Preparation method and application of nicotinamide mononucleotide co-crystal

Abstract

The invention belongs to the technical field of compound crystals, and particularly relates to a preparation method and application of a nicotinamide mononucleotide eutectic crystal. Raw materials of the nicotinamide mononucleotide eutectic comprise nicotinamide mononucleotide (NMN) and gallic acid (GA) in an equal molar ratio, and the nicotinamide mononucleotide eutectic is prepared by a liquid-assisted grinding method, a suspension method or a solvent evaporation method. The nicotinamide mononucleotide eutectic (NMN-GA) eutectic prepared by utilizing an intermolecular hydrogen bond between NMN and GA has relatively high purity, a certain crystal structure, a slow release characteristic and good cell protection and anti-oxidation effects, the bioavailability of nicotinamide mononucleotide (NMN) is remarkably improved, the efficiency of converting NMN into NAD < + > is enhanced, and the nicotinamide mononucleotide eutectic (NMN-GA) eutectic can be used for preparing a pharmaceutical preparation. The anti-aging characteristic is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of compound crystals, and more specifically relates to a preparation method of nicotinamide mononucleotide cocrystal and an application thereof. Background Art

[0002] Aging is an irreversible natural process accompanied by various biochemical degenerative changes in tissues. This gradual decline in biological functions leads to the occurrence of various chronic diseases and neurological diseases, such as neurodegenerative diseases, cancer, metabolic diseases, and cardiovascular diseases. The main aging-related factors include deoxyribonucleic acid (DNA) damage, inflammation, mitochondrial dysfunction, epigenetic disruption, and mitogen signaling. Moreover, senescent cells accelerate the production of reactive oxygen species (ROS), which induces the accumulation of superoxide radicals and accelerates aging. ROS mainly originate from mitochondria and play a vital role in regulating biological activities such as oxidative stress and cell apoptosis. Under normal circumstances, the mitochondrial antioxidant enzyme defense system effectively manages excessive cellular ROS generation and maintains dynamic balance. However, during the aging process, the massive accumulation of reactive oxygen species disrupts mitochondrial balance and impairs respiratory capacity. This accumulation can cause age-related diseases in the elderly.

[0003] Nicotinamide mononucleotide (NMN) is a key intermediate in the biosynthesis of nicotinamide adenine dinucleotide (NAD+), an essential coenzyme for cellular metabolism and energy production. NMN is converted to NAD+ after ingestion, and supplementation with NMN can improve mitochondrial oxidative stress and alleviate degenerative diseases by restoring mitochondrial function and enhancing NAD+ content. However, NAD+ levels tend to decline with age. NMN is widely present in foods such as vegetables, fruits, and meat, which are the main sources of NMN supplementation. However, due to the poor thermal stability of NMN, it is easily degraded in the presence of oxidation and gastric acid and enzymes, and its bioavailability is limited.

[0004] Gallic acid (GA) is a phytochemical found in various fruits and vegetables. It has free radical scavenging, antibacterial and anti-inflammatory effects and is widely used in food, pharmaceutical industry, herbal medicine and cosmetics. However, due to its chemical instability, its activity will decrease during long-term storage and exposure to air, light and temperature. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a nicotinamide mononucleotide cocrystal and its application, so as to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is to provide a nicotinamide mononucleotide co-crystal, wherein the raw materials include nicotinamide mononucleotide and gallic acid in an equal molar ratio.

[0008] Furthermore, the nicotinamide mononucleotide cocrystal belongs to P2 1 2 1 2 1 space group, and has unit cell parameters α=β=γ=90° and

[0009] The second technical solution of the present invention is to provide a method for preparing the above-mentioned nicotinamide mononucleotide cocrystal (liquid-assisted grinding method), comprising the following steps:

[0010] Nicotinamide mononucleotide and gallic acid are mixed, and water is used as a grinding medium. After ball milling, the mixture is dried to obtain the nicotinamide mononucleotide cocrystal.

[0011] Furthermore, the molar ratio of nicotinamide mononucleotide to gallic acid is 1:1.

[0012] Furthermore, the dosage ratio of the nicotinamide mononucleotide and water is 1 mmol: 0.05-0.15 mL.

[0013] Furthermore, the ball milling frequency is 25 Hz and the time is 30 min.

[0014] The third technical solution of the present invention is to provide a method for preparing the above-mentioned nicotinamide mononucleotide cocrystal (suspension method), the steps comprising:

[0015] After mixing nicotinamide mononucleotide and gallic acid, a mixed solvent of water and acetonitrile is added, and the mixture is suspended, filtered, and dried to obtain the nicotinamide mononucleotide cocrystal.

[0016] Furthermore, the molar ratio of nicotinamide mononucleotide to gallic acid is 1:1.

[0017] Furthermore, the volume ratio of water to acetonitrile in the mixed solvent is 1:1.

[0018] Furthermore, the dosage ratio of the nicotinamide mononucleotide to the mixed solvent is 1 mmol:6 mL.

[0019] Furthermore, the rotation speed of the suspension is 800 rpm and the time is 24 hours.

[0020] The fourth technical solution of the present invention is to provide a method for preparing the above-mentioned nicotinamide mononucleotide cocrystal (solvent evaporation method), comprising the following steps:

[0021] After mixing nicotinamide mononucleotide and gallic acid, a mixed solvent of water and acetonitrile is added to dissolve them, and crystals are precipitated by slowly evaporating the solvent to obtain nicotinamide mononucleotide cocrystals.

[0022] Furthermore, the molar ratio of nicotinamide mononucleotide to gallic acid is 1:1.

[0023] Furthermore, the volume ratio of water to acetonitrile in the mixed solvent is 1:1.

[0024] When preparing nicotinamide mononucleotide co-crystals by the solvent evaporation method, the minimum amount of the mixed solvent used is based on the amount that can completely dissolve nicotinamide mononucleotide and gallic acid.

[0025] The fifth technical solution of the present invention is to provide an application of the above-mentioned nicotinamide mononucleotide cocrystal in the preparation of anti-aging foods, anti-aging drugs or anti-aging cosmetics.

[0026] The present invention discloses the following technical effects:

[0027] The present invention utilizes the intermolecular interaction between nicotinamide mononucleotide and gallic acid to prepare nicotinamide mononucleotide cocrystal (NMN-GA) cocrystal, which has a specific crystal structure and high purity, certain drug sustained-release properties, and good cell protection and antioxidant effects.

[0028] The NMN-GA cocrystal prepared by the present invention significantly improves the bioavailability of nicotinamide mononucleotide, enhances the efficiency of converting nicotinamide mononucleotide into NAD+, and improves its anti-aging properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0030] Figure 1 Schematic diagram of the hydrogen bonding pattern of NMN-GA cocrystal.

[0031] Figure 2 Schematic diagram of π···π stacking in NMN-GA cocrystal.

[0032] Figure 3 Schematic diagram of NMN-GA eutectic stacking.

[0033] Figure 4 PXRD of NMN, GA and NMN-GA co-crystal.

[0034] Figure 5 DSC curves of NMN-GA cocrystal, NMN and GA.

[0035] Figure 6 FT-IR of NMN-GA cocrystal, NMN and GA.

[0036] Figure 7 Raman spectra of NMN-GA cocrystal, NMN and GA.

[0037] Figure 8 This is the hydrogen spectrum of NMN-GA eutectic.

[0038] Fig. 9 is the cumulative dissolution rate of NMN-GA cocrystal, NMN and GA.

[0039] Fig.10 The average concentration-time curve of NAD+ in plasma obtained under different conditions.

[0040] Fig.11 DPPH scavenging rates of NMN-GA cocrystal, NMN and GA.

[0041] Fig.12 ABTS clearance rates of NMN-GA cocrystal, NMN and GA.

[0042] Fig.13 This is the appearance of mice in each group at the 12th week of the experiment.

[0043] Fig.14 The figure shows the body weight change curves of mice in different groups.

[0044] Fig.15 Water maze test (a), escape latency time (b), escape latency distance (c), number of times crossing the platform (d), heat map of the trajectory of mice in search of the platform (e), and representative paths of mice in each group in search of the platform on the 5th day (f).

[0045] Fig.16 is the organ index of mice in each group.

[0046] Fig.17 The effects of different conditions on the levels of SOD, MDA, GSH, CAT and T-AOC in the serum of each group, where (a) is CAT, (b) is GSH, (c) is MDA, (d) is SOD, and (e) is T-AOC.

[0047] Fig.18 The viability of PC-12 cells after D-galactose modeling.

[0048] Fig.19 The cell viability of each group after administration of different concentrations of drugs in PC-12 model.

[0049] Fig. 20 The β-galactosidase staining images were obtained after drug administration and modeling in each group.

[0050] Fig.21 The percentage of senescent cells stained blue in each group.

[0051] Fig. 22 These are the ROS staining images after modeling for each group. DETAILED DESCRIPTION

[0052] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0053] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0054] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0055] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0056] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0057] The raw materials used in the specific embodiments of the present invention are all commercially available products.

[0058] Unless otherwise specified, the "room temperature" and "normal temperature" referred to in the specific embodiments of the present invention refer to 20-30°C.

[0059] Example 1

[0060] The steps of preparing nicotinamide mononucleotide cocrystals by liquid-assisted grinding include:

[0061] 334.22 mg of NMN and 170.12 mg of GA were mixed and placed in a grinding jar. 20 μL of water was added and ground using a ball mill at a rate of 25 Hz for 30 min. The ground product was dried at room temperature to obtain NMN-GA cocrystals with a yield of 96%.

[0062] Example 2

[0063] The steps of preparing nicotinamide mononucleotide cocrystals by suspension method include:

[0064] 334.22 mg of NMN and 170.12 mg of GA were mixed and placed in a beaker. 6 mL of a mixed solvent of water and acetonitrile (volume ratio 1:1) was added. The mixture was suspended at 800 rpm for 24 h. After filtration, light yellow NMN-GA cocrystals were obtained with a yield of 98%.

[0065] Example 3

[0066] The steps of preparing nicotinamide mononucleotide cocrystals by solvent evaporation method include:

[0067] Mix 334.22 mg of NMN and 170.12 mg of GA, put them into a beaker, dissolve the mixed powder with a mixed solvent of water and acetonitrile (volume ratio of 1:1), filter with filter paper, and then seal the mouth of the beaker with a sealing film and poke a small hole to allow the solvent to evaporate slowly. Crystals precipitate to obtain light yellow needle-shaped NMN-GA eutectic single crystals.

[0068] Test example

[0069] The single crystal diffraction test of NMN-GA eutectic single crystal was carried out using Rigaku X-ray single crystal diffractometer. The experiment used a graphite monochromator and a copper target ray with a wavelength of The data were collected by ω scanning and the data were corrected using the SADBAS program. The structure was solved using Olex2 and refined using the least squares method. The main crystallographic parameters and hydrogen bonding are shown in Tables 1 and 2.

[0070] Table 1 Main crystallographic parameters of NMN-GA cocrystal

[0071]

[0072] Table 2 Hydrogen bond lengths and bond angles of NMN-GA eutectic single crystals

[0073]

[0074] Symmetry codes: (i)+x,1+y,+Z; (ii)-1 / 2+x,3 / 2-y,1-z; (iii)2-x,1 / 2+y,1 / 2-z; (iv)1 / 2+x,3 / 2-y,1-z; (v)-1+x,+y,+z; (vi)1-x,-1 / 2+y,1 / 2-z.

[0075] X-ray single crystal diffraction can be used to obtain information about the three-dimensional structure of the compound. The analytical results show that the chemical composition of the NMN-GA cocrystal is C 18 H 27 N 2 O 16 P, P2 crystallized in the orthorhombic system 1 2 1 2 1 The space group of , and its asymmetric unit contains a nicotinamide mononucleotide, a gallic acid and three water molecules. There are a lot of hydrogen bonds in the crystal structure. NMN is hydrogen-bonded to the carboxylic acid group of GA through the amide group, and there is an intramolecular hydrogen bond between the phosphate group and the amide group.

[0076] Figure 1 Schematic diagram of the hydrogen bonding pattern of NMN-GA cocrystal; Figure 2 Schematic diagram of π···π stacking in NMN-GA cocrystal; Figure 3 Schematic diagram of NMN-GA eutectic stacking.

[0077] Unless otherwise specified, the NMN-GA co-crystal used in the following test examples of the present invention is the NMN-GA co-crystal prepared in Example 1.

[0078] Rigaku MiniFlex600 diffractometer was used to perform X-ray diffraction tests on NMN, GA and NMN-GA eutectics. A copper target was used as the light source. The test was performed under the conditions of voltage 40 kV, current 15 mA, scanning angle 3-50°, and 2° / min. The results are shown in Figure 2. Figure 4 shown.

[0079] Figure 4 This is the PXRD of NMN, GA and NMN-GA cocrystal. As can be seen from the figure, using the simulation diagram (NMN-GAsimulated) derived from SCXRD data as a comparison, it can be seen that the characteristic peaks of NMN-GA cocrystal are at 6.42°, 8.19°, 9.91°, 10.74°, and 13.23°, which are significantly different from the raw materials NMN and GA, and are consistent with the PXRD diagram simulated according to single crystal diffraction data, further confirming that a higher purity NMN-GA cocrystal was obtained.

[0080] Differential scanning calorimetry (DSC) analysis of NMN-GA cocrystals was performed using a NETZSCH STA449F5 synchronous analyzer. 5 mg of powder sample was heated from 25 °C to 400 °C at a heating rate of 10 °C / min under a continuously purged dry nitrogen atmosphere.

[0081] Figure 5 The DSC curves of NMN-GA cocrystal, NMN and GA show that the melting points of the cocrystals of each drug have changed significantly compared with the raw materials. The endothermic peak of gallic acid is at 176°C, and the exothermic peak is at 181°C. The endothermic peak of nicotinamide mononucleotide is at 253°C. The NMN-GA cocrystal has endothermic peaks at 77°C and 94°C, respectively, indicating that a new crystal phase has been formed rather than a simple physical mixture. The endothermic peak at 94°C is due to the removal of 3 molecules of water.

[0082] Fourier transform infrared spectroscopy (FT-IR) analysis of NMN-GA cocrystals was performed using a Nicolet Nexus470 infrared spectrometer. The raw materials (NMN and GA) and their NMN-GA cocrystal samples were added to dry KBr, fully ground and mixed, and then sliced. -1 , the spectral resolution is 2cm -1 , infrared spectrum analysis was carried out under the condition of an average of 64 scans.

[0083] Figure 6 FT-IR of NMN-GA cocrystal, NMN and GA. As shown in the figure, the infrared spectrum of GA is at 3495cm -1 is the stretching vibration absorption peak of hydroxyl -OH, 3275cm -1 is the stretching vibration absorption peak of the -OH group in -COOH, 1664 cm -1 C=O absorption peak, 1615cm -1 The 3442cm of NMN is the stretching vibration absorption peak of the benzene ring (C=C). -1 The stretching vibration absorption peak of the -NH group is at 1668 cm -1 It is the absorption peak of amide carbonyl (C=O). In the infrared spectrum of NMN-GA, the vibration attributed to C=O in the gallic acid molecule is from 1664cm -1 Move to 1691cm -1 , indicating that C=O participates in the formation of hydrogen bonds; the vibration of -NH in the NMN molecule increases from 3442cm -1 Move to 3447cm -1 , indicating that it participates in the formation of weak forces in the eutectic supramolecular.

[0084] The NMN-GA cocrystal was characterized by Raman spectroscopy (FT-Raman). Figure 7 shown.

[0085] RAMHR800 Raman spectrometer was used for testing. The scanning range of the eutectic sample was 2000-50cm -1 , at a resolution of 4cm -1 , the test was carried out under the condition of laser at 1064nm.

[0086] Figure 7 The Raman spectra of NMN-GA cocrystal, NMN and GA are used to characterize the changes in the molecular structure after the cocrystal is formed. Figure 7 It can be seen that in NMN, the characteristic peaks of C=O and -NH2 on the amide group appear at 807 cm -1 and 1591cm -1 In GA, the characteristic peaks of -OH on the benzene ring, -OH in -COOH, and C=O in -COOH appear at 1325 / 1594 cm -1 、1615cm -1 、723cm -1 , with the formation of the eutectic, 807cm -1 Transfer to 816cm -1 , 1591cm -1 Transfer to 1574cm -1 , indicating that C=O and -NH 2 Participates in the formation of hydrogen bonds. In GA, the characteristic peak of -COOH is from 723 / 1615cm -1 Move to 716 / 1604cm -1 The characteristic peaks of Ph-OH are from 1325 / 1594cm -1 Move to 1349 / 1580cm -1 , indicating that -COOH and Ph-OH in GA may be involved in the formation of hydrogen bonds. Raman spectroscopy showed that the hydrogen bond interaction between the acceptor and donor of hydrogen bonds in NMN and GA supported the cocrystal structure.

[0087] The NMN-GA cocrystal was subjected to nuclear magnetic resonance (NMR) analysis, and the results were as follows Figure 8 shown.

[0088] The results were performed on a Bruker Ascend 500 MHz NMR spectrometer. 1 H NMR analysis

[0089] Figure 8 This is the hydrogen spectrum of NMN-GA eutectic. 1 The integration of the characteristic peaks of each component in H NMR can determine the proportion of each component. As can be seen from the figure, the chemical shift of the characteristic peak of NMN in the cocrystal is as follows: 1H NMR (500MHz, DMSO-d6) δ9.92(d,J=19.1Hz,2H),9.24(d,J=6.2Hz,1H),9.00(d,J=8.1Hz,1H),8.27(dd,J=8.0,6.2Hz,1H),7.95(s ,1H),6.90(s,2H),6.09(d,J=6.1Hz,1H),4.33(dd,J=11.9,6.5Hz,2H),4.13(dd,J=16.3,3.9Hz,2H),3.90(dd,J=9.8,5.9Hz,3H). according to 1 The integration results of the characteristic peaks of NMN and GA in H NMR showed that the ratio of NMN to GA in the cocrystal was 1:1, which was consistent with the material ratio in the SXRD results.

[0090] In vitro dissolution behavior study

[0091] The release behavior of NMN from NMN-GA cocrystals was studied by dissolution experiments.

[0092] Methods: The small cup paddle method was used with a paddle speed of 75 rpm and a temperature of 37°C. PBS buffer (pH = 1.2) was selected as the dissolution medium, and the volume of the dissolution medium was 100 mL. Accurately weigh 576 mg of NMN-GA powder, 334 mg of NMN powder, and 170 mg of GA powder. After the temperature of the medium stabilized, the medium was added. Sampling was set at different time points, and 1 mL of buffer was added immediately after each sampling of 1 mL. After all sample solutions passed through a 0.45 μm membrane, the drug concentration was measured using UV (n = 3). The results are as follows Fig. 9 shown.

[0093] Fig. 9 The cumulative dissolution of NMN-GA cocrystal, NMN and GA. As can be seen from the figure, the API NMN dissolves well and is completely released after 15 minutes, while the cumulative release of the API GA is close to 100% at 45 minutes. After the formation of the cocrystal, GA is introduced into the cocrystal, which changes the cumulative release amount. Compared with the API, NMN in the cocrystal is released slowly and is not completely released until 45 minutes later, while GA in the cocrystal is completely released in 120 minutes, proving that the cocrystal has a sustained release effect compared to the API. The cumulative dissolution results show that compared with the API, by forming a cocrystal with gallic acid, the release of nicotinamide mononucleotide is delayed at the same time, indicating that the formation of the cocrystal has a sustained release effect.

[0094] The pharmacokinetic analysis of NMN-GA cocrystal was performed, and the results were as follows Fig.10 shown.

[0095] Methods: SD rats were divided into three groups, 3 rats in each group, namely blank group, NMN group and NMN-GA cocrystal group. Before blood collection, they were fasted for 8 hours, and then gavaged with distilled water (1 mL), NMN raw material (600 mg / kg) and NMN-GA cocrystal (NMN dose in cocrystal was 600 mg / kg). Blood samples were collected into anticoagulated centrifuge tubes at different time points (0h, 2h, 4h, 8h, 12h, 16h, 24h). The plasma was centrifuged at 8000rpm for 10min at 4°C, and the NAD+ content in serum was detected using a kit.

[0096] The bioavailability of NMN is indicated by measuring the concentration of NAD+ in plasma. Fig.10 The average concentration-time curve of NAD+ in plasma obtained under different conditions. It can be seen from the figure that after gavage of NMN and NMN-GA, the concentration of NAD+ in the plasma of the two groups reached its maximum value at 12h and then decreased. At 12h, the NAD+ concentration in the NMN-GA group was 162.45±13.77nmol / mL, and that in the NMN group was 77.73±7.12nmol / mL, which was 2.1 times that of the NMN-GA group. The results show that after NMN and GA form a eutectic, the bioavailability and anti-aging effect of NMN are significantly improved by promoting the generation of NAD+ in the blood.

[0097] The antioxidant capacity of NMN-GA cocrystals was tested by DPPH and ABTS. The results are as follows: Figure 11-Figure 12 shown.

[0098] DPPH free radical scavenging ability: Prepare 40 μg / mL DPPH ethanol solution, and its absorbance at 517 nm is 0.70±0.02. Mix 150 μL DPPH ethanol solution with 50 μL samples of different concentrations (such as Fig.11 The abscissa shows the concentration of the 50 μg / ml PBS or distilled water as a negative control, and the 96-well plate was added and incubated at 37°C in the dark for 30 min. The absorbance was measured at 517 nm using an ELISA reader.

[0099] The ability to scavenge DPPH free radicals is calculated as follows:

[0100] Clearance rate (%) = [1-(A 1 -A 2 ) / A 0 ]×100%, where A 0 is the absorbance of the negative control (no sample, distilled water group), A 1 is the absorbance after the sample and DPPH solution are mixed, A 2 is the absorbance of distilled water and sample.

[0101] Fig.11It is the DPPH clearance rate of NMN-GA cocrystal, NMN and GA. As can be seen from the figure, there is a certain dose-effect relationship between the scavenging effect of DPPH free radicals and the concentrations of GA and NMN, and the antioxidant activity increases with the increase of concentration. Through the DPPH clearance rate and ABTS clearance rate, it can be seen that when the concentration increases, the cocrystal is improved compared with the two raw materials (NMN and GA). When the concentration is 20ug / mL, the clearance rate of NMN is 11%, the clearance rate of GA is 55%, the clearance rate of cocrystal is 87%, and the DPPH clearance rate is increased by 21%.

[0102] ABTS free radical scavenging ability: Prepare a stock solution of 7 mmol / L ABTS aqueous solution and a stock solution of 140 mmol / L potassium persulfate aqueous solution, mix the ABTS stock solution (5 mL) with 88 μL potassium persulfate stock solution, react for 12 h in the dark, and dilute to an absorbance of 0.70 ± 0.02 at 734 nm;

[0103] 50 μL of samples of different concentrations (such as Fig.13 The distilled water (shown on the horizontal axis) or the negative control and 150 μL of ABTS dilution were added to the 96-well plate and mixed at 37°C for 20 min in the dark. The absorbance was measured at 734 nm using an ELISA reader.

[0104] The ability to scavenge ABTS free radicals was calculated as follows: scavenging rate (%) = [1-(A 1 -A 2 ) / A 0 ]×100%, where A 0 is the absorbance of the negative control (no sample, distilled water group), A 1 is the absorbance of the sample and ABTS solution mixed, A 2 is the absorbance of distilled water and sample.

[0105] Fig.12 is the ABTS clearance rate of NMN-GA cocrystal, NMN and GA. Fig.12 It can be seen that when the concentration is 12.5ug / mL, the clearance rate of NMN is 7%, the clearance rate of GA is 70%, the clearance rate of cocrystal is close to 100%, and the clearance rate of ABTS is increased by 23%.

[0106] By analyzing Figure 11-Figure 12 The results show that after the formation of cocrystal, it shows a significant synergistic effect on the scavenging of DPPH and ABTS free radicals.

[0107] The aging mouse model was established by intraperitoneal injection of D-gal (500 mg / kg / d), and the drug was administered by gavage. The mice were adapted to the environment for 7 days, and from the 8th day onwards, the mice were randomly divided into 6 groups, with 8 mice in each group.

[0108] The groups were as follows: control group (Control), intraperitoneal injection of 10% saline 1mL / kg / d; model group (Model), injection of D-galactose, 500mg / kg / d; positive group (Positive), injection of D-galactose + oral administration of vitamin C, 100mg / kg / d; NMN group, injection of D-galactose + oral administration of NMN, 100mg / kg / d; eutectic group, injection of D-galactose + oral administration of NMN-GA eutectic 172mg / kg / d; physical mixture group (PM), injection of D-galactose + oral administration of NMN+GA (150mg / kg / d). The modeling was continued for 12 weeks. During the experiment, the mental state, activity, coat color, skin, diet and water intake of mice in each group were observed regularly every day, and the weight of the mice was recorded to calculate the average value. The results are shown in Table 3.

[0109] Table 3 Body weight changes of mice in each group (g)

[0110]

[0111] It can be seen from the data in the table that there was no significant difference in the weight gain of mice at the beginning of the experiment, but as the experiment progressed, the weight of mice in the model group decreased significantly compared with other groups. This indicates that they aged and lost weight after modeling, and this aging state can be significantly improved after drug administration.

[0112] Fig.13 This is the appearance of mice in each group at the 12th week of the experiment.

[0113] Fig.14 The figure shows the body weight change curves of mice in different groups.

[0114] Morris water maze behavior was used to detect the spatial learning and memory function of mice, including the Place navigation test and the Probe trial test. Mice were trained to use an immersion platform in a circular tank (120 cm in diameter and 50 cm deep) filled with water (25±1°C). The maze was divided into four quadrants: northwest (I), northeast (II), southeast (III), and southwest (IV). There were several visual cues around the circular water tank as references for the spatial training of mice, and they remained unchanged during the experiment.

[0115] Positioning navigation experiment: Each mouse received a navigation training trajectory for 4 consecutive days at the starting position (north, south, east and west). The circular platform (12 cm in diameter) was located in the center of the northwest (I) quadrant, submerged 1 cm below the water surface. The positioning navigation experiment lasted for 4 days. Every day, the mice were randomly placed in the water facing the pool wall from the midpoint of the pool wall of the 4 quadrants. Mice that did not reach the platform during 60 seconds were placed on the platform for 15 seconds to consolidate memory. All tests were performed at approximately the same time every day to minimize performance changes due to the time of day. An infrared camera was installed directly above the water maze trough, and data was collected and analyzed by a computer video acquisition and analysis system to record whether the mouse could find the platform within 60 seconds, the time required to find the platform (Escape latency), and the movement distance (Escape path length).

[0116] Spatial exploration experiment: The memory retention ability of the platform space was examined. The exploration test was conducted 24 hours after the last training. During the exploration test, the platform was removed and the mice were allowed to swim freely for 60 seconds. The movement trajectory of the mice within 60 seconds, the number of platform crossings (Platform crossings), and the time spent in each quadrant (Time spent in each quadrant) were recorded as indicators for evaluating the spatial learning and memory function of the mice. The swimming path was recorded and analyzed.

[0117] Fig.15The water maze test (a), escape latency (b), escape latency distance (c), number of platform crossings (d), heat map of the trajectory of mice searching for the platform (e), and representative paths of mice searching for the platform on the fifth day in each group of mice (f). As can be seen from the figure, on the first day, there was no difference in escape latency and swimming distance between the groups. All six groups were able to successfully learn the task, which can be demonstrated by the gradually reduced escape latency and swimming distance in the 4-day navigation test, and the escape latency and swimming distance of the D-galactose-induced model group were significantly longer than those of the normal control group. At the same time, the changes in the escape latency and swimming distance of the mice in the drug-treated group were opposite to those observed in the D-galactose-induced model group, and the escape latency and swimming distance recovered to the same level as the positive control group. On the fifth day, the platform was removed and a probe test was performed. In the probe test, the time to enter the platform and the swimming distance of the target quadrant were significantly increased in the D-gal-induced model group compared with the normal control group. Compared with the D-gal-induced model group, the average number of times mice entered the platform was significantly increased after administration of NMN, GA, cocrystals, and physical mixtures. Figure (e) is a heat map of the trajectory of mice looking for the platform, and Figure (f) is a representative path of mice looking for the platform. These results show that D-gal-induced aging can lead to spatial learning and memory impairment, and treatment with NMN administration can better restore aging-related cognitive impairment caused by D-gal administration. In addition, aging model mice treated with NMN-GA cocrystals showed significant improvement.

[0118] After the behavioral test, the mice were fasted but not watered for 12 hours. After weighing, the eyeballs were removed and blood was collected. The blood was allowed to stand at 4°C until the serum was precipitated, and then centrifuged at 4°C and 3000rpm for 10 minutes. The supernatant was collected and packaged, and stored in liquid nitrogen for later use. After blood collection, the mice were quickly killed by dislocation, and the heart, liver, spleen, lung, kidney and other tissues were removed on ice. The blood stains were rinsed with ice saline, dried with filter paper, weighed, placed in cryopreservation tubes, and stored in liquid nitrogen.

[0119] The organ index was calculated according to the body weight and organ weight of each group of mice: organ index = (organ weight / body weight) × 100%. Fig.16 shown.

[0120] The organ index is an important indicator for judging the function of the organs from a macroscopic perspective. It is generally believed that aging will lead to the atrophy of organs such as the liver, spleen and thymus. Fig.16is the organ index of each group of mice. As can be seen from the figure, the visceral index of the liver, spleen and thymus of mice injected with D-galactose was significantly lower than that of the treatment group and the blank group, which indicates that D-galactose can reduce the visceral index. However, the visceral index of the treated mice has improved. The decline in physical function is the main manifestation of aging. The results showed that compared with the control group, the body weight, liver and brain indexes of the model group mice were significantly reduced, indicating that the mouse organs were atrophied or degenerative, and the aging mouse model was successfully established.

[0121] The antioxidant and intervention effects of NMN-GA were detected by analyzing the serum oxidative stress-related indicators of aged mice. MDA, SOD, GSH-Px, CAT, and T-AOC in the serum of each group of mice were detected according to the kit instructions. Fig.17 And as shown in Table 4.

[0122] Table 4

[0123]

[0124] Fig.17 The effects of different conditions on the levels of SOD, MDA, GSH, CAT and T-AOC in the serum of each group, where (a) is CAT, (b) is GSH, (c) is MDA, (d) is SOD, and (e) is T-AOC. Excessive D-galactose can induce the production of ROS, which Fig.17 As shown in Table 4, after D-gal induction, the activity of antioxidant substances GSH, SOD and CAT decreased sharply, indicating that the ability to scavenge oxygen free radicals during aging decreased and the generation of oxidation products accelerated. By detecting these antioxidant indicators, the antioxidant and intervention effects of NMN and GA after forming cocrystals were evaluated. A significant decrease in GSH expression was observed in the serum of mice in the model group injected with D-gal, indicating that D-gal induction can accelerate oxidative stress. Compared with the D-gal model group, the GSH content in the serum of the NMN-GA group increased by 54.3%, and increased by 42.5% compared with the bulk drug NMN group. The MDA level in the model group showed an increasing trend, while the serum MDA content of mice in the NMN-GA treatment group was significantly reduced by 53.2% compared with the bulk drug NMN group. In addition, compared with the bulk drug NMN group, the NMN-GA group decreased by 43.5%. The NMN-GA cocrystal enhanced the regulation of oxidative stress, indicating that NMN-GA is more effective than NMN in improving antioxidant activity. Compared with the model group, the SOD and CAT contents in the serum and the total antioxidant capacity T-AOC of the mice in the positive group, NMN group, PM group, and NMN-GA cocrystal group were increased. Compared with the NMN group, the SOD, CAT, and T-AOC of NMN-GA in the NMN-GA cocrystal group were significantly increased. Therefore, NMN-GA treatment showed excellent inhibitory effect on oxidative stress and had a good anti-aging effect.

[0125] PC12 cells were seeded in a 96-well plate at a density of 6000 cells / well, and 200 μL of the cells were placed in a 37°C incubator containing 5% CO. 2 After 24 hours of incubation in the incubator, the medium was changed. D-galactose was dissolved in complete culture medium, and incubated for 48 hours at 8, 16, 32, 64, and 128 mg / mL, respectively. The supernatant was discarded, 20 μL of MTT solution was added, and after incubation for 5 hours, the supernatant was aspirated and 150 μL of DMSO was added. The purple crystals were oscillated for 10 minutes to dissolve, and the absorbance of each well was measured at a wavelength of 490 nm using an enzyme marker. Each group of experiments was repeated 3 times.

[0126] Fig.18 The viability of PC-12 cells after D-galactose modeling. It can be found from the figure that when the concentration of D-galactose is above 32 mg / mL, the cell viability begins to decline and has obvious statistical significance. Compared with the blank group, the cell survival rate of PC12 cells in the model group gradually decreases after being damaged by different concentrations of D-galactose and is concentration-dependent, and the difference is statistically significant (P<0.01). When the concentration of D-galactose is 32 mg / mL, the cell survival rate drops to 50% to 60%. It can be seen that this concentration makes PC12 cells better induce senescence, so this concentration is determined as the dosage concentration that causes PC12 cell senescence in the later stage.

[0127] PC12 cells were seeded in a 96-well plate at a density of 6000 cells / well, and 200 μL of the cells were placed in a 37°C incubator containing 5% CO. 2 After 24 hours of incubation in the incubator, the medium was changed. A blank group, a model group, an NMN group, a GA group, a eutectic group, and a physical mixture group were set up. The blank group was given only blank culture medium, the model group was given a culture medium containing 32 mg / mL D-galactose, and the NMN group, the GA group, the NMN-GA eutectic group, and the NMN+GA physical mixture (PM) group were given a culture medium containing drugs and d-galactose at the same time. Each group was incubated for 48 hours at the same time. The supernatant was discarded, 20 μL of MTT solution was added, and after incubation for 4-6 hours, the supernatant was aspirated and 150 μL of DMSO was added. The purple crystals were oscillated for 10 minutes to dissolve, and the absorbance of each well was measured at a wavelength of 490 nm using an enzyme reader (n=3).

[0128] Fig.19 The figure shows the cell viability of each group after administration of different concentrations of drugs in PC-12 modeling. It can be found from the figure that compared with the blank group and the model group, the cell survival rate of the model group was significantly reduced, and the survival rate of PC12 cells after administration was improved. Moreover, the cocrystal group can significantly improve the cell survival rate at concentrations of 200μM and 400μM compared with other groups, proving that NMN and GA can improve the anti-aging effect after forming a cocrystal, and can resist the damage induced by D-galactose, which can alleviate the development of aging to a certain extent and prolong the life of PC12 cells.

[0129] The activity of SA-β-Gal, a marker of cell senescence, was determined by using a SA-β-Gal staining kit. The positive rate of β-galactosidase staining can better reflect the aging rate of cell populations or individual tissues and is an important biological indicator reflecting the degree of aging. Before staining, the culture medium was removed, the cells were washed once with PBS, and then fixed with β-Gal staining fixative at room temperature for 15 minutes. After fixation, the cells were washed three times with PBS and incubated at 37°C without CO 2 The cells were incubated with the prepared staining solution for 12 h under the same conditions. SA-β-gal positive cells appeared blue under visible light. The percentage of SA-β-gal stained cells in PC12 cells was counted under a microscope to detect the expression of SA-β-gal in PC12 cells.

[0130] Fig. 20 The β-galactosidase staining images of each group after drug administration and modeling; Fig.21 is the percentage of senescent cells stained blue in each group. Figure 20-21 It can be found that compared with the blank group, the positive staining rate of β-galactosidase in PC12 cells in the model group was significantly increased. Fig.21 It can be seen that the number of senescent cells stained blue was significantly higher than that of the blank group, and the difference was statistically significant. Compared with the model group, the positive staining rate of β-galactosidase in PC12 cells in the drug administration groups of different concentrations was significantly reduced, and it can be seen that the number of senescent cells stained blue was much less than that of the model group.

[0131] The cellular ROS concentration was measured using a ROS assay kit. Cells were seeded into 6-well plates (5 × 10 4 cells / well), cultured for 24 h, and then treated with D-gal for 24 h. The culture medium was then removed, and the cells were incubated with DCFH-DA (10 μM) at 37°C for 20 min in the dark, washed three times with blank culture medium, and photographed with an inverted fluorescence microscope.

[0132] Fig. 22 The ROS staining diagram after modeling for each group of treatment and administration is shown in the figure. It can be found that the fluorescence intensity of D-galactose-induced senescent cells is much higher than that of the control group, indicating that D-galactose accelerates the generation of intracellular ROS. In addition, the average fluorescence intensity of PC-12 cells after D-galactose treatment increased significantly compared with the control group. In addition, after incubation with NMN and GA, the cell fluorescence decreased. This shows that OFNPs loaded with NMN have better antioxidant effects.

[0133] The above experiments have verified that the NMN-GA cocrystal prepared by the present invention can change the dissolution of NMN and the level of NAD+ in the body and the anti-aging ability. The cocrystal has a certain crystal structure and sustained-release performance to improve the absorption and conversion efficiency of NMN in the body and improve the NAD+ level in the body as a whole. In vivo, mice treated with NMN-GA cocrystals have better physical fitness and spatial memory ability than aging mice treated with the raw material NMN. Moreover, NMN-GA cocrystals can reduce the generation of oxidative stress and increase the activity of related antioxidant enzymes. In vitro, NMN-GA cocrystals significantly increased the survival rate of PC-12 cells, reduced the number of senescent cells, and reduced the active oxygen of cells.

[0134] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0135] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A nicotinamide mononucleotide co-crystal, characterized in that: The raw materials include nicotinamide mononucleotide and gallic acid in an equimolar ratio.

2. The nicotinamide mononucleotide co-crystal according to claim 1, characterized in that The nicotinamide mononucleotide cocrystal belongs to the P212121 space group and has unit cell parameters α=β=γ=90° and 3. A method for preparing the nicotinamide mononucleotide cocrystal according to claim 1 or 2, characterized in that the steps include: Nicotinamide mononucleotide and gallic acid are mixed, and water is used as a grinding medium. After ball milling, the mixture is dried to obtain the nicotinamide mononucleotide cocrystal.

4. The preparation method according to claim 3, characterized in that: The molar ratio of nicotinamide mononucleotide to gallic acid is 1:1; and / or, The dosage ratio of nicotinamide mononucleotide to water is 1 mmol: 0.05-0.15 mL; and / or, The ball milling frequency is 25 Hz and the time is 30 min.

5. A method for preparing the nicotinamide mononucleotide cocrystal according to claim 1 or 2, characterized in that the steps include: After mixing nicotinamide mononucleotide and gallic acid, a mixed solvent of water and acetonitrile is added, and the mixture is suspended, filtered, and dried to obtain the nicotinamide mononucleotide cocrystal.

6. The preparation method according to claim 5, characterized in that: The molar ratio of nicotinamide mononucleotide to gallic acid is 1:1; and / or, The volume ratio of water to acetonitrile in the mixed solvent is 1:1; and / or, The dosage ratio of the nicotinamide mononucleotide to the mixed solvent is 1 mmol: 6 mL; and / or, The rotation speed of the suspension is 800 rpm and the time is 24 hours.

7. A method for preparing the nicotinamide mononucleotide cocrystal according to claim 1 or 2, characterized in that the steps include: After mixing nicotinamide mononucleotide and gallic acid, a mixed solvent of water and acetonitrile is added to dissolve them, and crystals are precipitated by evaporating the solvent to obtain nicotinamide mononucleotide cocrystals.

8. The preparation method according to claim 7, characterized in that: The molar ratio of nicotinamide mononucleotide to gallic acid is 1:1; and / or, The volume ratio of water to acetonitrile in the mixed solvent is 1:

1.

9. Use of the nicotinamide mononucleotide cocrystal according to claim 1 or 2 in the preparation of anti-aging foods, anti-aging drugs or anti-aging cosmetics.

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