Mitochondrial energy-activating multi-effect composition and use thereof

Abstract

The application discloses a kind of mitochondria energy activation composite multi-effect composition and its application, the composite multi-effect composition includes nicotinamide, nicotinamide adenine dinucleotide and lactobacillus fermentation lysate in it.In the present application, by the lactobacillus fermentation lysate of composite addition, the defect that nicotinamide and nicotinamide adenine dinucleotide cannot be combined in the prior art is realized.And, based on the addition of lactobacillus fermentation lysate, the three show better synergistic effect, can simultaneously produce antioxidant;Protect mitochondria;Promote skin barrier generation or enhance skin barrier defense;Anti-wrinkle;Tighten skin and other multiple effects, realize the new application of nicotinamide and nicotinamide adenine dinucleotide combination.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, and in particular to a mitochondrial energy-activating multi-effect composition and its application. Background Technology

[0002] In the field of skin anti-aging and barrier repair, existing composition systems often face several key drawbacks, including: limited selection of usable substances due to the inherent physicochemical properties of the materials themselves. For example, niacinamide, as an NAD+... + While the precursor can inhibit melanin transport, current technology indicates that its actual transdermal efficiency is only 0.3%-1%, and excessively high concentrations (e.g., >5%) can damage the stratum corneum barrier function. Nicotinamide adenine dinucleotide (NAD) + Although it possesses DNA repair capabilities, its large molecular weight (663.43 Daltons) leads to low skin permeability (difficulty in transdermal absorption), and NAD+... + It rapidly inactivates at pH < 5 (inactivation rate > 30% within 24 hours), significantly impacting its usability in cosmetic compositions. Furthermore, existing technologies rarely combine Lactobacillus fermentation lysates with other substances, and no synergistic effect with niacinamide has been disclosed; in fact, compositional analysis suggests potential inter-component interference. Moreover, the safety of fermentation products after compounding (potential skin irritation) is also a concern.

[0003] In existing technologies, nicotinamide adenine dinucleotide (NAD) + Nicotinamide and NAD+ are generally not used together because high concentrations of nicotinamide react with NAD+. + Combination therapy may conversely inhibit nicotinamide phosphoribosyltransferase activity and reduce NAD. + The conversion efficiency is reduced, resulting in antagonistic effects between components. Furthermore, the superposition of fermented products, which may contain enzymes (such as proteases), can accelerate the rapid decomposition of other components in the composition, thus affecting the overall effect. Therefore, how to obtain a product containing nicotinamide adenine dinucleotide (NAD) through reasonable compounding is a key challenge. + The combination of α and nicotinamide, and its ability to exert a synergistic effect of anti-aging and barrier repair, is a major challenge in the field. Summary of the Invention

[0004] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of this invention is to provide a multi-functional compound and its application in cosmetics. The composition of this invention contains nicotinamide, nicotinamide adenine dinucleotide, and lactobacillus fermentation lysate, which exhibit significant synergistic effects, effectively improving the skin's anti-wrinkle, firming, and antioxidant functions, and protecting or improving mitochondrial function, thus possessing extremely high commercial value.

[0005] In a first aspect, the present invention provides a composition comprising: nicotinamide, nicotinamide adenine dinucleotide, and lactobacillus fermentation lysate.

[0006] In some embodiments of the present invention, the composition comprises nicotinamide, nicotinamide adenine dinucleotide, and lactobacillus fermentation lysate.

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

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

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

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

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

[0012] In some embodiments of the present invention, the composition further includes cosmetically acceptable excipients.

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

[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 moisturizing agent includes, but is not limited to, glycerin and hyaluronic acid;

[0016] In some embodiments of the present invention, the acceptable excipients in the cosmetic also 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 adjusters, such as citric acid and sodium lactate.

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

[0018] In some embodiments of the present invention, the active ingredient for cosmetics refers to a substance having at least one of the following functions (1)-(4):

[0019] (1) Anti-wrinkle;

[0020] (2) Tightens skin;

[0021] (3) Antioxidant;

[0022] (4) Protect mitochondria or improve mitochondrial function.

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

[0024] The product is obtained by mixing nicotinamide, nicotinamide adenine dinucleotide, and lactobacillus fermentation lysate in the mass ratios described above.

[0025] A third aspect of the present invention provides a cosmetic product comprising the composition described in the above aspects.

[0026] In some embodiments of the present invention, the composition accounts for 0.6-5% of the total mass of the cosmetic.

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

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

[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 moisturizing agent includes, but is not limited to, glycerin and hyaluronic acid;

[0031] In some embodiments of the present invention, the acceptable excipients in the cosmetic also 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 adjusters, such as citric acid and sodium lactate.

[0032] A fourth aspect of the invention provides the use of the compositions described above in the preparation of cosmetics or pharmaceuticals.

[0033] In some embodiments of the present invention, the cosmetic or pharmaceutical product has at least one of the following functions (1)-(4):

[0034] (1) Anti-wrinkle;

[0035] (2) Tightens skin;

[0036] (3) Antioxidant;

[0037] (4) Protect mitochondria.

[0038] In some embodiments of the present invention, the composition comprises 0.6-5% by total mass in the cosmetic or pharmaceutical product.

[0039] In some embodiments of the present invention, the cosmetic or pharmaceutical product is a topical preparation.

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

[0041] In some embodiments of the present invention, the dosage form of the medicine includes: ointment, aerosol, and patch.

[0042] The beneficial effects of this invention are:

[0043] This invention provides a multi-functional compound and its application in cosmetics. The compound comprises niacinamide and niacinamide adenine dinucleotide, and overcomes the limitation of existing technologies where these two components cannot be used together by adding lactobacillus fermentation lysate. Furthermore, based on the addition of lactobacillus fermentation lysate, the three components exhibit a better synergistic effect, simultaneously producing multiple benefits such as antioxidant activity, mitochondrial protection, promotion of skin barrier formation or enhancement of skin barrier defense, anti-wrinkle effects, and skin firming. This represents a novel application of the combination of niacinamide and niacinamide adenine dinucleotide. Attached Figure Description

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

[0045] Figure 2 The ratio of red to green average fluorescence intensity of mitochondrial membrane potential after treatment in different experimental groups.

[0046] Figure 3 NAD after treatment in different experimental groups + / NADH ratio.

[0047] Figure 4 The relative expression levels of the PGC-1α gene after treatment in different experimental groups.

[0048] Figure 5 The relative expression levels of the FOXO1 gene after treatment in different experimental groups.

[0049] Figure 6 The content of type I collagen after treatment in different experimental groups.

[0050] Figure 7 The expression levels of elastin genes after treatment in different experimental groups.

[0051] Figure 8 The expression levels of the SIRT3 gene after treatment in different experimental groups are shown. Detailed Implementation

[0052] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0053] In the following embodiments, nicotinamide, nicotinamide adenine dinucleotide, and lactobacillus fermentation 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 this invention.

[0054] Example 1

[0055] This embodiment provides a multi-functional composite composition, the composition of which is as follows:

[0056] By weight, 0.009 parts nicotinamide, 0.0008 parts nicotinamide adenine dinucleotide, and 0.006 parts Lactobacillus fermentation lysate.

[0057] The preparation method is as follows:

[0058] According to the above-mentioned mass ratio, nicotinamide, nicotinamide adenine dinucleotide, and lactobacillus fermentation lysate are mixed evenly to obtain a cosmetic composition with multiple functions.

[0059] Example 2

[0060] This embodiment provides a multi-functional composite composition, the composition of which is as follows:

[0061] By weight, 0.15 parts nicotinamide, 0.0125 parts nicotinamide adenine dinucleotide, and 0.1 parts Lactobacillus fermentation lysate.

[0062] The preparation method is as follows:

[0063] According to the above-mentioned mass ratio, nicotinamide, nicotinamide adenine dinucleotide, and lactobacillus fermentation lysate are mixed evenly to obtain a cosmetic composition with multiple functions.

[0064] Example 3

[0065] This embodiment provides a multi-functional composite composition, the composition of which is as follows:

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

[0067] According to the above-mentioned mass ratio, nicotinamide, nicotinamide adenine dinucleotide, and lactobacillus fermentation lysate are mixed evenly to obtain a cosmetic composition with multiple functions.

[0068] Test Example 1

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

[0070] After resuscitating human immortalized epidermal cells (HaCaT), they were cultured routinely until the cell plating rate reached about 60%. The cells were then seeded into 6-well plates and incubated overnight at 37°C and 5% CO2.

[0071] When the cell deposition rate in the 6-well plates reached approximately 60%, the cells were divided into groups for drug administration. The cells were divided into a normal control group (NC) and a sample group. The normal control group (NC) received no treatment and continued culturing in fresh culture medium. The sample group was incubated with culture medium containing the multi-component cosmetic composition from Example 1, equal amounts of nicotinamide, nicotinamide adenine dinucleotide, Lactobacillus fermentation lysate, nicotinamide + Lactobacillus fermentation lysate, and nicotinamide + nicotinamide adenine dinucleotide, respectively, and incubated at 37°C and 5% CO2 for 24 hours.

[0072] The amounts of each substance added are as follows: based on the mass of the culture medium, the proportion of each substance is as follows:

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

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

[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% Lactobacillus fermentation lysate.

[0077] After incubation, the culture medium was replaced. The normal control group (NC) received no treatment and continued incubation for 24 hours using fresh culture medium. The sample groups were incubated with culture medium containing the multi-component cosmetic composition from Example 1, equal amounts of nicotinamide, nicotinamide adenine dinucleotide, Lactobacillus fermentation lysate, nicotinamide + Lactobacillus fermentation lysate, and nicotinamide + nicotinamide adenine dinucleotide, respectively, and incubated for 24 hours at 37°C and 5% CO2.

[0078] After incubation, the ATP content was detected using a commercially available kit.

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

[0080] Table 1 Energy Enhancement Results for 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, playing a central role in energy metabolism and physiological function regulation. When the body's energy demand increases, ATP releases high-energy phosphate bonds through hydrolysis, providing immediate energy support. Studies have shown that the rate of ATP synthesis and its reserve levels are directly related to cell viability and tissue fatigue resistance. By detecting ATP levels, an individual's energy metabolism efficiency can be assessed, providing a basis for energy enhancement strategies.

[0085] The results above show that, compared with the control group (NC), the combination (NAM+NAD+LF) significantly increased ATP content more than the single or dual components of NAM, NAD, and LF, indicating that the combination of NAM+NAD+LF produced a synergistic effect that was not achieved by using NAM, NAD, and LF alone.

[0086] Other embodiments were also tested and found to have effects that were basically similar to those of the composition in Example 1.

[0087] Test Example 2

[0088] In this test case, the mitochondrial activity improvement effect of Example 2 and the corresponding amounts of single-component and two-component substances was tested. The specific experimental steps are 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 seeding rate in the 6-well plates reached approximately 60%, the cells were divided into groups for drug administration. The cells were divided into three groups: a normal control group (NC), a model control group (MC), and a sample group. The NC and MC groups received no treatment and were cultured in fresh culture medium. The sample group was incubated with culture medium containing the multi-component cosmetic composition from Example 2, equal amounts of nicotinamide, nicotinamide adenine dinucleotide, Lactobacillus fermentation lysate, nicotinamide + Lactobacillus fermentation lysate, and nicotinamide + nicotinamide adenine dinucleotide, respectively, and incubated at 37°C and 5% CO2 for 24 hours.

[0091] The amounts of each substance added are as follows: based on the mass of the culture medium, the proportion of each substance is as follows:

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

[0093] NAM+LF group: 0.15% nicotinamide, 0.1% Lactobacillus fermentation lysate;

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

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

[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 group to UV light for 50 seconds (UVA, 9 mJ / cm²). 2 ).

[0097] After modeling, the normal control group (NC) and the model control group (MC) were not treated and were cultured for another 24 hours using fresh culture medium. The sample groups were respectively added to the culture medium containing the multi-component cosmetic composition of Example 2, equal amounts of nicotinamide, equal amounts of nicotinamide adenine dinucleotide, equal amounts of Lactobacillus fermentation lysate, equal amounts of nicotinamide + Lactobacillus fermentation lysate, and equal amounts of nicotinamide + nicotinamide adenine dinucleotide, and were incubated for another 24 hours at 37°C and 5% CO2.

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

[0099] The fluorescence intensity (S) of each group was analyzed using ImageJ, the average fluorescence intensity ratio of red to green at the mitochondrial membrane potential was calculated, and the improvement rate was calculated using the following formula.

[0100]

[0101] In the formula:

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

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

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

[0105]

[0106]

[0107] 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. Decreased MMP weakens ATP synthesis, leading to ROS accumulation and mitochondrial dysfunction; conversely, increased MMP enhances electron transport chain activity, promotes efficient energy production, reduces oxidative damage, and restores mitochondrial homeostasis, thereby directly improving mitochondrial vitality. Its levels can be detected using fluorescent probes, providing a basis for assessing mitochondrial health and intervening in energy metabolism issues.

[0109] The results above show that, compared with the control group (MC), the combination (NAM+NAD+LF) significantly enhances the mitochondrial membrane potential, which is more pronounced than that of single or dual components of NAM, NAD, and LF. This indicates that the combination of NAM+NAD+LF produces a synergistic effect that is not achieved by using NAM, NAD, and LF alone.

[0110] Other embodiments were also tested and found to have effects that were basically similar to those of the composition in Example 2.

[0111] Test Example 3

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

[0113] 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.

[0114] When the cell deposition rate in the 6-well plates reached approximately 60%, the cells were divided into groups for drug administration. The cells were divided into three groups: a normal control group (NC), a model control group (MC), and a sample group. The NC and MC groups received no treatment and were cultured in fresh culture medium. The sample group was incubated with culture medium containing the multi-component cosmetic composition from Example 3, equal amounts of nicotinamide, nicotinamide adenine dinucleotide, Lactobacillus fermentation lysate, nicotinamide + Lactobacillus fermentation lysate, and nicotinamide + nicotinamide adenine dinucleotide, respectively, and incubated at 37°C and 5% CO2 for 24 hours.

[0115] The amounts of each substance added are as follows: based on the mass of the culture medium, the proportion of each substance is as follows:

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

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

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

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

[0120] 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 group to UV light for 50 seconds (UVA, 9 mJ / cm²). 2 ).

[0121] After modeling, the normal control group (NC) and the model control group (MC) were not treated and were cultured for another 24 hours using fresh culture medium. The sample groups were respectively added to the culture medium containing the multi-component cosmetic composition of Example 3, equal amounts of nicotinamide, equal amounts of nicotinamide adenine dinucleotide, equal amounts of Lactobacillus fermentation lysate, equal amounts of nicotinamide + Lactobacillus fermentation lysate, and equal amounts of nicotinamide + nicotinamide adenine dinucleotide, and were incubated for another 24 hours at 37°C and 5% CO2.

[0122] After incubation, NAD was detected using a commercially available kit. + And NADH content, and calculate NAD + / NADH ratio.

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

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

[0125] Sample group Test results SD p-value Improvement Rate (vs. MC) Normal control (NC) 11.72 0.875 0.0012** 64％ Model Comparison (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] NAD + The NAD / NADH ratio affects a cell's ability to produce energy. In the cytoplasm, NAD+ is required to convert glucose into pyruvate via glycolysis.+ In mitochondria, the TCA cycle reduces NAD. + The molecules generate multiple NADH molecules, which are then oxidized by complex I of the electron transport chain (ETC) to produce ATP. + Decreased NAD levels can affect mitochondrial function, overall cellular health, and the development of age-related diseases. Therefore, NAD can be detected... + The NADH / NADH ratio level was used to assess whether the sample had an effect on improving mitochondrial dysfunction.

[0128] The results above show that, compared with the control group (MC), the composition (NAM+NAD+LF) increased NAD levels. + The effect of NADH is more significant than that of single or dual components of NAM, NAD, and LF, indicating that the combination of NAM+NAD+LF produces a synergistic effect that is not present when NAM, NAD, and LF are used alone.

[0129] Other embodiments were also tested and found to have effects that were basically similar to those of the composition in Example 3.

[0130] Test Example 4

[0131] In this test case, the effects of Example 1 and corresponding amounts of single-component 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 into 6-well plates and incubated overnight at 37°C and 5% CO2.

[0133] When the cell deposition rate in the 6-well plates reached approximately 60%, the cells were divided into groups for drug administration. The cells were divided into three groups: a normal control group (NC), a model control group (MC), and a sample group. The NC and MC groups received no treatment and were cultured in fresh culture medium. The sample group was incubated with culture medium containing the multi-component cosmetic composition from Example 1, equal amounts of nicotinamide, nicotinamide adenine dinucleotide, Lactobacillus fermentation lysate, nicotinamide + Lactobacillus fermentation lysate, and nicotinamide + nicotinamide adenine dinucleotide, respectively, and incubated at 37°C and 5% CO2 for 24 hours.

[0134] The amounts of each substance added are as follows: based on the mass of the culture medium, the proportion of each substance is as follows:

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

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

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

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

[0139] 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 group to UV light for 50 seconds (UVA, 9 mJ / cm²). 2 ).

[0140] After modeling, the normal control group (NC) and the model control group (MC) were not treated and were cultured for another 24 hours using fresh culture medium. The sample groups were respectively added to the culture medium containing the multi-component cosmetic composition of Example 1, equal amounts of nicotinamide, equal amounts of nicotinamide adenine dinucleotide, equal amounts of Lactobacillus fermentation lysate, equal amounts of nicotinamide + Lactobacillus fermentation lysate, and equal amounts of nicotinamide + nicotinamide adenine dinucleotide, and were incubated for another 24 hours at 37°C and 5% CO2.

[0141] After incubation, total RNA was extracted from each experimental group, and cDNA was synthesized by reverse transcription. The gene expression of β-actin (internal reference) and PGC-1α was detected by q-PCR.

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

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

[0144] Sample group Test results SD p-value Improvement Rate (vs. MC) Normal control (NC) 1.42 0.050 0.0203* 40％ Model Comparison (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 coactivator, PGC-1α promotes the transcription of a range of genes. In mitochondria, PGC-1α can enhance ATP production efficiency and reduce the accumulation of reactive oxygen species (ROS). Decreased PGC-1α levels lead to a reduction in mitochondrial numbers and energy metabolism imbalances, which are closely associated with metabolic syndrome, neurodegenerative diseases, and aging. Therefore, detecting PGC-1α expression levels can be used to assess the ameliorative effect of samples on mitochondrial dysfunction.

[0147] The results above show that, compared with the control group (MC), the combination (NAM+NAD+LF) significantly increased the expression level of PGC-1α, which is more significant than that of single or dual components of NAM, NAD, and LF. This indicates that the combination of NAM+NAD+LF produced a synergistic effect that is not present when NAM, NAD, and LF are used alone.

[0148] Other embodiments were also tested and found to have effects that were basically similar to those of the composition in Example 1.

[0149] Test Example 5

[0150] In this test case, the effects of Example 2 and corresponding amounts of single-component 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 into 6-well plates and incubated overnight at 37°C and 5% CO2.

[0152] When the cell seeding rate in the 6-well plates reached approximately 60%, the cells were divided into groups for drug administration. The cells were divided into three groups: a normal control group (NC), a model control group (MC), and a sample group. The NC and MC groups received no treatment and were cultured in fresh culture medium. The sample group was incubated with culture medium containing the multi-component cosmetic composition from Example 2, equal amounts of nicotinamide, nicotinamide adenine dinucleotide, Lactobacillus fermentation lysate, nicotinamide + Lactobacillus fermentation lysate, and nicotinamide + nicotinamide adenine dinucleotide, respectively, and incubated at 37°C and 5% CO2 for 24 hours.

[0153] The amounts of each substance added are as follows: based on the mass of the culture medium, the proportion of each substance is as follows:

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

[0155] NAM+LF group: 0.15% nicotinamide, 0.1% Lactobacillus fermentation lysate;

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

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

[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 group to UV light for 50 seconds (UVA, 9 mJ / cm²). 2 ).

[0159] After modeling, the normal control group (NC) and the model control group (MC) were not treated and were cultured for another 24 hours using fresh culture medium. The sample groups were respectively added to the culture medium containing the multi-component cosmetic composition of Example 2, equal amounts of nicotinamide, equal amounts of nicotinamide adenine dinucleotide, equal amounts of Lactobacillus fermentation lysate, equal amounts of nicotinamide + Lactobacillus fermentation lysate, and equal amounts of nicotinamide + nicotinamide adenine dinucleotide, and were incubated for another 24 hours at 37°C and 5% CO2.

[0160] After incubation, total RNA was extracted from each experimental group, and cDNA was synthesized by reverse transcription. The gene expression of β-actin (internal reference) and FOXO1 was detected by q-PCR.

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

[0162] Table 5. FOXO1 expression levels in each group

[0163] Sample group Test results SD p-value Improvement Rate (vs. MC) Normal control (NC) 1.44 0.044 0.0037** 43％ Model Comparison (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 and antioxidant enzyme genes, promoting the clearance of damaged mitochondria and reducing ROS accumulation; it also synergistically enhances the expression of genes related to mitochondrial biogenesis in conjunction with PGC-1α. Abnormal FOXO1 function leads to cumulative mitochondrial damage, which is associated with metabolic diseases and aging. Therefore, the effectiveness of FOXO1 in improving mitochondrial dysfunction can be assessed by detecting FOXO1 expression.

[0166] The results above show that, compared with the control group (MC), the combination (NAM+NAD+LF) significantly increased the expression level of FOXO1 compared with the single or dual components of NAM, NAD, and LF, indicating that the combination of NAM+NAD+LF produced a synergistic effect that was not achieved by using NAM, NAD, and LF alone.

[0167] Other embodiments were also tested and found to have effects that were basically similar to those of the composition in Example 2.

[0168] Test Example 6

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

[0170] 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.

[0171] When the cell deposition rate in the 6-well plates reached approximately 60%, the cells were divided into groups for drug administration. The cells were divided into three groups: a normal control group (NC), a model control group (MC), and a sample group. The NC and MC groups received no treatment and were cultured in fresh culture medium. The sample group was incubated with culture medium containing the multi-component cosmetic composition from Example 3, equal amounts of nicotinamide, nicotinamide adenine dinucleotide, Lactobacillus fermentation lysate, nicotinamide + Lactobacillus fermentation lysate, and nicotinamide + nicotinamide adenine dinucleotide, respectively, and incubated at 37°C and 5% CO2 for 24 hours.

[0172] The amounts of each substance added are as follows: based on the mass of the culture medium, the proportion of each substance is as follows:

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

[0174] NAM+LF group: 0.06% nicotinamide, 0.04% Lactobacillus fermentation lysate;

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

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

[0177] 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 group to UV light for 50 seconds (UVA, 30 mJ / cm²). 2 ).

[0178] After modeling, the normal control group (NC) and the model control group (MC) were not treated and were cultured for another 24 hours using fresh culture medium. The sample groups were respectively added to the culture medium containing the multi-component cosmetic composition of Example 3, equal amounts of nicotinamide, equal amounts of nicotinamide adenine dinucleotide, equal amounts of Lactobacillus fermentation lysate, equal amounts of nicotinamide + Lactobacillus fermentation lysate, and equal amounts of nicotinamide + nicotinamide adenine dinucleotide, and were incubated for another 24 hours at 37°C and 5% CO2.

[0179] After incubation, cells were fixed with 4% paraformaldehyde for 30 minutes. Immunofluorescence was then performed, and images were taken and analyzed under a microscope.

[0180] Total RNA was extracted from each experimental group, reverse transcribed into cDNA, and then detected by quantitative real-time PCR.

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

[0182] Table 6. Type I collagen content in each group

[0183] Sample group Test results SD p-value Improvement Rate (vs. MC) Normal control (NC) 1.00 0.120 0.0018** 120％ Model Comparison (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. Elastin gene expression levels in each group

[0186]

[0187]

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

[0189] Type I collagen and elastin exert their anti-wrinkle effects by maintaining the integrity and elasticity of skin structure. In the dermis, type I collagen forms a dense fibrous network providing mechanical support and inhibiting skin sagging; it also stimulates fibroblasts to secrete more extracellular matrix components, strengthening the skin barrier. Elastin imparts elasticity to the skin through a cross-linked network, reducing permanent wrinkles caused by repeated stretching. Both synergistically regulate the balance of MMPs / TIMPs (e.g., inhibiting MMP-1 and activating TIMP-1), delaying collagen degradation. Loss of function leads to collagen fiber breakage and elastin fiber fragmentation, causing skin laxity and wrinkles. This experiment assessed the anti-wrinkle effect by detecting type I collagen content and elastin gene expression levels.

[0190] The results above show that, compared with the control group, the anti-wrinkle effect of the composition (NAM+NAD+LF) is more significant than that of single or dual components of NAM, NAD, and LF, indicating that the combination of NAM+NAD+LF produces a synergistic effect that is not achieved by using NAM, NAD, and LF alone.

[0191] Other embodiments were also tested and found to have effects that were basically similar to those of the composition in Example 3.

[0192] Test Example 7

[0193] In this test case, the anti-aging effects of Example 3, the corresponding amounts of single-component substances, 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 deposition rate in the 6-well plates reached approximately 60%, the cells were divided into groups for drug administration. The cells were divided into a normal control group (NC) and a sample group. The normal control group (NC) received no treatment and continued culturing in fresh culture medium. The sample group was incubated with culture medium containing the multi-component cosmetic composition from Example 3, equal amounts of nicotinamide, nicotinamide adenine dinucleotide, Lactobacillus fermentation lysate, nicotinamide + Lactobacillus fermentation lysate, and nicotinamide + nicotinamide adenine dinucleotide, respectively, and incubated at 37°C and 5% CO2 for 24 hours.

[0196] The amounts of each substance added are as follows: based on the mass of the culture medium, the proportion of each substance is as follows:

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

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

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

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

[0201] After incubation, the culture medium was replaced. The normal control group (NC) received no treatment and continued incubation for 24 hours using fresh culture medium. The sample groups were incubated with culture medium containing the multi-component cosmetic composition from Example 3, equal amounts of nicotinamide, equal amounts of nicotinamide adenine dinucleotide, equal amounts of Lactobacillus fermentation lysate, equal amounts of nicotinamide + Lactobacillus fermentation lysate, and equal amounts of nicotinamide + nicotinamide adenine dinucleotide, respectively, and incubated for 24 hours at 37°C and 5% CO2.

[0202] After incubation, total RNA was extracted from each experimental group, reverse transcribed into cDNA, and then detected by quantitative real-time PCR.

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

[0204] Table 8. SIRT3 gene expression results for each group

[0205] Sample group Test results 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 an NAD+-dependent mitochondrial enzyme that slows aging by enhancing mitochondrial function, scavenging reactive oxygen species (ROS), and promoting autophagy in damaged mitochondria. It regulates metabolic homeostasis, inhibits inflammation, and enhances DNA repair, while also improving cellular stress response by modulating histones and mitochondrial biosynthesis-related proteins (such as PGC-1α). Decreased SIRT3 activity leads to mitochondrial damage, ROS accumulation, and increased aging markers, accelerating age-related diseases. Therefore, SIRT3 gene expression results can be used to assess the anti-aging effects of samples.

[0208] The results above show that, compared with the control group (NC), the combination (NAM+NAD+LF) significantly enhanced SIRT3 gene expression compared with single or dual components of NAM, NAD, and LF, indicating that the combination of NAM+NAD+LF produced a synergistic effect that was not achieved by using NAM, NAD, and LF alone.

[0209] Other embodiments were also tested and found to have effects that 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 changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A composition, characterized in that, The composition comprises: nicotinamide, nicotinamide adenine dinucleotide and lactobacillus fermentation lysate; In the composition, the mass ratio of nicotinamide, nicotinamide adenine dinucleotide, and lactobacillus fermentation lysate is 0.009:0.0008:0.006, 0.15:0.0125:0.1, or 0.06:0.005:0.

04.

2. A method for preparing the composition according to claim 1, comprising the following steps: The product is obtained by mixing nicotinamide, nicotinamide adenine dinucleotide, and lactobacillus fermentation lysate according to the mass ratio described in claim 1.

3. The use of the composition according to claim 1 in the preparation of cosmetics; The cosmetic product has at least one of the following functions: (1)-(2) (1) Antioxidant; (2) Protect mitochondria.

4. The application according to claim 3, characterized in that, In the cosmetic product, the composition accounts for 0.6-5% of the total mass.

5. The application according to claim 3, characterized in that, The dosage forms of the cosmetics include: emulsions, aqueous solutions, oils, gels, and powders.

6. The application according to claim 3, characterized in that, The cosmetics also include cosmetic excipients.

7. The application according to claim 6, characterized in that, Acceptable excipients in the cosmetics include: solvents, emulsifiers, stabilizers, thickeners, preservatives, fragrances, pigments, fillers, and moisturizers.

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