Mitochondrial energy activated composite multi-effect composition and application thereof

Abstract

The invention discloses a mitochondrial energy activated composite multi-effect composition and application thereof. The composite multi-effect composition comprises nicotinamide, nicotinamide adenine dinucleotide and a lactobacillus fermentation lysate. According to the invention, the defect that nicotinamide and nicotinamide adenine dinucleotide cannot be combined in the prior art is overcome through the lactobacillus fermentation lysate which is added in a composite manner. Moreover, based on the addition of the lactobacillus fermentation lysate, the lactobacillus fermentation lysate, the lactobacillus fermentation lysate and the lactobacillus fermentation lysate show a better synergistic effect, and oxidation resistance can be generated at the same time; mitochondria is protected; promoting skin barrier generation or enhancing skin barrier defense; wrinkle resistance; therefore, the novel application of the combination of the nicotinamide and the nicotinamide adenine dinucleotide is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and particularly relates to a mitochondrial energy activation composite multi-effect composition and its application. Background Art

[0002] In the fields of skin anti-aging and barrier repair, the composition systems in the prior art often face various key defects, including: affected by the physical and chemical properties of the substances themselves, there are too few available substance selections. For example, niacinamide, as a NAD + precursor, although it can inhibit melanin transport, it is pointed out in the prior art that its actual transdermal efficiency is only 0.3%-1%, and once the use concentration is too high (such as >5%), it will also damage the stratum corneum barrier function. And nicotinamide adenine dinucleotide (NAD + ) has the ability to repair DNA, but its large molecular weight (663.43 daltons) also results in low skin permeability (difficult to penetrate the skin), and NAD + will be rapidly inactivated (inactivation rate >30% in 24 hours) in an environment with pH < 5, thus greatly affecting its availability in cosmetic compositions. For the lysate of Lactobacillus fermentation, it is rarely used in combination with other substances in the prior art, and currently, it has not been disclosed that it has a synergistic effect with nicotinamide substances. Moreover, in terms of composition, there may even be interference between components. Moreover, the safety of the fermentation product after compounding (skin irritation reaction may occur) is also a concern.

[0003] In the prior art, nicotinamide adenine dinucleotide (NAD + ) and nicotinamide are usually not used in combination because high-concentration nicotinamide and NAD + in combination may reversely inhibit the activity of nicotinamide phosphoribosyltransferase and reduce the conversion efficiency of NAD + , thus producing an antagonistic effect of components. Moreover, after adding the fermented product, since the fermented product may generally contain enzyme substances (such as protease), it will also accelerate the rapid decomposition of other components in the composition, thus affecting the overall effect of the composition. Therefore, how to obtain a composition containing nicotinamide adenine dinucleotide (NAD + ) and nicotinamide through reasonable compounding and enable it to simultaneously exert the synergistic anti-aging and barrier repair effects is a major problem in this field. Summary of the Invention

[0004] The present invention aims to solve at least one of the above-mentioned technical problems in the prior art. To this end, the object of the present invention is to provide a composite multi-effect composition and its application in cosmetics. The composition of the present invention contains niacinamide, nicotinamide adenine dinucleotide, and lysate of Lactobacillus fermentation. There is a significant synergistic effect among the three, which can effectively improve the anti-wrinkle, firming, and antioxidant functions of the skin, and can protect mitochondria or improve mitochondrial function, having extremely high commercial value.

[0005] In a first aspect of the present invention, there is provided a composition, which comprises: niacinamide, nicotinamide adenine dinucleotide, and lysate of Lactobacillus fermentation.

[0006] In some embodiments of the present invention, the composition consists of niacinamide, nicotinamide adenine dinucleotide, and lysate of Lactobacillus fermentation.

[0007] In some embodiments of the present invention, in the composition, the mass ratio of niacinamide, nicotinamide adenine dinucleotide, and lysate of Lactobacillus fermentation is 0.005-0.2:0.0005-0.02:0.005-0.2.

[0008] In some embodiments of the present invention, in the composition, the mass ratio of niacinamide, nicotinamide adenine dinucleotide, and lysate of Lactobacillus fermentation is 0.005-0.15:0.0005-0.02:0.005-0.1.

[0009] In some embodiments of the present invention, in the composition, the mass ratio of niacinamide, nicotinamide adenine dinucleotide, and lysate of Lactobacillus fermentation is 0.009:0.0008:0.006.

[0010] In some embodiments of the present invention, in the composition, the mass ratio of niacinamide, nicotinamide adenine dinucleotide, and lysate of Lactobacillus fermentation is 0.15:0.0125:0.1.

[0011] In some embodiments of the present invention, in the composition, the mass ratio of niacinamide, nicotinamide adenine dinucleotide, and lysate of Lactobacillus fermentation is 0.06:0.005:0.04.

[0012] In some embodiments of the present invention, the composition further comprises excipients acceptable in cosmetics.

[0013] In some embodiments of the present invention, the excipients acceptable in cosmetics include but are not limited to: solvents, emulsifiers, stabilizers, thickeners, preservatives, fragrances, pigments, fillers, and humectants.

[0014] In some embodiments of the present invention, the preservatives include, but are not limited to, phenoxyethanol and parabens.

[0015] In some embodiments of the present invention, the humectants include, but are not limited to, glycerol and hyaluronic acid;

[0016] In some embodiments of the present invention, the excipients acceptable in the cosmetics further include antioxidants such as vitamin E and butylated hydroxytoluene; chelating agents such as sodium ethylenediaminetetraacetate; surfactants such as sodium lauryl sulfate and cocamidopropyl betaine; and pH regulators such as citric acid and sodium lactate.

[0017] In some embodiments of the present invention, the composition further includes other active ingredients for cosmetics.

[0018] In some embodiments of the present invention, the active ingredients for cosmetics refer to substances having at least one of the following functions (1)-(4):

[0019] (1) Anti-wrinkle;

[0020] (2) Skin tightening;

[0021] (3) Antioxidation;

[0022] (4) Protecting mitochondria or improving mitochondrial function.

[0023] The second aspect of the present invention provides a method for preparing the composition described in the above aspect, including the following steps:

[0024] Mix nicotinamide, nicotinamide adenine dinucleotide and the lysate of Lactobacillus fermentum according to the mass ratio described in the above aspect to obtain the composition.

[0025] The third aspect of the present invention provides a cosmetic, which contains the composition described in the above aspect.

[0026] In some embodiments of the present invention, in the cosmetic, calculated based on the total mass of the cosmetic, the proportion of the composition is 0.6-5%.

[0027] In some embodiments of the present invention, the cosmetic may further include excipients acceptable in the cosmetic.

[0028] In some embodiments of the present invention, the excipients acceptable in the cosmetic include, but are not limited to: solvents, emulsifiers, stabilizers, thickeners, preservatives, fragrances, pigments, fillers and humectants.

[0029] In some embodiments of the present invention, the preservatives include, but are not limited to, phenoxyethanol and parabens.

[0030] In some embodiments of the present invention, the humectant includes, but is not limited to, glycerol and hyaluronic acid;

[0031] In some embodiments of the present invention, the excipients acceptable in the cosmetics further include antioxidants such as vitamin E and butylated hydroxytoluene; chelating agents such as sodium ethylenediaminetetraacetate; surfactants such as sodium lauryl sulfate and cocamidopropyl betaine; and pH regulators such as citric acid and sodium lactate.

[0032] The fourth aspect of the present invention provides an application of the composition described in the above aspects in the preparation of cosmetics or pharmaceuticals.

[0033] In some embodiments of the present invention, the cosmetics or pharmaceuticals have at least one of the following functions (1)-(4):

[0034] (1) Anti-wrinkle;

[0035] (2) Skin tightening;

[0036] (3) Antioxidation;

[0037] (4) Protect mitochondria.

[0038] In some embodiments of the present invention, in the cosmetics or pharmaceuticals, by total mass, the proportion of the composition is 0.6-5%%.

[0039] In some embodiments of the present invention, the cosmetics or pharmaceuticals are topical preparations.

[0040] In some embodiments of the present invention, the dosage forms of the cosmetics include: emulsions, aqueous solutions, oils, gels, and powders.

[0041] In some embodiments of the present invention, the dosage forms of the pharmaceuticals include: ointments, aerosols, and patches.

[0042] The beneficial effects of the present invention are:

[0043] The present invention provides a composite multi-effect composition and its application in cosmetics. Its components include nicotinamide and nicotinamide adenine dinucleotide, and the defect that the two cannot be used in combination in the prior art is overcome by the composite addition of the lysate of Lactobacillus fermentation. Moreover, due to the addition of the lysate of Lactobacillus fermentation, the three show better synergistic effects, and can simultaneously produce antioxidant effects; protect mitochondria; promote the generation of skin barrier or enhance skin barrier defense; anti-wrinkle; skin tightening and other multiple effects, realizing a new application of the combination of nicotinamide and nicotinamide adenine dinucleotide. Description of the Drawings

[0044] Figure 1ATP content after treatment of different experimental groups.

[0045] Figure 2 Red / green average fluorescence intensity ratio of mitochondrial membrane potential after treatment of different experimental groups.

[0046] Figure 3 For NAD + / NADH ratio.

[0047] Figure 4 Relative expression level of PGC-1α gene after treatment of different experimental groups.

[0048] Figure 5 Relative expression level of FOXO1 gene after treatment of different experimental groups.

[0049] Figure 6 Content of type I collagen after treatment of different experimental groups.

[0050] Figure 7 Elastin gene expression level after treatment of different experimental groups.

[0051] Figure 8 SIRT3 gene expression level after treatment of different experimental groups. Detailed implementation manners

[0052] The content of the present invention will be further described in detail below through specific examples. The raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial channels or by existing technical methods without special instructions. Unless otherwise specified, the test or measurement methods are conventional methods in the art.

[0053] In the following examples, nicotinamide, nicotinamide adenine dinucleotide, and Lactobacillus ferment lysate are all commercially available products. Of course, those skilled in the art can also prepare the above components based on the preparation methods disclosed in the prior art, which are also included in the protection scope of the present invention.

[0054] Example 1

[0055] A composite multi-effect composition is provided in this example, and its composition is as follows:

[0056] By mass, 0.009 parts of nicotinamide, 0.0008 parts of nicotinamide adenine dinucleotide, and 0.006 parts of Lactobacillus ferment lysate.

[0057] The preparation method is as follows:

[0058] According to the above mass ratio, mix nicotinamide, nicotinamide adenine dinucleotide, and Lactobacillus ferment lysate evenly to obtain a cosmetic composition with multiple effects.

[0059] Example 2

[0060] In this example, a composite multi-effect composition is provided, and its composition components are as follows:

[0061] By mass, 0.15 parts of nicotinamide, 0.0125 parts of nicotinamide adenine dinucleotide, and 0.1 part of Lactobacillus ferment lysate.

[0062] The preparation method is as follows:

[0063] According to the above mass ratio, mix nicotinamide, nicotinamide adenine dinucleotide, and Lactobacillus ferment lysate evenly to obtain a cosmetic composition with multiple effects.

[0064] Example 3

[0065] In this example, a composite multi-effect composition is provided, and its composition components are as follows:

[0066] By mass, 0.06 parts of nicotinamide, 0.005 parts of nicotinamide adenine dinucleotide, and 0.04 parts of Lactobacillus ferment lysate. The preparation method is as follows:

[0067] According to the above mass ratio, mix nicotinamide, nicotinamide adenine dinucleotide, and Lactobacillus ferment lysate evenly to obtain a cosmetic composition with multiple effects.

[0068] Test Example 1

[0069] In this test example, the energy boosting effects of Example 1, single-component substances with corresponding contents, and two-component substances were tested. The specific experimental steps are as follows:

[0070] After resuscitating human immortalized epidermal cells (HaCaT), conduct routine culture. When the cell plating rate reaches about 60%, inoculate the cells into a 6-well plate and incubate overnight at 37°C and 5% CO2.

[0071] When the cell plating rate of the 6-well plate reaches about 60%, conduct grouped drug administration. Conduct experimental grouping: Divide the plated cells into a normal control group (NC) and a sample group. Among them, the normal control group (NC) is not treated with anything and continues to be cultured with fresh medium. The sample group is respectively added with a medium containing the multi-component composite cosmetic composition in Example 1, nicotinamide with the same content, nicotinamide adenine dinucleotide with the same content, Lactobacillus ferment lysate with the same content, nicotinamide + Lactobacillus ferment lysate with the same content, and nicotinamide + nicotinamide adenine dinucleotide with the same content, and continue to incubate at 37°C and 5% CO2 for 24 h.

[0072] Among them, the addition amounts of various substances are as follows: based on the mass of the culture medium, the proportions of the corresponding substances are as follows:

[0073] NAM + NAD + LF group (i.e., Example 1): 0.009% nicotinamide, 0.0008% nicotinamide adenine dinucleotide, 0.006% lysate of Lactobacillus fermentation;

[0074] NAM + LF group: 0.009% nicotinamide, 0.006% lysate of Lactobacillus fermentation;

[0075] NAM + NAD group: 0.009% nicotinamide, 0.0008% nicotinamide adenine dinucleotide;

[0076] NAM group: 0.009% nicotinamide; NAD group: 0.0008% nicotinamide adenine dinucleotide; LF group: 0.006% lysate of Lactobacillus fermentation.

[0077] After the incubation, the culture medium was changed. The normal control group (NC) was not treated with anything and continued to be cultured with fresh culture medium for 24 h. The sample groups were respectively added with culture media containing the multi-component composite cosmetic composition in Example 1, equal amounts of nicotinamide, equal amounts of nicotinamide adenine dinucleotide, equal amounts of lysate of Lactobacillus fermentation, equal amounts of nicotinamide + lysate of Lactobacillus fermentation, and equal amounts of nicotinamide + nicotinamide adenine dinucleotide, and continued to be incubated at 37 °C and 5% CO2 for 24 h.

[0078] After the incubation, a commercially available kit was used to detect the ATP content.

[0079] The results are shown in Table 1 and Figure 1 as follows.

[0080] Table 1 Energy enhancement results of each group

[0081]

[0082]

[0083] Among them, compared with NC, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; three biological replicates (N = 3).

[0084] ATP (adenosine triphosphate) is a universal molecule that directly provides energy within cells and plays a central role in energy metabolism and the regulation of physiological functions. When the body's energy demand increases, ATP releases high-energy phosphate bonds through hydrolysis reactions to provide immediate energy support for the body. Research shows that the synthesis rate and reserve level of ATP are directly related to cell viability and the anti-fatigue ability of tissues. By detecting the ATP content, the energy metabolism efficiency of an individual can be evaluated, providing a basis for energy enhancement strategies.

[0085] As can be seen from the above results, compared with the control group (NC), the composition (NAM + NAD + LF) increased the ATP content more significantly than the single-component or two-component groups of NAM, NAD, and LF, indicating that the combination of NAM + NAD + LF produced a synergistic effect that was not possessed by the single use of NAM, NAD, or LF.

[0086] Related tests were also conducted on other embodiments, and it was found that their effects were basically similar to those of the composition in Example 1.

[0087] Test Example 2

[0088] In this test example, the effects of Example 2 and the corresponding single-component substances and two-component substances on improving mitochondrial viability were tested. The specific experimental steps were as follows:

[0089] After resuscitating human dermal fibroblasts, they were cultured routinely. When the cell plating rate reached about 60%, the cells were seeded into 6-well plates and incubated overnight at 37°C and 5% CO2.

[0090] When the cell plating rate of the 6-well plates reached about 60%, grouping and drug administration were carried out. The experimental groups were divided as follows: the seeded cells were divided into a normal control group (NC), a model control group (MC), and a sample group. Among them, the normal control group (NC) and the model control group (MC) were not treated with anything and were continued to be cultured with fresh medium. The sample group was respectively added with a medium containing the multi-component composite cosmetic composition in Example 2, equal amounts of nicotinamide, equal amounts of nicotinamide adenine dinucleotide, equal amounts of Lactobacillus ferment lysate, equal amounts of nicotinamide + Lactobacillus ferment lysate, and equal amounts of nicotinamide + nicotinamide adenine dinucleotide, and continued to be incubated at 37°C and 5% CO2 for 24 h.

[0091] Among them, the addition amounts of each substance were as follows: based on the mass of the medium, the proportion of the corresponding substance was:

[0092] NAM + NAD + LF group (i.e., Example 2): 0.15% nicotinamide, 0.0125% nicotinamide adenine dinucleotide, 0.1% Lactobacillus ferment lysate;

[0093] NAM+LF group: 0.15% niacinamide, 0.1% lysate of Lactobacillus fermentum;

[0094] NAM+NAD group: 0.15% niacinamide, 0.0125% nicotinamide adenine dinucleotide;

[0095] NAM group: 0.15% niacinamide; NAD group: 0.0125% nicotinamide adenine dinucleotide; LF group: 0.1% lysate of Lactobacillus fermentum.

[0096] After incubation, discard the culture medium and gently rinse the cells 1-2 times with D-Hanks balanced salt solution (D-HBSS). Then expose the model control group (MC) and the sample groups to ultraviolet light for 50 s (UVA, 9 mJ / cm 2 ).

[0097] After modeling, the normal control group (NC) and the model control group (MC) were not treated with anything and continued to be cultured with fresh culture medium for 24 h. The sample groups were respectively added with the culture media containing the multi-component composite cosmetic composition in Example 2, equal contents of niacinamide, equal contents of nicotinamide adenine dinucleotide, equal contents of lysate of Lactobacillus fermentum, equal contents of niacinamide + lysate of Lactobacillus fermentum, and equal contents of niacinamide + nicotinamide adenine dinucleotide, and continued to be incubated at 37 °C and 5% CO2 for 24 h.

[0098] After incubation, JC-1 staining was performed and observed and photographed under a fluorescence microscope.

[0099] Use Image J to analyze the fluorescence intensity (S) of each group, calculate the ratio of the average red / green fluorescence intensity of the mitochondrial membrane potential, and calculate the improvement rate through the following formula.

[0100]

[0101] In the formula:

[0102] P represents the ratio of the average red / green fluorescence intensity of the mitochondrial membrane potential.

[0103] The results are shown in Table 2 and Figure 2 as follows.

[0104] Table 2 Results of improvement of mitochondrial viability in each group

[0105]

[0106]

[0107] Among them, compared with MC, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; three biological replicates (N = 3).

[0108] Mitochondrial membrane potential (MMP) is a core indicator of mitochondrial energy conversion efficiency. A decrease in MMP weakens ATP synthesis, triggers ROS accumulation and mitochondrial dysfunction; while an increase in MMP can enhance the activity of the electron transport chain, promote efficient energy production, reduce oxidative damage and restore mitochondrial dynamic balance, thereby directly enhancing mitochondrial vitality. Its level can be detected by fluorescence probes, providing a basis for evaluating mitochondrial health and intervening in energy metabolism problems.

[0109] As can be seen from the above results, compared with the control group (MC), the effect of the composition (NAM + NAD + LF) in enhancing mitochondrial membrane potential is more significant than that of the single-component or two-component of NAM, NAD, and LF, indicating that the combination of NAM + NAD + LF produces a synergistic effect that is not possessed by the single use of NAM, NAD, and LF.

[0110] Related tests were also carried out on other embodiments, and it was found that their effects were basically similar to those of the composition in Example 2.

[0111] Test Example 3

[0112] In this test example, the effects of Example 3 and the corresponding single-component substances and two-component substances in improving mitochondrial dysfunction were tested. The specific experimental steps are as follows:

[0113] After resuscitating human dermal fibroblasts, they were cultured routinely. When the cell plating rate reached about 60%, the cells were seeded in 6-well plates and incubated overnight at 37°C and 5% CO2.

[0114] When the cell plating rate of the 6-well plate reached about 60%, grouping and drug administration were carried out. The experimental groups were divided as follows: the plated cells were divided into a normal control group (NC), a model control group (MC), and a sample group. Among them, the normal control group (NC) and the model control group (MC) were not treated with anything and continued to be cultured with fresh medium. The sample group was respectively added with a medium containing the multi-component composite cosmetic composition in Example 3, equal amounts of nicotinamide, equal amounts of nicotinamide adenine dinucleotide, equal amounts of Lactobacillus ferment lysate, equal amounts of nicotinamide + Lactobacillus ferment lysate, and equal amounts of nicotinamide + nicotinamide adenine dinucleotide, and continued to be incubated at 37°C and 5% CO2 for 24 h.

[0115] Among them, the addition amount of each substance is: calculated by the mass of the medium, the proportion of the corresponding substance is:

[0116] NAM + NAD + LF group (i.e., Example 3): 0.06% niacinamide, 0.005% nicotinamide adenine dinucleotide, 0.04% lysate of Lactobacillus fermentation;

[0117] NAM + LF group: 0.06% niacinamide, 0.04% lysate of Lactobacillus fermentation;

[0118] NAM + NAD group: 0.06% niacinamide, 0.005% nicotinamide adenine dinucleotide;

[0119] NAM group: 0.06% niacinamide; NAD group: 0.005% nicotinamide adenine dinucleotide; LF group: 0.04% lysate of Lactobacillus fermentation.

[0120] After incubation, discard the medium and gently rinse the cells 1 - 2 times with D - Hanks balanced salt solution (D - HBSS). Then expose the model control group (MC) and the sample groups to ultraviolet light for 50 s (UVA, 9 mJ / cm 2 )

[0121] After model establishment, the normal control group (NC) and the model control group (MC) are not treated with anything and continue to be cultured with fresh medium for 24 h. The sample groups are respectively added with the above - mentioned media containing the multi - component composite cosmetic composition in Example 3, equal contents of niacinamide, equal contents of nicotinamide adenine dinucleotide, equal contents of lysate of Lactobacillus fermentation, equal contents of niacinamide + lysate of Lactobacillus fermentation, and equal contents of niacinamide + nicotinamide adenine dinucleotide, and continue to be incubated at 37 °C and 5% CO2 for 24 h.

[0122] After incubation, detect the contents of NAD + and NADH using a commercially available kit, and calculate the NAD + / NADH ratio.

[0123] The results are shown in Table 3 and Figure 3 as follows.

[0124] Table 3 Improvement effects of mitochondrial dysfunction in each group

[0125] Sample Group Test Result SD P - value Improvement Rate (vs MC) Normal Control (NC) 11.72 0.875 0.0012** 64％ Model Control (MC) 7.14 0.387 / / NAM 8.17 0.413 0.0340* 15％ NAD 9.25 0.559 0.0057** 30％ LF 7.69 0.398 0.1603 8％ NAM + LF 8.59 0.483 0.0153* 20％ NAM + NAD 9.00 0.533 0.0081** 26％ NAM + NAD + LF 9.90 0.589 0.0024** 39％

[0126] Among them, compared with MC, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; three biological replicates (N = 3).

[0127] The NAD + / NADH ratio affects the energy - generating ability of cells. In the cytoplasm, the conversion of glucose to pyruvate through glycolysis requires NAD+ In mitochondria, the TCA cycle reduces NAD + molecules to produce multiple NADH molecules, which are oxidized by Complex I of the electron transport chain (ETC) to generate ATP. A decrease in NAD + levels affects mitochondrial function, overall cellular health, and the development of age-related diseases. Therefore, it is possible to evaluate whether a sample has an effect on improving mitochondrial dysfunction by detecting the level of the NAD + / NADH ratio.

[0128] As can be seen from the above results, compared with the control group (MC), the composition (NAM + NAD + LF) has a more significant effect on increasing the NAD + / NADH ratio than the single-component or two-component substances of NAM, NAD, and LF, indicating that the combination of NAM + NAD + LF produces a synergistic effect that is not possessed by the single use of NAM, NAD, and LF.

[0129] Related tests were also carried out on other examples, and it was found that their effects were basically similar to those of the composition in Example 3.

[0130] Test Example 4

[0131] In this test example, the effects of Example 1 and the corresponding single-component substances and two-component substances on improving mitochondrial dysfunction were tested. The specific experimental steps were as follows:

[0132] After resuscitating human dermal fibroblasts, they were cultured routinely. When the cell plating rate reached about 60%, the cells were seeded in a 6-well plate and incubated overnight at 37°C and 5% CO2.

[0133] When the cell plating rate of the 6-well plate reached about 60%, grouping and drug administration were carried out. The experimental groups were divided as follows: the plated cells were divided into a normal control group (NC), a model control group (MC), and a sample group. Among them, the normal control group (NC) and the model control group (MC) were not treated with anything and were continuously cultured with fresh medium. The sample group was respectively added with a medium containing the multi-component composite cosmetic composition in Example 1, nicotinamide with the same content, nicotinamide adenine dinucleotide with the same content, the lysate of Lactobacillus fermentum with the same content, nicotinamide + the lysate of Lactobacillus fermentum with the same content, and nicotinamide + nicotinamide adenine dinucleotide with the same content, and incubated continuously at 37°C and 5% CO2 for 24 h.

[0134] Among them, the addition amount of each substance was as follows: based on the mass of the medium, the proportion of the corresponding substance was:

[0135] NAM + NAD+ LF group (i.e., Example 1): 0.009% niacinamide, 0.0008% nicotinamide adenine dinucleotide, 0.006% Lactobacillus fermentation lysate;

[0136] NAM + LF group: 0.009% niacinamide, 0.006% Lactobacillus fermentation lysate;

[0137] NAM + NAD group: 0.009% niacinamide, 0.0008% nicotinamide adenine dinucleotide;

[0138] NAM group: 0.009% niacinamide; NAD group: 0.0008% nicotinamide adenine dinucleotide; LF group: 0.006% Lactobacillus fermentation lysate.

[0139] After the incubation, discard the medium and gently rinse the cells 1 - 2 times with D - Hanks balanced salt solution (D - HBSS). Then expose the model control group (MC) and the sample groups to ultraviolet light for 50 s (UVA, 9 mJ / cm 2 )

[0140] After model establishment, the normal control group (NC) and the model control group (MC) are not treated with anything and continue to be cultured with fresh medium for 24 h. The sample groups are respectively added with the above - mentioned media containing the multi - component composite cosmetic composition in Example 1, equal amounts of niacinamide, equal amounts of nicotinamide adenine dinucleotide, equal amounts of Lactobacillus fermentation lysate, equal amounts of niacinamide + Lactobacillus fermentation lysate, and equal amounts of niacinamide + nicotinamide adenine dinucleotide, and continue to be incubated at 37 °C and 5% CO2 for 24 h.

[0141] After the incubation, extract the total RNA of each experimental group, reverse - transcribe it into cDNA, and use q - PCR to detect the gene expression levels of β - actin (internal reference) and PGC - 1α.

[0142] The results are shown in Table 4 and Figure 4 as follows.

[0143] Table 4 PGC - 1α expression levels in each group

[0144] Sample Group Test Result SD P - value Improvement Rate (vs MC) Normal Control (NC) 1.42 0.050 0.0203* 40％ Model Control (MC) 1.01 0.183 / / NAM 1.27 0.257 0.2310 25％ NAD 1.38 0.138 0.0498* 36％ LF 1.23 0.151 0.1902 21％ NAM + LF 1.44 0.119 0.0270* 42％ NAM + NAD 1.27 0.152 0.1316 26％ NAM + NAD + LF 1.72 0.222 0.0132* 70％

[0145] Among them, compared with MC, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; three biological replicates (N = 3).

[0146] PGC-1α improves mitochondrial dysfunction by regulating mitochondrial biogenesis and function. As a transcriptional co-activator, PGC-1α can promote the transcription of a series of genes. In mitochondria, PGC-1α can enhance the efficiency of ATP generation and reduce the accumulation of reactive oxygen species (ROS). A decrease in the level of PGC-1α leads to a reduction in the number of mitochondria and an imbalance in energy metabolism, which is closely related to metabolic syndrome, neurodegenerative diseases, and aging. Therefore, the improvement effect of a sample on mitochondrial dysfunction can be evaluated by detecting the expression level of PGC-1α.

[0147] As can be seen from the above results, compared with the control group (MC), the combination (NAM+NAD+LF) significantly increased the expression level of PGC-1α compared with the single-component or two-component groups of NAM, NAD, and LF, indicating that the combination of NAM+NAD+LF produced a synergistic effect that was not possessed by NAM, NAD, and LF used alone.

[0148] Related tests were also carried out on other examples, and it was found that their effects were basically similar to those of the composition in Example 1.

[0149] Test Example 5

[0150] In this test example, the effects of Example 2 and the corresponding single-component substances and two-component substances on improving mitochondrial dysfunction were tested. The specific experimental steps were as follows:

[0151] After resuscitating human dermal fibroblasts, they were cultured routinely. When the cell plating rate reached about 60%, the cells were seeded in 6-well plates and incubated overnight at 37°C and 5% CO2.

[0152] When the cell plating rate of the 6-well plates reached about 60%, grouping and drug administration were carried out. The experimental groups were divided as follows: the plated cells were divided into a normal control group (NC), a model control group (MC), and a sample group. Among them, the normal control group (NC) and the model control group (MC) were not treated with anything and were continued to be cultured with fresh medium. The sample group was added with a medium containing the multi-component compound cosmetic composition in Example 2, equal amounts of nicotinamide, equal amounts of nicotinamide adenine dinucleotide, equal amounts of Lactobacillus ferment lysate, equal amounts of nicotinamide + Lactobacillus ferment lysate, and equal amounts of nicotinamide + nicotinamide adenine dinucleotide, and continued to be incubated at 37°C and 5% CO2 for 24 h.

[0153] Among them, the addition amounts of each substance were as follows: based on the mass of the medium, the proportion of the corresponding substance was:

[0154] The NAM+NAD+LF group (i.e., Example 2): 0.15% nicotinamide, 0.0125% nicotinamide adenine dinucleotide, 0.1% Lactobacillus ferment lysate;

[0155] NAM + LF group: 0.15% niacinamide, 0.1% lysate of Lactobacillus fermentum;

[0156] NAM + NAD group: 0.15% niacinamide, 0.0125% nicotinamide adenine dinucleotide;

[0157] NAM group: 0.15% niacinamide; NAD group: 0.0125% nicotinamide adenine dinucleotide; LF group: 0.1% lysate of Lactobacillus fermentum.

[0158] After incubation, discard the culture medium, and gently rinse the cells 1 - 2 times with D-Hanks balanced salt solution (D-HBSS). Then expose the model control group (MC) and the sample groups to ultraviolet light for 50 s (UVA, 9 mJ / cm 2 )

[0159] After model establishment, the normal control group (NC) and the model control group (MC) are not treated with anything and continue to be cultured with fresh culture medium for 24 h. The sample groups are respectively added with the above-mentioned culture media containing the multi-component composite cosmetic composition in Example 2, equal amounts of niacinamide, equal amounts of nicotinamide adenine dinucleotide, equal amounts of lysate of Lactobacillus fermentum, equal amounts of niacinamide + lysate of Lactobacillus fermentum, and equal amounts of niacinamide + nicotinamide adenine dinucleotide, and continue to be incubated at 37 °C and 5% CO2 for 24 h.

[0160] After incubation, extract the total RNA of each experimental group, reverse transcribe it into cDNA, and use q-PCR to detect the gene expression of β-actin (internal reference) and FOXO1.

[0161] The results are shown in Table 5 and Figure 5 as follows.

[0162] Table 5 FOXO1 expression levels in each group

[0163] Sample Group Test Result SD P - value Improvement Rate (vs MC) Normal Control (NC) 1.44 0.044 0.0037** 43％ Model Control (MC) 1.00 0.114 / / NAM 1.30 0.076 0.0209* 29％ NAD 1.48 0.041 0.0024** 48％ LF 1.34 0.128 0.0273* 34％ NAM + LF 1.45 0.188 0.0244* 45％ NAM + NAD 1.38 0.170 0.0343* 37％ NAM + NAD + LF 1.68 0.040 0.0007*** 67％

[0164] Among them, compared with MC, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; three biological replicates (N = 3).

[0165] FOXO1 improves mitochondrial dysfunction by enhancing mitophagy and antioxidant capacity. FOXO1 activates mitophagy genes and antioxidant enzyme genes, promotes the clearance of damaged mitochondria and reduces ROS accumulation; at the same time, it synergistically enhances the expression of genes related to mitochondrial biogenesis with PGC-1α. Abnormal FOXO1 function can lead to the accumulation of mitochondrial damage, which is related to metabolic diseases and aging. Therefore, the effect of improving mitochondrial dysfunction can be evaluated by detecting FOXO1 expression.

[0166] As can be seen from the above results, compared with the control group (MC), the combination (NAM + NAD + LF) significantly increased the expression level of FOXO1 compared with the single component or binary combination of NAM, NAD, and LF, indicating that the combination of NAM + NAD + LF produced a synergistic effect that was not possessed by the single use of NAM, NAD, and LF.

[0167] Related tests were also carried out on other embodiments, and it was found that their effects were basically similar to those of the composition in Example 2.

[0168] Test Example 6

[0169] In this test example, the anti-wrinkle effects of Example 3 and the corresponding single-component substances and binary-component substances were tested. The specific experimental steps were as follows:

[0170] After resuscitating human dermal fibroblasts, they were cultured routinely. When the cell seeding rate reached about 60%, the cells were inoculated into 6-well plates and incubated overnight at 37°C and 5% CO2.

[0171] When the cell seeding rate of the 6-well plates reached about 60%, grouping and drug administration were carried out. The experimental groups were divided as follows: the cells after plating were divided into a normal control group (NC), a model control group (MC), and a sample group. Among them, the normal control group (NC) and the model control group (MC) were not treated with anything and continued to be cultured with fresh medium. The sample group was respectively added with a medium containing the multi-component composite cosmetic composition in Example 3, equal amounts of nicotinamide, equal amounts of nicotinamide adenine dinucleotide, equal amounts of Lactobacillus ferment lysate, equal amounts of nicotinamide + Lactobacillus ferment lysate, and equal amounts of nicotinamide + nicotinamide adenine dinucleotide, and continued to be incubated at 37°C and 5% CO2 for 24 h.

[0172] Among them, the addition amounts of each substance were as follows: based on the mass of the medium, the proportion of the corresponding substance was:

[0173] NAM + NAD + LF group (i.e., Example 3): 0.06% nicotinamide, 0.005% nicotinamide adenine dinucleotide, 0.04% Lactobacillus ferment lysate;

[0174] NAM + LF group: 0.06% niacinamide, 0.04% lysate of Lactobacillus ferment;

[0175] NAM + NAD group: 0.06% niacinamide, 0.005% nicotinamide adenine dinucleotide;

[0176] NAM group: 0.06% niacinamide; NAD group: 0.005% nicotinamide adenine dinucleotide; LF group: 0.04% lysate of Lactobacillus ferment.

[0177] After incubation, discard the medium and gently rinse the cells 1 - 2 times with D - Hanks balanced salt solution (D - HBSS). Then expose the model control group (MC) and the sample groups to ultraviolet light for 50 s (UVA, 30 mJ / cm 2 ).

[0178] After model establishment, the normal control group (NC) and the model control group (MC) are not treated with anything and continue to be cultured with fresh medium for 24 h. The sample groups are respectively added with the media containing the multi - component composite cosmetic composition in Example 3, equal contents of niacinamide, equal contents of nicotinamide adenine dinucleotide, equal contents of lysate of Lactobacillus ferment, equal contents of niacinamide + lysate of Lactobacillus ferment, and equal contents of niacinamide + nicotinamide adenine dinucleotide, and continue to be incubated at 37 °C and 5% CO2 for 24 h.

[0179] After incubation, fix the cells with 4% paraformaldehyde. After 30 min of fixation, perform immunofluorescence detection, take pictures and observe under a microscope, and collect and analyze the pictures.

[0180] At the same time, extract the total RNA of each experimental group, reverse - transcribe it into cDNA, and perform fluorescence quantitative PCR detection.

[0181] The results are shown in Table 6, Table 7 and Figure 6 , Figure 7 .

[0182] Table 6 Content of type I collagen in each group

[0183] Sample Group Test Result SD P - value Improvement Rate (vs MC) Normal Control (NC) 1.00 0.120 0.0018** 120％ Model Control (MC) 0.45 0.045 / / NAM 0.61 0.031 0.0082** 34％ NAD 0.63 0.033 0.0051** 40％ LF 0.63 0.035 0.0061** 39％ NAM + LF 0.66 0.040 0.0045** 45％ NAM + NAD 0.60 0.041 0.0151* 32％ NAM + NAD + LF 0.77 0.062 0.0021** 70％

[0184] Among them, compared with MC, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; three biological replicates (N = 3).

[0185] Table 7 Expression level of elastin gene in each group

[0186]

[0187]

[0188] Among them, compared with the NC group, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; three biological replicates (N = 3).

[0189] Type I collagen and elastin play an anti-wrinkle role by maintaining the structural integrity and elasticity of the skin. In the dermis, type I collagen forms a dense fiber network to provide mechanical support and inhibit skin collapse; at the same time, it stimulates fibroblasts to secrete more extracellular matrix components and strengthen the skin barrier. Elastin endows the skin with resilience through a cross-linked network and reduces permanent wrinkles caused by repeated stretching. The two work together to regulate the MMPs / TIMPs balance (such as inhibiting MMP-1 and activating TIMP-1) and delay collagen degradation. Loss of function will lead to broken collagen fibers and fragmented elastic fibers, causing skin relaxation and wrinkles. In this experiment, the anti-wrinkle effect was evaluated by detecting the content of type I collagen and the expression level of the elastin gene.

[0190] It can be seen from the above results that compared with the control group, the anti-wrinkle effect of the composition (NAM + NAD + LF) is more significant than that of the single-component or two-component of NAM, NAD, and LF, indicating that the combination of NAM + NAD + LF produces a synergistic effect that is not possessed by the single use of NAM, NAD, and LF.

[0191] Related tests were also carried out on other embodiments, and it was found that their effects were basically similar to those of the composition in Example 3.

[0192] Test Example 7

[0193] In this test example, the anti-aging effects of Example 3, the single-component substances with corresponding contents, and the two-component substances were tested. The specific experimental steps were as follows:

[0194] After resuscitating human fibroblasts, they were cultured routinely. When the cell plating rate reached about 60%, the cells were seeded into 6-well plates and incubated overnight at 37°C and 5% CO2.

[0195] When the cell plating rate of the 6-well plates reached about 60%, grouping and drug administration were carried out. The experimental groups were divided as follows: the cells after plating were divided into a normal control group (NC) and a sample group. Among them, the normal control group (NC) was not treated with anything and was continued to be cultured with fresh medium. The sample group was respectively added with media containing the multi-component composite cosmetic composition in Example 3, nicotinamide with the same content, nicotinamide adenine dinucleotide with the same content, the lysate of Lactobacillus fermentum with the same content, nicotinamide + the lysate of Lactobacillus fermentum with the same content, and nicotinamide + nicotinamide adenine dinucleotide with the same content, and continued to be incubated at 37°C and 5% CO2 for 24 h.

[0196] Among them, the addition amounts of various substances are as follows: based on the mass of the culture medium, the proportions of the corresponding substances are:

[0197] NAM + NAD + LF group (i.e., Example 3): 0.06% niacinamide, 0.005% nicotinamide adenine dinucleotide, 0.04% lysate of Lactobacillus fermentation;

[0198] NAM + LF group: 0.06% niacinamide, 0.04% lysate of Lactobacillus fermentation;

[0199] NAM + NAD group: 0.06% niacinamide, 0.005% nicotinamide adenine dinucleotide;

[0200] NAM group: 0.06% niacinamide; NAD group: 0.005% nicotinamide adenine dinucleotide; LF group: 0.04% lysate of Lactobacillus fermentation.

[0201] After the incubation is completed, the culture medium is replaced. The normal control group (NC) is not subjected to any treatment and is continued to be cultured with fresh culture medium for 24 h. The sample groups are respectively added with culture media containing the multi-component composite cosmetic composition in Example 3, equal contents of niacinamide, equal contents of nicotinamide adenine dinucleotide, equal contents of lysate of Lactobacillus fermentation, equal contents of niacinamide + lysate of Lactobacillus fermentation, and equal contents of niacinamide + nicotinamide adenine dinucleotide, and are continued to be incubated at 37 °C and 5% CO2 for 24 h.

[0202] After the incubation is completed, the total RNA of each experimental group is extracted, reverse transcribed into cDNA, and fluorescence quantitative PCR detection is carried out.

[0203] The results are shown in Table 8 and Figure 8 as follows.

[0204] Table 8 SIRT3 gene expression results of each group

[0205] Sample Group Test Result SD P - value Improvement Rate (vs NC) Normal Control (NC) 1.00 0.054 / / NAM 1.46 0.043 0.0003*** 45％ NAD 1.53 0.042 0.0002*** 52％ LF 1.30 0.064 0.0034** 30％ NAM + LF 1.51 0.042 0.0002*** 51％ NAM + NAD 1.40 0.048 0.0006*** 40％ NAM + NAD + LF 1.75 0.010 0.0000**** 75％

[0206] Among them, compared with NC, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; three biological replicates (N = 3).

[0207] SIRT3 is a NAD+-dependent mitochondrial enzyme that delays aging by enhancing mitochondrial function, scavenging reactive oxygen species, and promoting damaged mitochondrial autophagy. It regulates metabolic balance, inhibits inflammation, and enhances DNA repair, while improving cellular stress resistance by regulating histones and proteins related to mitochondrial biogenesis (such as PGC-1α). A decrease in its activity leads to mitochondrial damage, ROS accumulation, and an increase in aging markers, accelerating aging-related diseases. Therefore, the anti-aging effect of a sample can be detected by the expression results of the SIRT3 gene.

[0208] As can be seen from the above results, compared with the control group (NC), the combination (NAM+NAD+LF) significantly increased the expression of the SIRT3 gene compared to the single components or binary combinations of NAM, NAD, and LF, indicating that the combination of NAM+NAD+LF produced a synergistic effect that was not present when NAM, NAD, and LF were used alone.

[0209] Related tests were also conducted on other embodiments, and it was found that their effects were basically similar to those of the composition in Example 3.

[0210] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A composition, characterized in that, The composition comprises: niacinamide, nicotinamide adenine dinucleotide, and lysate of Lactobacillus fermentation.

2. The composition according to claim 1, wherein In the composition, the mass ratio of niacinamide, nicotinamide adenine dinucleotide, and lysate of Lactobacillus fermentation is 0.005 - 0.2:0.0005 - 0.02:0.005 - 0.2; Preferably, in the composition, the mass ratio of niacinamide, nicotinamide adenine dinucleotide, and lysate of Lactobacillus fermentation is 0.005 - 0.15:0.0005 - 0.02:0.005 - 0.

1.

3. The composition according to claim 1 or 2, characterized in that, The composition further comprises excipients acceptable in cosmetics; Preferably, the excipients acceptable in cosmetics include: solvents, emulsifiers, stabilizers, thickeners, preservatives, fragrances, pigments, fillers, and humectants.

4. The composition according to claim 3, wherein The composition further comprises other active ingredients for cosmetics; The active ingredients for cosmetics refer to substances having at least one of the following functions (1)-(4): (1) Anti-wrinkle; (2) Tightening the skin; (3) Antioxidant; (4) Protecting mitochondria or improving mitochondrial function.

5. The preparation method of the composition according to any one of claims 1 - 4, comprising the following steps: Mix niacinamide, nicotinamide adenine dinucleotide, and lysate of Lactobacillus fermentation according to the mass ratio described in claim 2, and that is it.

6. A cosmetic, characterized in that, The cosmetics contain the composition according to any one of claims 1 - 4; Preferably, in the cosmetics, calculated by the total mass of the cosmetics, the proportion of the composition is 0.6 - 5%.

7. The application of the composition according to any one of claims 1 - 4 in the preparation of cosmetics or drugs.

8. The application according to claim 7, wherein The cosmetics or drugs have at least one of the following functions (1)-(4): (1) Anti-wrinkle; (2) Tightening the skin; (3) Antioxidant; (4) Protecting mitochondria.

9. The application according to claim 7, characterized in that In the cosmetics or drugs, calculated by the total mass, the proportion of the composition is 0.6 - 5%.

10. The application according to claim 7, wherein The dosage forms of the cosmetics include: emulsions, aqueous solutions, oils, gels, and powders; the dosage forms of the drugs include: ointments, aerosols, and patches.

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