Application of nicotinamide ribose malate in anti-aging

Abstract

The invention belongs to the technical field of biochemistry, and particularly relates to application of nicotinamide ribose malate in anti-aging. The 10 [mu] g / ml nicotinamide ribose malate is used for resisting aging, and especially the 10 [mu] g / ml nicotinamide ribose malate is used for resisting aging of zebra fish, and the anti-aging effect is obvious. The invention discloses application of nicotinamide ribose malate in preparation of anti-aging skin care products, health care products and drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemistry, and particularly relates to the application of nicotinamide riboside malate in anti-aging. Background Art

[0002] Nicotinamide riboside malate, with the English name Nicotinamide Riboside Malate, abbreviated as NRHM. As a precursor of NAD+, nicotinamide riboside malate has multiple physiological effects such as increasing the concentration of NAD+ in tissues, inducing insulin sensitivity, and enhancing the function of deacetylase. Its sensitivity to light, air, and humidity is significantly reduced, and it can maintain good chemical stability even in an environment exposed to adverse factors. At the same time, it has low hygroscopicity and is not prone to absorb moisture and deteriorate in a humid environment. In addition, it can still maintain good stability at higher temperatures, which enables nicotinamide riboside malate to withstand higher temperature conditions during the preparation process, thus helping to optimize the production process and improve production efficiency. It can be used as a pharmaceutical ingredient for increasing the concentration of NAD+ in tissues, inducing insulin sensitivity, and enhancing the function of deacetylase, and can also be used as a high-quality raw material for health products to improve the stability and bioavailability of products.

[0003] According to existing research references, NAD+ is closely related to aging inhibition. The higher the level of NAD+ in the body, the more obvious the anti-aging effect. Hydrogen peroxide H2O2 can induce the production of excessive reactive oxygen species (ROS) in zebrafish, damage biomacromolecules in cells, reduce the level of NAD+ in the body, lead to cell aging, and thus make the whole zebrafish show an aging state.

[0004] According to publicly available information, nicotinamide riboside malate can increase the level of NAD+ in the body. NAD+ plays a key role in cell energy metabolism and repair processes, and can promote cell repair and energy production, thus helping to resist the effects of aging. So far, there is no report on the concentration at which nicotinamide riboside malate exerts an anti-aging effect in cells, and there is no quantitative anti-aging application of nicotinamide riboside malate in zebrafish. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides an application of nicotinamide riboside malate in anti-aging, and the application of 10 μg / ml nicotinamide riboside malate in anti-aging of zebrafish.

[0006] The application of nicotinamide riboside malate in anti-aging, where nicotinamide riboside malate is used for anti-aging.

[0007] As a preferred technical solution, the use of nicotinamide riboside malate in the preparation of anti-aging health products.

[0008] As a preferred technical solution, the use of nicotinamide riboside malate in the preparation of anti-aging skin care products.

[0009] As a preferred technical solution, the use of nicotinamide riboside malate in the preparation of anti-aging drugs.

[0010] As a preferred technical solution, 10 μg / ml nicotinamide riboside malate is used for anti-aging.

[0011] As a preferred technical solution, the use of 10 μg / ml nicotinamide riboside malate in the preparation of anti-aging skin care products.

[0012] As a preferred technical solution, the use of 10 μg / ml nicotinamide riboside malate in the preparation of anti-aging health care products.

[0013] As a preferred technical solution, the use of 10 μg / ml nicotinamide riboside malate in the preparation of anti-aging drugs.

[0014] As a preferred technical solution, nicotinamide riboside malate is used for anti-aging of zebrafish.

[0015] As a preferred technical solution, 10 μg / ml nicotinamide riboside malate is used for anti-aging of zebrafish.

[0016] Experimental results show that nicotinamide riboside malate can effectively increase the NAD+ level in zebrafish with aging models, and has a relatively obvious anti-aging effect on zebrafish. Beneficial effects

[0017] 1. The present invention first provides that 10 μg / ml nicotinamide riboside malate is used for anti-aging.

[0018] 2. The present invention provides that 10 μg / ml nicotinamide riboside malate is used for anti-aging of zebrafish, and its anti-aging effect is obvious. As time goes on, the NAD+ level in zebrafish continues to increase. Brief description of the drawings

[0019] Figure 1 Relative fluorescence intensity graph.

[0020] Figure 2 Side view of zebrafish incubated for 30 minutes.

[0021] Figure 3 Side view of zebrafish incubated for 60 minutes.

[0022] Figure 4 Side view of zebrafish incubated for 120 minutes. Detailed description of the invention

[0023] To make the present invention easy to understand, the present invention will be described in detail below in conjunction with specific embodiments. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terms used herein are only for describing the specific embodiments and do not represent limitations.

[0024] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice of the present invention, the preferred methods and materials are now described.

[0025] Experimental instruments Artificial climate incubator (RGX-70ES), microscope (B Braun), stereoscopic fluorescence microscope.

[0026] Experimental consumables 60 mm culture dishes, 90 mm culture dishes, six-well plates, 3 mL plastic droppers, 15 mL EP tubes, etc.

[0027] Experimental reagents 60×E3 solution (NaCl 17.4 g / L; CaCl•2H2O 2.9 g / L; KCl 0.8 g / L; MgCl2•6H2O 4.89 g / L); DMSO (CAS: 67-68-5); methylene blue (CAS: 7220-79-3); nicotinamide ribose malate (BT22-CYSC006); NAD+ red fluorescent probe (50 μg), etc.

[0028] Test system: wild-type AB strain zebrafish.

[0029] Example 1 Sample treatment group for 30 minutes Select 10 healthy adult wild-type (AB) zebrafish and obtain fertilized embryos according to the conventional breeding method. Cultivate them at a water temperature of 28 ± 1 °C, pH 7.0 - 7.5, and keep the water quality clean until 6 hpf. Treat them in a culture solution containing H2O2 (500 μM) for 24 hours to induce the establishment of an aging model. Transfer the zebrafish larvae to a culture solution containing 10 μg / ml nicotinamide ribose malate and culture them continuously until 72 hpf.

[0030] Fix zebrafish larvae with 4% paraformaldehyde for 2 - 4 hours, then rinse with PBS 3 times, 5 minutes each time. Next, permeabilize with PBS solution containing 0.2% Triton X-100 for 15 - 20 minutes, and then rinse with PBS 3 times, 5 minutes each time. Add the treated zebrafish larvae to the NAD+ red fluorescent probe solution and incubate at a constant temperature of 32 °C for 30 minutes. After incubation, wash away the unbound probe with PBS.

[0031] Fluorescence detection and analysis: Observe the labeled zebrafish larvae through a fluorescence microscope and take pictures for record. Use image analysis software (Imagel) to quantitatively analyze the fluorescence intensity in the pictures. Select the same area for measurement in each sample, and take the average value as the fluorescence intensity value of the sample at the corresponding incubation time, which represents the NAD+ level in zebrafish larvae.

[0032] Example 2 sample treatment group for 60 minutes Select 10 healthy adult wild-type (AB) zebrafish and obtain fertilized embryos according to the conventional breeding method. Cultivate them at a water temperature of 28 ± 1 °C, pH 7.0 - 7.5, and keep the water quality clean until 6 hpf. Treat them in a culture solution containing H2O2 (500 μM) for 24 hours to induce the establishment of an aging model. Transfer the zebrafish larvae to a culture solution containing 10 μg / ml nicotinamide ribose malate and culture continuously until 72 hpf.

[0033] Fix zebrafish larvae with 4% paraformaldehyde for 2 - 4 hours, then rinse with PBS 3 times, 5 minutes each time. Next, permeabilize with PBS solution containing 0.2% Triton X-100 for 15 - 20 minutes, and then rinse with PBS 3 times, 5 minutes each time. Add the treated zebrafish larvae to the NAD+ red fluorescent probe solution and incubate at 32 °C for 60 minutes. After incubation, wash away the unbound probe with PBS.

[0034] Fluorescence detection and analysis: Observe the labeled zebrafish larvae through a fluorescence microscope and take pictures for record. Use image analysis software (Imagel) to quantitatively analyze the fluorescence intensity in the pictures. Select the same area for measurement in each sample, and take the average value as the fluorescence intensity value of the sample at the corresponding incubation time, which represents the NAD+ level in zebrafish larvae.

[0035] Example 3 sample treatment group for 120 minutes Select 10 healthy adult wild-type (AB) zebrafish, and obtain fertilized embryos according to the conventional breeding method. Cultivate them at a water temperature of 28 ± 1 °C, pH 7.0 - 7.5, and keep the water quality clean until 6 hpf. Treat them in a culture medium containing H2O2 (500 μM) for 24 hours to induce the establishment of an aging model. Transfer the zebrafish larvae to a culture medium containing 10 μg / ml nicotinamide ribose malate and culture them continuously until 72 hpf.

[0036] Fix the zebrafish larvae with 4% paraformaldehyde for 2 - 4 hours, then rinse them 3 times with PBS for 5 minutes each time. Next, permeabilize them with a PBS solution containing 0.2% Triton X-100 for 15 - 20 minutes, and then rinse them 3 times with PBS for 5 minutes each time. Add the treated zebrafish larvae to the NAD+ red fluorescent probe solution and incubate them at a constant temperature of 32 °C for 120 minutes. After the incubation, wash away the unbound probe with PBS.

[0037] Fluorescence detection and analysis: Observe the labeled zebrafish larvae through a fluorescence microscope and take pictures for record. Use image analysis software (Imagel) to quantitatively analyze the fluorescence intensity in the pictures. Select the same area for measurement in each sample, and take the average value as the fluorescence intensity value of the sample at the corresponding incubation time, which represents the NAD+ level in the zebrafish larvae.

[0038] Control group of the model in Comparative Example 1 for 30 minutes Select 10 healthy adult wild-type (AB) zebrafish, and obtain fertilized embryos according to the conventional breeding method. Cultivate them at a water temperature of 28 ± 1 °C, pH 7.0 - 7.5, and keep the water quality clean until 6 hpf. Treat them in a culture medium containing H2O2 (500 μM) for 24 hours to induce the establishment of an aging model. The zebrafish larvae continue to be cultured in a normal culture medium and are continuously treated until 72 hpf.

[0039] Fix the zebrafish larvae with 4% paraformaldehyde for 2 - 4 hours, then rinse them 3 times with PBS for 5 minutes each time. Next, permeabilize them with a PBS solution containing 0.2% Triton X-100 for 15 - 20 minutes, and then rinse them 3 times with PBS for 5 minutes each time. Add the treated zebrafish larvae to the NAD+ red fluorescent probe solution and incubate them at a constant temperature of 32 °C for 30 minutes. After the incubation, wash away the unbound probe with PBS.

[0040] Fluorescence detection and analysis: Observe the labeled zebrafish larvae under a fluorescence microscope and take pictures for recording. Use image analysis software (ImageJ) to quantitatively analyze the fluorescence intensity in the pictures. Select the same area for measurement in each sample, and take the average value as the fluorescence intensity value of the sample at the corresponding incubation time, which represents the NAD+ level in the zebrafish larvae.

[0041] Model control group of Comparative Example 2, 60 minutes Select 10 healthy adult wild-type (AB) zebrafish, and obtain fertilized embryos according to the conventional breeding method. Cultivate them at a water temperature of 28 ± 1 °C, pH 7.0 - 7.5, and keep the water quality clean until 6 hpf. Treat them in a culture solution containing H2O2 (500 μM) for 24 hours to induce the establishment of an aging model. The zebrafish larvae continue to be cultured in normal culture solution and are continuously treated until 72 hpf.

[0042] Fix the zebrafish larvae with 4% paraformaldehyde for 2 - 4 hours, then rinse them 3 times with PBS, 5 minutes each time. Then permeabilize them with a PBS solution containing 0.2% Triton X-100 for 15 - 20 minutes, and rinse them 3 times with PBS, 5 minutes each time. Add the NAD+ red fluorescent probe solution to the treated zebrafish larvae and incubate them at a constant temperature of 32 °C for 60 minutes. After the incubation, wash away the unbound probe with PBS.

[0043] Fluorescence detection and analysis: Observe the labeled zebrafish larvae under a fluorescence microscope and take pictures for recording. Use image analysis software (ImageJ) to quantitatively analyze the fluorescence intensity in the pictures. Select the same area for measurement in each sample, and take the average value as the fluorescence intensity value of the sample at the corresponding incubation time, which represents the NAD+ level in the zebrafish larvae.

[0044] Model control group of Comparative Example 3, 120 minutes Select 10 healthy adult wild-type (AB) zebrafish, and obtain fertilized embryos according to the conventional breeding method. Cultivate them at a water temperature of 28 ± 1 °C, pH 7.0 - 7.5, and keep the water quality clean until 6 hpf. Treat them in a culture solution containing H2O2 (500 μM) for 24 hours to induce the establishment of an aging model. The zebrafish larvae continue to be cultured in normal culture solution and are continuously treated until 72 hpf.

[0045] The zebrafish larvae were fixed with 4% paraformaldehyde for 2 - 4 hours, and then rinsed three times with PBS for 5 minutes each time. Subsequently, they were permeabilized with a PBS solution containing 0.2% Triton X-100 for 15 - 20 minutes, and then rinsed three times with PBS for 5 minutes each time. The treated zebrafish larvae were added to the NAD+ red fluorescent probe solution and incubated at a constant temperature of 32 °C for 120 minutes. After incubation, the unbound probe was washed away with PBS.

[0046] Fluorescence detection and analysis: The labeled zebrafish larvae were observed through a fluorescence microscope and photographed. The fluorescence intensity in the photos was quantitatively analyzed using image analysis software (ImageJ). The same area was selected for measurement in each sample, and the average value was taken as the fluorescence intensity value of the sample at the corresponding incubation time, representing the NAD+ level in the zebrafish larvae.

[0047] Data analysis: The data were analyzed using statistical software (SPSS). Repeated measures analysis of variance was performed on the fluorescence intensity of each group at different incubation times to observe the effect of incubation time on the fluorescence intensity. P < 0.05 was considered statistically significant.

[0048] Detection results: The relative fluorescence intensity of the sample treatment group in Example 1 after incubation for 30 minutes was 0.38, the relative fluorescence intensity of the sample treatment group in Example 2 after incubation for 60 minutes was 0.60, and the relative fluorescence intensity of the sample treatment group in Example 3 after incubation for 120 minutes was 0.71; the relative fluorescence intensity of the model control group in Comparative Example 1 after incubation for 30 minutes was 0.27, the relative fluorescence intensity of the model control group in Comparative Example 2 after incubation for 60 minutes was 0.33, and the relative fluorescence intensity of the model control group in Comparative Example 3 after incubation for 120 minutes was 0.38.

[0049] From the detection results, it can be seen that after treating zebrafish with nicotinamide ribose malate, as time went on, the staining fluorescence intensity in the zebrafish increased continuously, indicating that the NAD+ level in the zebrafish increased continuously after treatment with nicotinamide ribose malate, and the effect of delaying aging was obvious. Nicotinamide ribose malate has a significant anti-aging effect.

Claims

1. Use of nicotinamide ribose malate in anti-aging, characterized in that, Nicotinamide riboside malate is used for anti-aging.

2. Use of nicotinamide riboside malate according to claim 1 in anti-aging, characterized in that, Use of nicotinamide riboside malate in the preparation of health products for anti-aging.

3. Use of nicotinamide riboside malate according to claim 1 in anti-aging, characterized in that, Use of nicotinamide riboside malate in the preparation of skin care products for anti-aging.

4. Use of nicotinamide riboside malate according to claim 1 in anti-aging, characterized in that, Use of nicotinamide riboside malate in the preparation of drugs for anti-aging.

5. The application of nicotinamide riboside malate according to claim 1 in anti-aging, characterized in that, 10 μg / ml of nicotinamide riboside malate is used for anti-aging.

6. Use of nicotinamide riboside malate according to claim 5 in anti-aging, characterized in that, Use of 10 μg / ml of nicotinamide riboside malate in the preparation of skin care products for anti-aging.

7. Use of nicotinamide riboside malate according to claim 5 in anti-aging, characterized in that, Use of 10 μg / ml of nicotinamide riboside malate in the preparation of health products for anti-aging.

8. Use of nicotinamide riboside malate according to claim 5 in anti-aging, characterized in that, Use of 10 μg / ml of nicotinamide riboside malate in the preparation of drugs for anti-aging.

9. Use of nicotinamide riboside malate according to claim 1 in anti-aging, characterized in that, Nicotinamide riboside malate is used for anti-aging of zebrafish.

10. Use of nicotinamide riboside malate according to claim 9 in anti-aging, characterized in that, 10 μg / ml of nicotinamide riboside malate is used for anti-aging of zebrafish.

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