Application of nicotinamide ribose malate in light aging resistance

Abstract

The invention belongs to the technical field of biochemistry, and particularly relates to application of nicotinamide ribose malate in light aging resistance. The invention provides an application of nicotinamide ribose malate in light aging resistance, and particularly relates to an application of nicotinamide ribose malate in quantitative light aging resistance of human immortalized keratinocytes. According to the invention, 0.375 mg / mL to 1.50 mg / mL of nicotinamide ribose malate is used for resisting light aging of the skin, and the light aging resisting effect is improved by 12.13% to 35.09%. Particularly, 1.50 mg / mL of nicotinamide ribose malate is used for resisting light aging of human immortalized keratinocytes, the light aging resisting effect is improved by 35.09%, and the light aging resisting effect is obvious.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemistry, and in particular relates to the application of nicotinamide ribosylmalate in anti-photoaging. Background Art

[0002] Nicotinamide riboside malate, also known as Nicotinamide Riboside Malate, or NRHM for short. 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 easy 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, thereby helping to optimize the production process and improve production efficiency. It can be used as a pharmaceutical ingredient to increase the concentration of NAD+ in tissues, induce insulin sensitivity, and enhance the function of deacetylase, and it can also be used as a high-quality raw material for health products to improve product stability and bioavailability.

[0003] UV rays enter the skin to varying degrees and interact with skin cells. Among ultraviolet rays, UVA (320-400nm) can penetrate the epidermis to reach the dermis, mainly causing photoaging of skin cells. UVA radiation acts on melanocytes and other skin cells, such as keratinocytes, causing DNA damage through oxidative stress and the production of reactive oxygen species (ROS), which activate signaling pathways related to cell and tissue growth, differentiation, aging, and photoaging. In addition, ROS produced in UVA-irradiated human skin cells are the main cause of photoaging. UV exposure can also induce the expression of ROS production, leading to skin inflammation and downregulation of SIRT1.

[0004] According to public information, nicotinamide riboside is a precursor of NAD+, which can be absorbed by the skin through oral or topical forms, thereby increasing NAD+ levels. NAD+ plays a key role in cellular energy metabolism and repair, and can promote cell repair and energy production, thereby helping to resist the effects of photoaging. So far, there have been no reports on the anti-photoaging effects of nicotinamide riboside malate in the skin, let alone the quantitative anti-photoaging application of nicotinamide riboside malate in human immortalized keratinocytes. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides the use of nicotinamide riboside malate in anti-photoaging, and nicotinamide riboside is used for skin anti-photoaging.

[0006] As a preferred technical solution, the use of nicotinamide riboside in the preparation of skin care products for anti-photoaging.

[0007] As a preferred technical solution, nicotinamide riboside malate is used for skin anti-photoaging.

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

[0009] As a preferred technical solution, nicotinamide riboside malate with a concentration of 0.375 mg / mL - 1.50 mg / mL is used for skin anti-photoaging.

[0010] As a preferred technical solution, nicotinamide riboside malate with a concentration of 1.50 mg / mL is used for skin anti-photoaging.

[0011] As a preferred technical solution, the use of nicotinamide riboside malate with a concentration of 1.50 mg / mL in the preparation of skin care products for anti-photoaging.

[0012] As a preferred technical solution, nicotinamide riboside malate is used for anti-photoaging of human immortalized keratinocytes.

[0013] As a preferred technical solution, nicotinamide riboside malate with a concentration of 0.375 mg / mL - 1.50 mg / mL is used for anti-photoaging of human immortalized keratinocytes.

[0014] As a preferred technical solution, nicotinamide riboside malate with a concentration of 1.50 mg / mL is used for anti-photoaging of human immortalized keratinocytes.

[0015] Nicotinamide riboside malate at a concentration of 0.375 mg / mL - 1.50 mg / mL up-regulates the relative expression level of SIRT1 gene, and the promotion rate of gene relative expression level is 12.13% - 35.09%, showing an anti-photoaging effect. Nicotinamide riboside malate at a concentration of 1.50 mg / mL significantly up-regulates the relative expression level of SIRT1 gene, showing an anti-photoaging effect. Beneficial effects

[0016] 1. The present invention first uses nicotinamide riboside malate for skin anti-photoaging.

[0017] 2. The present invention uses nicotinamide riboside malate with a concentration of 0.375 mg / mL - 1.50 mg / mL for skin anti-photoaging.

[0018] 3. The present invention uses nicotinamide riboside malate with a concentration of 0.375 mg / mL - 1.50 mg / mL for anti-photoaging of human immortalized keratinocytes, and the anti-photoaging effect is improved by 12.13% - 35.09%.

[0019] 4. The nicotinamide riboside malate at 1.50 mg / mL of the present invention is used for anti-photoaging of human immortalized keratinocytes, and the anti-photoaging effect is improved by 35.09%, and its anti-photoaging effect is obvious. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Graph of the results of detecting the cell viability of the test samples Figure 2 Column graph of the results of relative gene expression Figure 3 Cell detection graph of sample group 1 Figure 4 Cell detection graph of sample group 2 Figure 5 Cell detection graph of sample group 3 Figure 6 Cell detection graph of the blank control group Figure 7 Cell detection graph of the model control group DETAILED DESCRIPTION OF THE INVENTION

[0021] In order 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 restrictive.

[0022] 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 implementation of the present invention, the preferred methods and materials are now described.

[0023] I. Main experimental reagents Nicotinamide riboside malate (BONTAC, BT23N1241.001) Human immortalized keratinocytes (HaCaT) DMEM medium (LOT: 2537078) Fetal bovine serum (LOT: BC20230111) Trypsin (LOT: 2523119) PBS buffer (LOT: 2309011) TRIzol reagent (LOT: 041223230824).

[0024] II. Main experimental instruments Biological safety cabinet (SafeFast Classic 212 A) Carbon Dioxide Incubator (SFEGROW 188 PRO) Inverted Phase Contrast Microscope (MI52-N) Microplate Reader (SpectraMax 340PC384) Analytical Balance (FA2204N) Flow Cytometer (JIMBIO iCytal S1) Cell Phototoxicity Irradiator (LUYOR-3450) Quantitative PCR Instrument (ABI PRISM, 7500 Sequence Detection System).

[0025] Test Example 1: Zero Adjustment Group Add 100 μL of cell culture medium to a 96-well cell culture plate, and place the 96-well plate in a carbon dioxide incubator at 37 °C and 5% CO 2 , and culture for 24 hours in an environment with a humidity > 90%. Discard the supernatant, add medium containing MTT to each well, and incubate in a carbon dioxide incubator at 37 °C and 5% CO 2 , and incubate for 3 hours in an environment with a humidity > 90%. After incubation, discard the supernatant, add 100 μL of dimethyl sulfoxide (DMSO) to each well, and measure the absorbance at a wavelength of 570 nm using a microplate reader.

[0026] Test Example 2: Blank Control Group Inoculate the cell suspension at 100 μL / well into a 96-well cell culture plate, and place it in a carbon dioxide incubator at 37 °C and 5% CO 2 , and culture for 18 - 24 hours in an environment with a humidity > 90%. Add 100 μL of cell culture medium to each well, and place the 96-well plate in a carbon dioxide incubator at 37 °C and 5% CO 2 , and culture for 24 hours in an environment with a humidity > 90%. Discard the supernatant, add medium containing MTT to each well, and incubate in a carbon dioxide incubator at 37 °C and 5% CO 2 , and incubate for 3 hours in an environment with a humidity > 90%. After incubation, discard the supernatant, add 100 μL of dimethyl sulfoxide (DMSO) to each well, and measure the absorbance at a wavelength of 570 nm using a microplate reader.

[0027] Test Example 3: Sample Group 1 Inoculate the cell suspension at 100 μL / well into a 96-well cell culture plate, and place it in a carbon dioxide incubator at 37 °C and 5% CO 2 , and culture for 18 - 24 hours in an environment with a humidity > 90%. Add 100 μL of cell culture medium containing 5.000 mg / mL nicotinamide ribose malate to each well, and place the 96-well plate in a carbon dioxide incubator at 37 °C and 5% CO 2, culture for 24 hours in an environment with humidity > 90%. Discard the supernatant, add medium containing MTT to each well, and place it in a carbon dioxide incubator at 37 °C with 5% CO 2 , incubate for 3 hours in an environment with humidity > 90%. After incubation, discard the supernatant, add 100 μl of dimethyl sulfoxide (DMSO) to each well, and measure the absorbance at a wavelength of 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0028] Test Example 4, Sample Group 2 Inoculate the cell suspension at 100 μl / well into a 96-well cell culture plate, and place it in a carbon dioxide incubator at 37 °C with 5% CO 2 , culture for 18 - 24 hours in an environment with humidity > 90%. Add 100 μl of cell culture medium containing 1.582 mg / mL of nicotinamide riboside malate to each well, and place the 96-well plate in a carbon dioxide incubator at 37 °C with 5% CO 2 , culture for 24 hours in an environment with humidity > 90%. Discard the supernatant, add medium containing MTT to each well, and place it in a carbon dioxide incubator at 37 °C with 5% CO 2 , incubate for 3 hours in an environment with humidity > 90%. After incubation, discard the supernatant, add 100 μl of dimethyl sulfoxide (DMSO) to each well, and measure the absorbance at a wavelength of 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0029] Test Example 5, Sample Group 3 Inoculate the cell suspension at 100 μl / well into a 96-well cell culture plate, and place it in a carbon dioxide incubator at 37 °C with 5% CO 2 , culture for 18 - 24 hours in an environment with humidity > 90%. Add 100 μl of cell culture medium containing 0.501 mg / mL of nicotinamide riboside malate to each well, and place the 96-well plate in a carbon dioxide incubator at 37 °C with 5% CO 2 , culture for 24 hours in an environment with humidity > 90%. Discard the supernatant, add medium containing MTT to each well, and place it in a carbon dioxide incubator at 37 °C with 5% CO 2 , incubate for 3 hours in an environment with humidity > 90%. After incubation, discard the supernatant, add 100 μl of dimethyl sulfoxide (DMSO) to each well, and measure the absorbance at a wavelength of 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0030] Test Example 6, Sample Group 4 Inoculate the cell suspension at 100 μl / well into a 96-well cell culture plate, and place it in a carbon dioxide incubator at 37 °C with 5% CO 2 , culture for 18 - 24 hours in an environment with humidity > 90%. Add 100 μl of cell culture medium containing 0.158 mg / mL of nicotinamide riboside malate to each well, and place the 96-well plate in a carbon dioxide incubator at 37 °C with 5% CO 2, incubate for 24 hours in an environment with humidity > 90%. Discard the supernatant, add medium containing MTT to each well, and place it in a CO₂ incubator at 37°C with 5% CO 2 , incubate for 3 hours in an environment with humidity > 90%. After incubation, discard the supernatant, add 100 μl of dimethyl sulfoxide (DMSO) to each well, and measure the absorbance at a wavelength of 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0031] Test Example 7, Sample Group 5 Inoculate the cell suspension at 100 μl / well into a 96-well cell culture plate, and place it in a CO₂ incubator at 37°C with 5% CO 2 , incubate for 18 - 24 hours in an environment with humidity > 90%. Add 100 μl of cell culture medium containing 0.050 mg / mL of nicotinamide riboside malate to each well, and place the 96-well plate in a CO₂ incubator at 37°C with 5% CO 2 , incubate for 24 hours in an environment with humidity > 90%. Discard the supernatant, add medium containing MTT to each well, and place it in a CO₂ incubator at 37°C with 5% CO 2 , incubate for 3 hours in an environment with humidity > 90%. After incubation, discard the supernatant, add 100 μl of dimethyl sulfoxide (DMSO) to each well, and measure the absorbance at a wavelength of 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0032] Test Example 8, Sample Group 6 Inoculate the cell suspension at 100 μl / well into a 96-well cell culture plate, and place it in a CO₂ incubator at 37°C with 5% CO 2 , incubate for 18 - 24 hours in an environment with humidity > 90%. Add 100 μl of cell culture medium containing 0.016 mg / mL of nicotinamide riboside malate to each well, and place the 96-well plate in a CO₂ incubator at 37°C with 5% CO 2 , incubate for 24 hours in an environment with humidity > 90%. Discard the supernatant, add medium containing MTT to each well, and place it in a CO₂ incubator at 37°C with 5% CO 2 , incubate for 3 hours in an environment with humidity > 90%. After incubation, discard the supernatant, add 100 μl of dimethyl sulfoxide (DMSO) to each well, and measure the absorbance at a wavelength of 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0033] Test Example 9, Sample Group 7 Inoculate the cell suspension at 100 μl / well into a 96-well cell culture plate, and place it in a CO₂ incubator at 37°C with 5% CO 2 , incubate for 18 - 24 hours in an environment with humidity > 90%. Add 100 μl of cell culture medium containing 0.005 mg / mL of nicotinamide riboside malate to each well, and place the 96-well plate in a CO₂ incubator at 37°C with 5% CO 2, incubate for 24 hours in an environment with humidity > 90%. Discard the supernatant, add medium containing MTT to each well, and place it in a carbon dioxide incubator at 37 °C, 5% CO 2 , incubate for 3 hours in an environment with humidity > 90%. After incubation, discard the supernatant, add 100 μl of dimethyl sulfoxide (DMSO) to each well, and measure the absorbance at a wavelength of 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0034] Test Example 10, Sample Group 8 Inoculate the cell suspension at 100 μl / well into a 96-well cell culture plate, and place it in a carbon dioxide incubator at 37 °C, 5% CO 2 , incubate for 18 - 24 hours in an environment with humidity > 90%. Add 100 μl of cell culture medium containing 0.002 mg / mL of nicotinamide ribose malate to each well, and place the 96-well plate in a carbon dioxide incubator at 37 °C, 5% CO 2 , incubate for 24 hours in an environment with humidity > 90%. Discard the supernatant, add medium containing MTT to each well, and place it in a carbon dioxide incubator at 37 °C, 5% CO 2 , incubate for 3 hours in an environment with humidity > 90%. After incubation, discard the supernatant, add 100 μl of dimethyl sulfoxide (DMSO) to each well, and measure the absorbance at a wavelength of 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0035] Calculation of cell viability: Cell activity (%) = (OD of test example sample group - OD of test example zero adjustment group) ÷ (OD of test example blank control group - OD of test example zero adjustment group) × 100% Experimental results, cell viability, and MTT test results are shown in the following table.

[0036]

[0037] Example 1, Sample Group 1 Inoculate the cell suspension at 2 mL / well into a 6-well cell culture plate, and place it in a carbon dioxide incubator at 37 °C, 5% CO 2 , incubate for 18 - 24 hours in an environment with humidity > 90%. Add 2 mL of medium containing 0.375 mg / mL of nicotinamide ribose malate, and expose the cells for 24 hours. After completion, wash the well plate with PBS and perform UVA irradiation treatment. After irradiation, add 2 mL of medium containing 0.375 mg / mL of nicotinamide ribose malate and incubate for another 4 hours. Wash twice with 1 mL of PBS. After washing, add 1 mL of Trizol reagent to each well and pipette to fully lyse the cells. Extract RNA, reverse transcribe it to cDNA, and then perform fluorescence quantitative PCR detection.

[0038] Example 2, Sample Group 2 Inoculate the cell suspension into a 6-well cell culture plate at 2 mL / well and place it in a carbon dioxide incubator at 37 °C and 5% CO 2 , and culture it for 18 - 24 hours in an environment with a humidity > 90%. Add 2 mL of a medium containing 0.75 mg / mL nicotinamide ribose malate, and expose the cells for 24 hours. After completion, wash the well plate with PBS and perform UVA irradiation treatment. After the irradiation is completed, add 2 mL of a medium containing 0.75 mg / mL nicotinamide ribose malate and incubate for another 4 hours. Wash twice with 1 mL of PBS. After the washing is completed, add 1 mL of Trizol reagent to each well and pipette to fully lyse the cells. Extract RNA, reverse transcribe it into cDNA, and then perform fluorescence quantitative PCR detection.

[0039] Example 3 Sample Group 3 Inoculate the cell suspension into a 6-well cell culture plate at 2 mL / well and place it in a carbon dioxide incubator at 37 °C and 5% CO 2 , and culture it for 18 - 24 hours in an environment with a humidity > 90%. Add 2 mL of a medium containing 1.50 mg / mL nicotinamide ribose malate, and expose the cells for 24 hours. After completion, wash the well plate with PBS and perform UVA irradiation treatment. After the irradiation is completed, add 2 mL of a medium containing 1.50 mg / mL nicotinamide ribose malate and incubate for another 4 hours. Wash twice with 1 mL of PBS. After the washing is completed, add 1 mL of Trizol reagent to each well and pipette to fully lyse the cells. Extract RNA, reverse transcribe it into cDNA, and then perform fluorescence quantitative PCR detection.

[0040] Control Group 1 Blank Control Group Inoculate the cell suspension into a 6-well cell culture plate at 2 mL / well and place it in a carbon dioxide incubator at 37 °C and 5% CO 2 , and culture it for 18 - 24 hours in an environment with a humidity > 90%. Add 2 mL of a medium, and expose the cells for 24 hours. After completion, wash the well plate with PBS. Add 2 mL of a medium and incubate for another 4 hours. Wash twice with 1 mL of PBS. After the washing is completed, add 1 mL of Trizol reagent to each well and pipette to fully lyse the cells. Extract RNA, reverse transcribe it into cDNA, and then perform fluorescence quantitative PCR detection.

[0041] Control Group 2 Model Control Group Inoculate the cell suspension into a 6-well cell culture plate at 2 mL / well and place it in a carbon dioxide incubator at 37 °C and 5% CO 2, culture for 18 - 24 hours in an environment with humidity > 90%. Add 2 mL of culture medium and expose the cells for 24 hours. After completion, wash the well plate with PBS and perform UVA irradiation treatment. After the irradiation ends, add 2 mL of culture medium and incubate for another 4 hours. Wash twice with 1 mL of PBS. After the washing is completed, add 1 mL of Trizol reagent to each well and pipette to fully lyse the cells. Extract RNA, reverse transcribe it into cDNA, and then perform fluorescence quantitative PCR detection.

[0042] The experimental data are normalized to the blank control group, and statistical analysis is performed using the t - test method. If the SIRT1 gene is significantly down - regulated in the model control group compared to the blank control group, it indicates that the modeling is successful; if the SIRT1 gene is significantly up - regulated in the sample group compared to the model control group, it indicates that the sample has an anti - photoaging effect.

[0043]

[0044] The relative expression levels of the SIRT1 gene are shown in the following table.

[0045]

[0046] Experimental conclusion: Compared with the blank control group, the relative expression level of the SIRT1 gene in the model control group is significantly down - regulated, proving that the aging model is successfully established; compared with the model control group, at the concentration of 0.375 mg / mL - 1.50 mg / mL of nicotinamide riboside malate, the relative expression level of the SIRT1 gene is up - regulated, and the promotion rate of the relative gene expression level is 12.13% - 35.09%, showing an anti - photoaging effect. At the concentration of 1.50 mg / mL of nicotinamide riboside malate, the relative expression level of the SIRT1 gene is significantly up - regulated, showing an anti - photoaging effect.

Claims

1. The use of nicotinamide ribosylmalate in anti-photoaging, characterized in that: Nicotinamide riboside is used to protect the skin against photoaging.

2. The use of nicotinamide ribosylmalate in anti-photoaging according to claim 1, characterized in that: Use of nicotinamide riboside in preparing anti-photoaging skin care products.

3. The use of nicotinamide ribosylmalate in anti-photoaging according to claim 1, characterized in that: Nicotinamide ribosylmalate for skin anti-photoaging.

4. The use of nicotinamide ribosylmalate in anti-photoaging according to claim 3, characterized in that: Use of nicotinamide ribosylmalate in preparing anti-photoaging skin care products.

5. The use of nicotinamide ribosylmalate in anti-photoaging according to claim 1, characterized in that: Nicotinamide ribosylmalate at a concentration of 0.375 mg / mL-1.50 mg / mL is used for skin anti-photoaging.

6. The use of nicotinamide ribosylmalate in anti-photoaging according to claim 5, characterized in that: Nicotinamide ribosylmalate at a concentration of 1.50 mg / mL is used for skin anti-photoaging.

7. The use of nicotinamide ribosylmalate in anti-photoaging according to claim 6, characterized in that: Use of nicotinamide ribosylmalate at a concentration of 1.50 mg / mL in the preparation of anti-photoaging skin care products.

8. The use of nicotinamide ribosylmalate in anti-photoaging according to claim 1, characterized in that: Nicotinamide ribosylmalate for anti-photoaging of human immortalized keratinocytes.

9. The use of nicotinamide ribosylmalate in anti-photoaging according to claim 8, characterized in that: Nicotinamide ribosylmalate at a concentration of 0.375 mg / mL-1.50 mg / mL was used to protect human immortalized keratinocytes against photoaging.

10. The use of nicotinamide ribosylmalate in anti-photoaging according to claim 9, characterized in that: Nicotinamide ribosylmalate at a concentration of 1.50 mg / mL was used to protect human immortalized keratinocytes against photoaging.

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