5 minutes to collect the supernatant. An appropriate amount of ×10 PB or the like is added to prepare a sample for HPLC, and the sample is subjected to HPLC to measure the amount of NAD per certain number of cells.

<Method for determining the degree of aging>
The present invention uses the method for measuring the NAD synthesis activity of a test substance containing eNAMPT-EVs of the present invention to measure the NAD synthesis activity of a subject's plasma or eNAMPT-EVs purified from the subject's plasma. Also included is a method of determining the degree of aging in a subject comprising the steps. According to the method for measuring the NAD synthesis activity of a test substance containing eNAMPT-EVs of the present invention, the NAD synthesis activity of the subject's plasma can be easily measured. Since the NAD synthesis activity of the subject's plasma is proportional to the amount of eNAMPT-EVs contained in the plasma, the amount of eNAMPT-EVs contained in the plasma from the measurement results of the NAD synthesis activity of the subject's plasma (relative amount and / or absolute amount ) can be known. Since the amount of eNAMPT-EVs in plasma is known to decrease with aging, it is possible to determine the degree of aging of a subject from the amount of eNAMPT-EVs in plasma. Also, when the degree of aging is large (when the amount of eNAMPT-EVs in plasma is less than the average amount of eNAMPT-EVs in plasma at actual age), a composition containing eNAMPT-EVs (NMN and By ingesting a composition for increasing NAD biosynthesis or an anti-aging composition, it is possible to improve aging and improve symptoms, dysfunction, etc. due to aging.

EXAMPLES The present invention will be described in more detail below with reference to examples, but the present invention is not limited by these examples.

(Test 1) Detection of eNAMPT-EVs in human plasma by Western blotting Human plasma collected by heparinization (preserved frozen) was thawed in a 37°C constant temperature bath and centrifuged at 2,000 xg for 15 minutes. The supernatant was carefully collected and used as a human plasma sample for Western blotting (whole human plasma; hP). This sample was subjected to Western blotting, and detection was attempted with an anti-NAMPT antibody (manufactured by Bethyl, polyclonal anti-NAMPT antibody). As a result, as shown in FIG. 1 (left figure), in the human plasma sample, the NAMPT band that should be visible around 70 kDa was not detected.

Next, the human plasma sample was passed through a 0.22 μm pore filter (Millex GP, Merck Millipore) blocked with 5% BSA (fatty acid-free) PBS solution, placed in a similarly blocked centrifuge tube, and washed with PBS. Diluted twice. Ultracentrifugation was performed at 210,000×g for 101 minutes at 4° C. (ultracentrifuge; Beckman, XE-90, rotor; Beckman, 70.Ti), the supernatant was removed, and the pellet was recovered. For further ultracentrifugation, the pellet was dissolved in PBS and subjected to a second ultracentrifugation at 210,000 xg for 101 minutes at 4°C. A RIPA buffer was added to the collected pellets to dissolve them, a loading buffer containing 2ME (mercaptoethanol) was added, and the pellets were heated at 95° C. for 10 minutes to prepare samples for Western blotting. Electrophoresis was performed on these samples, and detection was attempted with an anti-NAMPT antibody (manufactured by Bethyl, polyclonal anti-NAMPT antibody). As a result, as shown in FIG. 1 (right figure), no NAMPT band was detected in the sample after one ultracentrifugation. A NAMPT band was detected in the sample that had been ultracentrifuged twice.

Based on the above results, it was considered possible that human plasma contains substances that inhibit the detection of eNAMPT by Western blotting.

(Test 2) Purification of mouse eNAMPT-EVs by ultracentrifugation Blood was collected from mice (C57BL/6J) and centrifuged at 2,000 x g for 5 minutes using sodium heparin as an anticoagulant to obtain plasma. rice field. Mouse plasma (mP) was treated in the same manner as in Test 1 above, and subjected to ultracentrifugation at 210,000×g for different times. The obtained pellet was treated in the same manner and subjected to Western blotting. The results are shown in FIG.

As shown in FIG. 2, in mouse plasma, eNAMPT detected increased depending on the time of ultracentrifugation. Moreover, it was not observed that the extension of the ultracentrifugation time adversely affected the detection by Western blotting.

(Test 3) Purification of human eNAMPT-EVs by size exclusion column eNAMPT-EVs were purified from human plasma by size exclusion chromatography. Specifically, after purifying human plasma with a size exclusion column (qEV35, Meiwaphoresis) for extracting extracellular vesicles of 35 to 350 nm, RIPA buffer was added to the EV-containing solution concentrated with Amicon Ultra ultracel, 100 kDa (Millipore). After addition and dissolution, a loading buffer containing 2ME (mercaptoethanol) was added and heated at 95° C. for 10 minutes to prepare a sample for Western blotting. The results are shown in FIG.

As shown in FIG. 3, when purified from human plasma by size exclusion chromatography, only a band near 70 kDa (human eNAMPT) was clearly detected in the plasma of all subjects (upper diagram in FIG. 3). In addition, TSG101, an exosome marker, was also detected (Figure 3, bottom), confirming that the purified sample contained EVs.

As described above, purification of human eNAMPT-EVs by a size exclusion column yields highly pure human eNAMPT-EVs, but there is a limit to the amount that can be processed at one time, and the yield is low. It cannot be said that it is suitable as a purification method when administration to living bodies is considered.

(Test 4) Investigation of purification conditions of eNAMPT-EVs from human plasma by ultracentrifugation Human plasma collected by heparinization (refrigerated storage) was thawed in a 37°C constant temperature bath, and 2,000 x g for 15 minutes. After centrifugation under these conditions, the supernatant was recovered and used as a human plasma sample for Western blotting (whole human plasma; hP). The human plasma sample was passed through a 0.22 μm pore filter (Millex GP, Merck Millipore) blocked with 5% BSA (fatty acid-free) PBS solution, placed in a similarly blocked centrifuge tube, and multiplied 8 times with PBS. It was diluted and used as a sample for ultracentrifugation.

The ultracentrifugal force was fixed at 210,000×g, the centrifugation time was changed from 30 minutes to 125 minutes, and the other conditions were the same as in Test 1 above. Western blotting was performed. The results are shown in FIG.

As shown in FIG. 4, for human plasma, the amount of eNAMPT detected was greatest when the centrifugation time was 50 minutes. Although the intensity of the eNAMPT band was slightly weakened at 75 minutes of centrifugation, it was still clear. In the case of human plasma, it was found that by setting the optimum centrifugation time, the amount of contamination with impurities such as lipids can be reduced and a sufficient amount of eNAMPT-EVs can be recovered. In the case of purification of eNAMPT-EVs from mouse plasma, when ultracentrifugation was used, the yield of eNAMPT-EVs increased as the centrifugation time was lengthened (Test 2). In the case of purification of eNAMPT-EVs using ultracentrifugation, it is necessary to set fairly limited conditions such as an ultracentrifugal force of 210,000 × g and a centrifugation time of about 40 to 75 minutes. revealed for the first time by Using the k factor representing centrifugal efficiency, the optimal centrifugal condition is t(min)/k=0.56 to 1.06. Note that the k factor is a value calculated by the following formula. (r _max : maximum radius, r _min : minimum radius, RPM: revolutions per minute)

(Test 5) Confirmation of purification conditions for eNAMPT-EVs from human plasma by ultracentrifugation Conditions for purification of eNAMPT-EVs from human plasma by ultracentrifugation established in Test 4 above (ultracentrifugal force 210,000 x g , centrifugation time 50 min) were validated using human plasma samples of various sexes and ages. As a control, an ultracentrifugal force of 210,000×g and a centrifugation time of 101 minutes were used. The samples used were plasma from 5 different subjects and a pool of plasma from 10 humans of various ages and sexes. FIG. 5 shows the results of Western blotting performed in the same manner as in Test 4 above.

As shown in FIG. 5, under conditions of ultracentrifugal force of 210,000×g and centrifugation time of 50 minutes, a specific band of eNAMPT-EVs could be confirmed around 70 kDa in all human plasma samples. On the other hand, under conditions of an ultracentrifugal force of 210,000×g and a centrifugation time of 101 minutes, a specific band of eNAMPT-EVs could not be confirmed, probably because substances that inhibit detection by Western blotting are also precipitated. As described above, the purification conditions of eNAMPT-EVs from human plasma by the ultracentrifugation method established in Test 4 above (ultracentrifugal force 210,000 × g, centrifugation time 50 minutes) are for any human plasma sample found to be applicable.

(Test 6) Measurement of NAD synthesis activity of eNAMPT-EVs using human cultured HEK293 cells HEK293 cells, which had been grown in advance in a growth medium (GM; growth medium; 10% FBS in DMEM), were treated with 0.05% trypsin. /EDTA and stripped from the plate. GM was added, and the cells were washed by centrifugation at 200×g for 5 minutes, suspended in GM, and passed through a cell strainer with a pore size of 100 μm. 5×10 ⁴ cells/well were seeded on a 24-well plate coated with Type I collagen. After 24 hours, the plate was washed once with αMEM heated to 37° C., and 500 μL of αMEM containing 10% mouse plasma or 500 μL of αMEM alone (negative control) was added and incubated for 20 minutes. As a positive control, the culture in GM was continued without washing with αMEM. After incubation for 20 minutes, the cells were washed three times with PBS heated to 37° C. Finally, the PBS was completely aspirated, and H ₂ ClO ₄ was added at 300 μL/well. The 24-well plate was completely sealed with parafilm and stored at -80°C. A portion of the cells after the 20-minute incubation was collected for Western blotting to confirm the uptake of mouse eNAMPT into the cells. The results are shown in FIG. 6 (left).

As shown in FIG. 6 (left), mouse eNAMPT was not detected in cells when the medium was changed from GM containing 10% FBS to αMEM, but when incubated with 10% mouse plasma, mouse eNAMPT was confirmed to be incorporated into cells.

The 24-well plate was then removed from −80° C. and sonicated to completely disrupt the cells. After centrifugation at 500×g for 1 minute with a plate centrifuge, the cells were scraped off with a cell scraper while confirming that the cells had been scraped off under a stereoscopic microscope, and transferred to a 1.5 mL tube. Centrifuged at 20,000×g for 5 minutes and collected 250 μL of supernatant. 83 μL of 3M K2CO3 was added thereto, and after vortexing, incubated on ice for 15 minutes. After centrifugation at 20,000×g for 5 minutes, the supernatant was collected. 17 μL of ×10 PB (Phosphate buffer) was added to 113 μL of the supernatant to obtain a sample for HPLC. HPLC was performed to quantify NAD. The results are shown in FIG. 6 (right figure).

When the GM medium was changed to αMEM medium, intracellular NAD in HEK293 cells was greatly reduced, but when the cells were cultured with 10% mouse plasma added, a marked increase in NAD (NAD synthesis) was observed.

(Test 7) Measurement of NAD synthesis activity of human eNAMPT-EVs using human cultured cells HEK293 Replace mouse plasma with human plasma or eNAMPT-EVs obtained by purifying human plasma by ultracentrifugation, and incubate for 1 hour. The test was conducted in the same manner as Test 6 above, except for the time. The results for human plasma (whole plasma) are shown in FIG. 7 (left), and the results for eNAMPT-EVs obtained by ultracentrifugation purification from human plasma are shown in FIG. 7 (right). As eNAMPT-EVs obtained by purifying human plasma by ultracentrifugation, human plasma (subjects A to D, plasma of 4 people) equivalent to 10% of the medium was ultracentrifuged (ultracentrifugal force 210,000 × g , centrifugation time of 50 minutes) were used.

As shown in FIG. 7 (left panel), similar to the results of the mouse plasma test, when human plasma was added to 10% of the medium volume, NAD synthesis was significantly increased compared to αMEM. rice field. In addition, as shown in FIG. 7 (right figure), almost the same increase in NAD synthesis was observed in purified eNAMPT-EVs of all four subjects. It is conceivable that under these test conditions, the amount of synthesized NAD peaked out, and thus the difference in the synthesized amount of NAD for each subject could not be detected.

(Test 8) Measurement of NAD synthesis activity of human eNAMPT-EVs using human cultured cells HEK293 The test above except that mouse plasma was replaced with human plasma or eNAMPT-EVs obtained by purifying human plasma by ultracentrifugation. The test was performed in the same manner as in 6. The results for human plasma (whole plasma) are shown in FIG. 8 (left), and the results for eNAMPT-EVs obtained by ultracentrifugation purification from human plasma are shown in FIG. 8 (right). As eNAMPT-EVs obtained by purifying human plasma by ultracentrifugation, human plasma (subjects B and C, plasma of 2 subjects) equivalent to 5% of the medium was ultracentrifuged (ultracentrifugal force 210,000 ×g, centrifugation time 50 minutes) purified eNAMPT-EVs were used. In addition, the result of the group in which FK866, an inhibitor of NAMPT activity, was reacted with human plasma and eNAMPT-EVs for 1 hour in advance and added to the medium (FK866 final concentration: 100 nM) is shown by a hatched bar.

As shown in FIG. 8 (left panel), similar to the results of the mouse plasma test, when human plasma was added at 1% to the medium volume, NAD synthesis was significantly increased compared to αMEM. rice field. In addition, as shown in FIG. 8 (left and right diagrams), differences in NAD synthesis levels were detected between the plasma and purified eNAMPT-EVs of the two subjects. Therefore, under these test conditions, it is possible to detect differences in the amount of NAD synthesis between subjects, and thus it is possible to identify the amount of eNAMPT-EVs contained in the plasma of subjects. Also, in the group using the NAMPT inhibitor (FK866), no increase in NAD was observed due to addition of human plasma and eNAMPT-EVs. Human plasma and purified EVs contain various physiologically active substances other than eNAMPT, and we show that the increase in NAD observed when added to cell culture media is due to the activity of NAMPT. In addition, according to this method, NAD synthesis activity can be detected in as little as about 5 μL of plasma, so eNAMPT-EVs activity can be determined with blood at a level obtained by pricking a fingertip with a needle, for example.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a simple and efficient purification method for obtaining human eNAMPT-EVs with few impurities. In addition, human eNAMPT-EVs obtained by this purification method have excellent NAD synthesis activity, etc., and are suitable for use in compositions for increasing NMN and/or NAD biosynthesis and anti-aging compositions. can do. Furthermore, according to the novel method for measuring the NAD synthesis activity of eNAMPT-EVs in biological samples, it is possible to determine the degree of aging of subjects using biological samples.

Claims (13)

(A) Purification of human eNAMPT-EVs, including a step of ultracentrifuging a solution containing a human biological sample at 180,000 x g to 250,000 x g for 30 minutes to 80 minutes at 4 ° C to 37 ° C. Method. 2. The purification method according to claim 1, wherein the biological sample is serum or plasma. 3. The purification method according to claim 1, wherein the solution containing the biological sample is a solution obtained by diluting a human biological sample with a buffer. The ultracentrifugation conditions in the step (A) are 190,000×g to 220,000×g, 40 minutes to 75 minutes, and 4° C. to 37° C., according to any one of claims 1 to 3. purification method. The purification method according to any one of claims 1 to 4, wherein the ultracentrifugation conditions in step (A) are 210,000 xg, 50 minutes, and 4°C to 37°C. A composition for increasing NMN and/or NAD biosynthesis, comprising human eNAMPT-EVs obtained by the purification method according to any one of claims 1 to 5. 7. The composition of claim 6, further comprising carriers and/or excipients. An anti-aging composition comprising human eNAMPT-EVs obtained by the purification method according to any one of claims 1 to 5. 9. The anti-aging composition according to claim 8, further comprising carriers and/or excipients. (a) culturing human cultured cells in bovine serum-containing medium for a certain period of time;
(b) changing the medium of the human cultured cells obtained in step (a) to a serum-free medium and culturing for a certain period of time;
(c) adding a test substance or negative control substance containing eNAMPT-EVs to the human cultured cells obtained in step (b) and culturing for a certain period of time; and (d) obtained in step (c), A test containing eNAMPT-EVs, including the step of measuring and comparing the amount of NAD per certain number of cells of human cultured cells added with a test substance containing eNAMPT-EVs and human cultured cells with a negative control substance added A method for measuring the NAD synthesis activity of a substance. The method for measuring NAD synthesis activity according to claim 10, wherein the test substance containing eNAMPT-EVs is a subject's biological sample. The test substance containing the eNAMPT-EVs is eNAMPT-EVs purified from the subject's plasma or the subject's plasma by the method according to any one of claims 1 to 5, according to claim 10 or 11. A method for measuring the NAD synthesis activity of Using the method according to any one of claims 10 to 12, the subject's plasma, or eNAMPT-EVs purified from the subject's plasma, the subject's aging degree including the step of measuring the NAD synthesis activity how to judge.

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