Cosmetic composition comprising NAD-liposome complex

Abstract

The present invention relates to a cosmetic composition comprising an NAD-liposome complex including nicotinamide adenine dinucleotide and liposomes. More specifically, the present invention relates to a technology in which a complex is formed by ionically bonding nicotinamide adenine dinucleotide with anionic elastic liposomes, thereby enhancing skin permeability and inhibiting the expression of aging-related genes.

Description

Cosmetic composition comprising NAD liposome complex

[0001] The present invention relates to a cosmetic composition comprising an NAD liposome complex, and more particularly, to a technology for forming a complex by ionic bonding of nicotinamide adenine dinucleotide with anionic elastic liposomes, thereby improving the skin permeability of nicotinamide adenine dinucleotide and inhibiting the expression of aging genes.

[0002]

[0003] Nicotinamide adenine dinucleotide (NAD) + ) is an essential biological molecule that regulates body metabolism. In previous studies, NAD + NAD plays an important role in the progression of aging and degenerative diseases. + Alternatively, important results have been revealed that supplementation of the precursor may improve or inhibit the aging process.

[0004] But NAD + The pharmacological activity or mechanism of action of NAD has not yet been elucidated. + is chemically unstable, making it difficult to apply widely for cosmetic purposes, and NAD + It has a short half-life in vivo, low skin permeability that makes it difficult to penetrate the outermost stratum corneum of the skin as a water-soluble ingredient, and problems with aggregation in formulations containing anionic polymers due to its strong cationicity (M. Lukic et al., Tenside Surfactants Detergents, 2016, 53, 7-19; I. Yuli et al., Cosmetics, 2023, 10, 45).

[0005] To solve this problem, NAD was added to liposomes using conventional techniques. +There have been attempts to encapsulate it (S. Miyagawa et al., Journal of Chemical Engineering of Japan, 2001, 34 (1), 30-35; AU Khan et al., American Journal of Physiology-Lung Cellular and Molecular Physiology, 2002, 282, L1082-L1091; R. Matsumoto et al., Physical Chemistry Chemical Physics, 2010, 12, 13904-13906). However, these methods use organic solvents that are harmful to the human body, making them difficult to apply as cosmetic materials and difficult to mass-produce and use industrially. In addition, delivery of it involves very complex and difficult methods, such as direct injection into the bloodstream using a syringe, etc. A method for mass-producing NAD+ into liposomes has also been proposed, but it is difficult to store and use in large quantities industrially, only unsaturated lipids that are easily oxidized were used, and there is a problem of aggregation when saturated lipids are used to encapsulate NAD+ into liposomes (Korean Patent Publication No. 10-2024-0009434). In addition, the previously known polyol dilution method cannot be used to encapsulate NAD+. + It is difficult to apply because there is a problem that stable liposomes are not formed due to aggregation with anionic lipids during encapsulation (K. ohishi et al., Collids and Surfaces A: Physicochemical and Engineering Aspects, 2022, 650, 129592; K. ohishi et al., Collids and Surfaces A: Physicochemical and Engineering Aspects, 656, 130509).

[0006] Under this background, the inventors of the present invention have developed NAD + It is stabilized by physical fixation through ionic bonding on the elastic liposome surface, and NAD +The present invention was completed by confirming its own stability, improved skin penetration, and skin aging inhibition effect.

[0007]

[0008] One object of the present invention is to provide a composition comprising hydrogenated lecithin, fatty acid, and nicotinamide adenine dinucleotide (NAD). + ) to provide an NAD liposome complex.

[0009] Another object of the present invention is to provide a cosmetic composition comprising the NAD liposome complex of the present invention.

[0010] Another object of the present invention is to provide a method for preparing an NAD liposome complex, comprising the steps of: forming a liposome with a solution containing a lipid and a solvent; forming a mixture containing nicotinamide adenine dinucleotide and a solvent; and combining the liposome with the mixture.

[0011]

[0012] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions described below.

[0013]

[0014] A first aspect of the present invention for achieving the aforementioned object is to provide an NAD liposome complex comprising hydrogenated lecithin, a fatty acid, and nicotinamide adenine dinucleotide (NAD+).

[0015]

[0016] Hereinafter, the present invention will be described in more detail.

[0017]

[0018] In the present invention, “Nicotinamide adenine dinucleotide (NAD) + )” is an effective ingredient of the NAD liposome complex of the present invention, which can promote cell metabolism and DNA damage repair, improve skin barrier function, and play an anti-aging role.

[0019] The above nicotinamide adenine dinucleotide structure is linked through the phosphate groups of two nucleotides, one of which may have an adenine nucleobase and the other may have nicotinamide.

[0020] In the present invention, the NAD+ may be included in an amount of 0.01 to 30 wt% relative to the total weight of the NAD liposome complex. Preferably, it may be included in an amount of 0.05 to 25 wt%, and more preferably, it may be included in an amount of 0.07 to 20 wt%. Within the above content range, the stability of the complex may be improved, and its delivery efficiency to the skin may be improved.

[0021]

[0022] As mentioned above, NAD + It has a very short half-life in vivo and is known to be a water-soluble substance that has difficulty penetrating the stratum corneum of the skin.

[0023] Accordingly, the present invention provides NAD + The present invention aims to solve the above problem by providing an NAD liposome complex to deliver the NAD liposome complex to the dermis of the skin and thereby provide an anti-aging effect.

[0024]

[0025] In the present invention, the term “effective ingredient” may be an ingredient that helps with wrinkles, elasticity, lifting, skin texture, skin transparency, antioxidant, gloss, moisturizing, improving skin barrier damage, and caring for skin protection and intrinsic ability.

[0026] In the present invention, the term "liposome" refers to a lipid vesicle designed to carry an active ingredient inside. The liposome formation described above can help deliver the active ingredient deep into the skin.

[0027] In the present invention, the term "liposome complex" may be a form in which the cationic portion of the "nicotinamide adenine dinucleotide" and the anionic surface of the "liposome" are associated through ionic or hydrogen bonds. In addition, the liposome complex may be a nanocomplex from the perspective of ease of skin delivery.

[0028]

[0029] The above ionic bond is an electrostatic bond, and NAD on the liposome surface + By stably and uniformly attaching NAD + It is possible to form a complex that improves stability and carrying capacity.

[0030]

[0031] The liposome according to the present invention can use hydrogenated lecithin, which corresponds to a saturated lipid, as the main lipid (backbone) of the liposome.

[0032] The above saturated lipid may be more advantageous in the present invention than unsaturated lipid, which is prone to oxidation. Preferably, the liposome may be a nanoliposome.

[0033] The above “hydrogenated lecithin” is a component with CAS No. 92128-87-5, and is a saturated lipid, unlike regular lecithin, which is an unsaturated lipid. When hydrogenated lecithin, a saturated lipid, is used as the main lipid of liposomes, it has the advantage of increasing skin permeability compared to liposomes that use lecithin, an unsaturated lipid, as the main lipid.

[0034] In the present invention, the hydrogenated lecithin may be included in an amount of 0.005 to 5 wt% relative to the total weight of the NAD liposome complex. Preferably, it may be included in an amount of 0.01 to 2.5 wt%, and more preferably, it may be included in an amount of 0.04 to 2 wt%. Within the above content range, the complex may exhibit better stability.

[0035] When NAD+ is liposomally encapsulated using conventional liposomal techniques utilizing saturated lipids, there is a problem in that NAD+ tends to aggregate during encapsulation. However, the present invention overcomes this problem by adopting a configuration in which NAD+ is ionically bound to the liposome surface.

[0036]

[0037] In the present invention, the term “fatty acid” may be an anionic fatty acid, having a saturated or unsaturated long aliphatic chain with an even number of carbon atoms, and having a monovalent, divalent, or trivalent or higher negative charge at one end. Specifically, it may be included to strengthen the negative charge of the liposome according to the present invention, so that NAD+ is associated with the surface of the liposome through ionic bonding.

[0038] More specifically, fatty acids refer to carboxylic acids having long aliphatic chains (4 to 28 carbon atoms) that are saturated or unsaturated.

[0039] Anionic fatty acids having a single charge may be, for example, at least one selected from among oleic acid, stearic acid, myristic acid, palmitic acid, stearic acid, oleic acid, and behenic acid, but are not limited thereto.

[0040] In addition, the anionic fatty acid having a divalent charge may be, for example, at least one selected from sodium lauroyl glutamate, sodium myristoyl glutamate, magnesium palmitoyl glutamate, and sodium stearoyl glutamate, but is not limited thereto.

[0041] To prevent aggregation of liposomes, it is preferable to use an anionic fatty acid with a divalent charge, more preferably sodium stearoyl glutamate.

[0042] In the present invention, the fatty acid may be included in an amount of 0.001 to 1 wt% relative to the total weight of the NAD liposome complex. Preferably, the fatty acid may be included in an amount of 0.003 to 0.5 wt%, and more preferably, the fatty acid may be included in an amount of 0.005 to 0.1 wt%. Within the above content range, the complex may exhibit better stability.

[0043] In the present invention, the NAD liposome complex according to the present invention comprises NAD of the core + NAD on surface compared to content + The content may be higher. This is a technology that is different from the conventional simple NAD liposome complex, and it is more efficient than the conventional NAD + can be conveyed.

[0044] The above nicotinamide adenine dinucleotide is supported on the surface or core of the liposome, and the content ratio of nicotinamide adenine dinucleotide supported on the core:surface may be 50:50 to 0:100. Preferably, it may be 49:51 to 1:99.

[0045] For example, hydrogenated lecithin and fatty acids form single or multilamellar vesicles with lamellar layers, and NAD + It can exist mainly in the form of ion bonding on the surface of anionic vesicles or partially encapsulated inside the vesicles.

[0046]

[0047] The above complex can prevent aggregation of the complex by controlling the surface charge of the complex.

[0048] Specifically, the surface charge of the complex may be -60 to 5 mV, preferably -55 to -10 mV, and more preferably -50 to -15 mV. Within this range, the complexes may be stably maintained without aggregation within the formulation.

[0049]

[0050] The nicotinamide adenine dinucleotide may be supported on the hydrogenated lecithin at a weight ratio of 1:0.5 to 200. Within the above range, the efficiency of the NAD complex may be better.

[0051]

[0052] The size of the above complex may have an average diameter of 50 nm to 500 nm. Within the above range, the complex may be more stable.

[0053]

[0054] The above NAD liposome complex may further comprise a solvent, preferably dipropylene glycol.

[0055]

[0056] The NAD liposome complex according to the present invention may additionally include a nonionic surfactant to impart elasticity to the liposome.

[0057] The nonionic surfactant may include, for example, one or more of polyoxyethylene alkyl ether, polyoxyethylene propylene alkyl ether, alkyl polyalkylene glycol, alkyl aryl polyalkylene glycol, fatty alcohol polyoxyethylene glycol, fatty acid polyoxyethylene glycol, alkyl dimethyl amine oxide, di-alkyl methyl amine oxide, alkyl amidopropyl amine oxide, alkyl glucamide, alkyl polyglucoside oxylated petroleum acid, alkyl amine, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, sorbitan laurate, sorbitan stearate, sorbitan oleate, glycyrrhizic acid, and dipotassium glycyrrhizate. Preferably, the nonionic surfactant may include sorbitan oleate.

[0058] The above nonionic surfactant can further increase skin permeability by imparting flexibility and elasticity to liposomes.

[0059] In the present invention, the nonionic surfactant may be included in an amount of 0.001 to 1 wt% relative to the total weight of the NAD liposome complex. Preferably, the nonionic surfactant may be included in an amount of 0.003 to 0.5 wt%, and more preferably, the nonionic surfactant may be included in an amount of 0.005 to 0.1 wt%. Within the above content range, skin permeability may be improved.

[0060]

[0061] Another aspect of the present invention for achieving the above object is to provide a cosmetic composition comprising the NAD liposome complex of the present invention.

[0062] The above cosmetic composition is specifically intended to prevent skin aging and may be used on the skin topically or parenterally.

[0063] Specifically, the NAD liposome complex may be included in an amount of 0.1 to 100% based on the total weight of the cosmetic composition.

[0064] More specifically, the formulation of the cosmetic composition may be in the form of a toner, essence, serum, emulsion, or cream, and more preferably, it may be provided as an essence serum, but is not limited thereto.

[0065]

[0066] Another aspect of the present invention for achieving the above object provides a method for preparing an NAD liposome complex, comprising the steps of: forming a liposome with a solution containing a lipid, an anionic fatty acid, and a solvent; forming a mixture containing nicotinamide adenine dinucleotide and a solvent; and combining the liposome and the mixture.

[0067] The above lipid may include, for example, hydrogenated lecithin.

[0068] Specifically, the lipid may be a saturated lipid of the present invention, and provides an effect of improving skin permeability compared to unsaturated lipid.

[0069] The anionic fatty acid may be an anionic fatty acid having a monovalent, divalent, trivalent or higher charge. For example, it may include stearic acid or sodium stearoyl glutamate.

[0070] The above liposome may further contain cholesterol and a non-ionic surfactant.

[0071] Specifically, the nonionic surfactant may impart flexibility and elasticity to liposomes and further increase skin permeability.

[0072] The solvent may include, for example, one or more of 1,2-hexanediol, dipropylene glycol, and water.

[0073] Specifically, by including the dipropylene glycol, it is advantageous in supplying moisture to the skin, thereby improving dryness, and by improving stickiness properties, it can provide excellent spreadability.

[0074]

[0075] In a specific embodiment, the NAD liposome complex of the present invention is applied to the skin to provide NAD + The anti-aging effect of human fibroblast cells was confirmed by increasing the penetration rate into the skin dermis.

[0076] Therefore, through the above specific example, the cosmetic composition including the NAD liposome complex of the present invention is NAD + NAD provides stable and high skin penetration rate of active ingredients to the skin dermis. + It suggests that it can be used as a source technology for inhibiting skin aging by transmitting and suppressing the expression of aging genes.

[0077]

[0078] The present invention provides a complex stably containing nicotinamide adenine dinucleotide, thereby improving skin permeability and imparting an effect of inhibiting the expression of aging genes.

[0079]

[0080] Figure 1 is a schematic diagram showing a manufacturing method of an embodiment of the present invention.

[0081] Figure 2 is a schematic diagram showing the complex formation reaction of the present invention.

[0082] Figure 3 is a photograph showing the stability of the complex of the present invention according to temperature and time.

[0083] Figure 4 is a graph showing the reverse analysis of an embodiment of the present invention.

[0084] Figure 5 is a graph showing a comparison of skin permeability of composite raw materials manufactured as an example of the present invention.

[0085] Figure 6 is a graph showing a comparison of skin permeability of essences manufactured as an example of the present invention.

[0086] Figure 7 is a graph showing the effect of reducing cell aging-promoting genes in an embodiment of the present invention.

[0087] Figure 8 is a graph showing the reduction rate of the expressed aging gene P16 by the treatment of the examples and comparative examples of the present invention.

[0088] Figure 9 is a graph showing the reduction rate of the expressed aging gene CAV1 by the treatment of the examples and comparative examples of the present invention.

[0089] Figure 10 is a graph showing the reduction rate of the expressed aging gene P21 by the treatment of the examples and comparative examples of the present invention.

[0090] Figure 11 is a graph showing the oxidative stress inhibition effect of examples and comparative examples of the present invention.

[0091] Figure 12 is a photograph showing the cell regeneration effect after UV irradiation of examples and comparative examples of the present invention.

[0092] Figure 13 is a graph showing the cell regeneration effect after UV irradiation of examples and comparative examples of the present invention.

[0093] Figure 14 is a graph showing the cell regeneration effect of examples and comparative examples of the present invention.

[0094] Figure 15 is a photograph showing the cell expression and distribution of cell growth markers Ki67 and DAPI after treatment with examples and comparative examples of the present invention.

[0095] Figure 16 is a graph showing cell expression of the cell growth marker Ki67 after treatment with examples and comparative examples of the present invention.

[0096] Figure 17 is a graph showing cell expression of the cell growth marker DAPI after treatment with examples and comparative examples of the present invention.

[0097] Figure 18 is a photograph showing fibrillin expression in examples and comparative examples of the present invention.

[0098] Figure 19 is a graph showing fibrillin expression of examples and comparative examples of the present invention.

[0099] Figure 20 is a photograph showing the amount of collagen produced in the skin dermis of examples and comparative examples of the present invention.

[0100] Figure 21 is a graph showing the amount of collagen produced in the skin dermis of examples and comparative examples of the present invention.

[0101]

[0102] Hereinafter, the present invention will be described in more detail through examples. These examples are intended to illustrate the present invention more concretely, but the scope of the present invention is not limited by these examples. The raw materials used in the present invention were purchased from conventional commercial sources.

[0103]

[0104] Examples 1 to 2 and Comparative Examples 1 to 2: Preparation of liposome complexes

[0105] In order to manufacture the liposome complex of the present invention and compare the aggregation state according to lipid, Examples 1 and 2 and Comparative Examples 1 and 2 were manufactured with the contents and weight ratios shown in Table 1 below.

[0106] Specifically, as shown in FIGS. 1 and 2, liposome complexes were prepared by the following methods ① to ⑤.

[0107] ① Dipropylene glycol, hydrogenated lecithin, cholesterol, nonionic surfactant, and fatty acid were completely dissolved at 80℃ (phase A).

[0108] ② A mixture was prepared by adding ① solution completely dissolved in water (B phase) at 80℃.

[0109] ③ The above mixture was stirred at 80°C for 30 minutes to prepare a liposome solution.

[0110] ④ ③After lowering the temperature of the solution to room temperature, water (phase C) was added and stirred for 10 minutes.

[0111] ⑤ NAD at 30 to 40℃ + A second mixture (phase D) was prepared by completely dispersing 1,2-hexanediol in water, and the complex was prepared by adding it to the ④ solution and stirring it at room temperature for 10 minutes.

[0112] As shown in Table 1 above, in Examples 1 and 2, nicotinamide adenine dinucleotide (NAD) was used as the active ingredient of the liposome complex. + ), hydrogenated lecithin as the main lipid (backbone) of the liposome, stearic acid (pKa = 4.7) as a -1-valent charged fatty acid (Example 1), sodium stearoyl glutamate as a -2-valent charged fatty acid to reinforce the negative charge (Example 2), sorbitan oleate as a nonionic surfactant to impart elasticity to the liposome, cholesterol as a liposome lipid membrane stabilizer, 1,2-hexanediol as a preservative, and water were added to prepare the liposome.

[0113] Unit (%) Comparative Example 1 Comparative Example 2 Example 1 Example 2 Purified water to 100 to 100 to 100 to 100 Glycerin 76.500 Dipropylene glycol 2.000 2.000 Lecithin 4.000 Hydrogenated lecithin 0.1000.100 Sorbitan oleate 0.020 0.020 Stearic acid 0.020 Sodium stearoyl glutamate 0.020 Cholesterol 0.020 0.020 1,2-hexadiol 2.500 2.500 2.500 2.500 Sodium hydroxide 0.300 Nicotinamide dinucleotide 0.1000 0.1000.1000.100 Manufacturing method Simple mixing Korean published patent publication 10-2024-0009434 This patented manufacturing method This patented manufacturing method Average diameter (nm) 402.2 ± 40.7 237.5 ± 2.5 268.1 ± 3.5 Zeta potential (mV) -4.8 ± 0.2 - 24.0 ± 0.4 - 33.3 ± 1.7 Appearance

[0114] To compare the properties of NAD liposomes according to the manufacturing method, each liposome was manufactured according to Table 1. As a result of the manufacturing, as shown in the property image in the table, the liposome manufactured in Comparative Example 2 had problems such as being difficult to store and use in large quantities industrially due to the use of unsaturated lecithin, being prone to rancidity, and solidifying at low temperatures.

[0115] However, Examples 1 and 2 manufactured by the manufacturing method of the present invention are NAD + It was confirmed that NAD liposome complex was produced in the form of NAD liposome complex without aggregation by assembling with liposomes through ionic bonds.

[0116] Experimental Example 1: Stability of raw materials and stability within formulations

[0117] The raw material stability and formulation stability of the liposome complex of the present invention were evaluated.

[0118] For stability evaluation under harsh conditions, the NAD liposome complex prepared as an example of the present invention was diluted to the cosmetic application content (0.01% NAD + The changes over time were observed at temperatures ranging from 0 to 50°C for 0 to 4 weeks. As a result, as shown in Fig. 3, it was confirmed that the properties were stably maintained even with the passage of time and changes in temperature.

[0119] Additionally, for long-term stability evaluation under harsh conditions, NAD + NAD of NAD liposome complex prepared in aqueous solution (Comparative Example 1) and Example 2 + The content was compared with the 4-week potency analysis. As a result, as shown in Figure 4, NAD + The aqueous solution is NAD + While the content decreases significantly as the temperature increases, the NAD in the NAD liposome complex of the present invention +It was confirmed that the content was clearly reduced. More specifically, the NAD liposome complex of the present invention was about 40% at a temperature of 40°C and about 50% at a temperature of 50°C. + It was confirmed that the content was maintained and the carrying capacity was excellent.

[0120] Experimental Example 2: Skin Permeability

[0121] To evaluate the skin permeability of the liposome complex of the present invention, essences were prepared with the contents shown in Table 2 below.

[0122] To evaluate the skin penetration ability of the liposome complex of the present invention, NAD + The skin permeability was compared by checking the keratin, epidermis, and total permeation amount of the NAD liposome complex prepared in the aqueous solution (Comparative Example 1) and Example 2. As a result, as shown in Fig. 5, NAD + The aqueous solution had high penetration into the stratum corneum, but low epidermal and dermal permeability and total NAD + Although the penetration amount remained at 38.9 μg, it was confirmed that the NAD liposome complex of the present invention penetrated all the amount penetrating the stratum corneum to the skin dermis, and the total NAD + The permeation amount was also 63.7 μg NAD + It was confirmed that it increased by 1.6 times (60%) compared to the aqueous solution.

[0123] Unit (%) Comparative Example 1 Containing Essence Example 2 Containing Essence Purified Water to 100 to 100 Comparative Example 1 10-Example 2-10 Glycerin 55 Dipropylene Glycol 55 Dimethicone 55 1,2-Hexanediol 22 Panthenol 11 Polysorbate 60 11 Acrylate / C10-30 Alkyl Acrylate Crosspolymer 0.2 0.2 Tromethamine 0.2 0.2 EDTA 3NA 0.0 2 0.02

[0124]

[0125] Additionally, to evaluate the skin penetration ability of a cosmetic product comprising the liposome complex of the present invention, NAD +The NAD skin permeation amount of the control essence including an aqueous solution and the essence including the NAD liposome complex of the present invention was compared.

[0126] As a result, as shown in Fig. 6, simple NAD + Compared to the control essence (Comparative Example 1) prepared by including a 0.01% aqueous solution, the essence of the present invention (Example 2) has NAD + It was confirmed that the penetration amount increased by 1.9 times (90%).

[0127]

[0128] Experimental Example 3: Cell aging

[0129] The effect of the liposome complex of the present invention on reducing cell aging genes was confirmed.

[0130] To confirm the skin aging inhibition effect, the aging degree of human fibroblasts and the expression levels of aging genes P16, CAV1, and P21 were compared.

[0131] As a result, as shown in Fig. 7, the NAD liposome complex of the present invention contains NAD + It was confirmed that the production of β-galactosidase, a cell senescence enzyme, was reduced by approximately 24.83% compared to the control alone (Comparative Example 1).

[0132] In addition, as shown in Figures 8, 9 and 10, the aging genes P16, CAV1 and P21 expressed by oxidative stress are NAD + Compared to the single (comparative example 1), it was reduced by 12.58%, 6.06%, and 16.75%, respectively.

[0133]

[0134] Experimental Example 4: Inhibitory Effect of Oxidative Stress

[0135] To evaluate the inhibitory effect of NAD liposome complex on cellular oxidative stress, dichlorofluorescein diacetate (DCFDA) assay was used.

[0136] As a result, as shown in Fig. 11, the NAD liposome complex of the present invention (Experimental Example 2) contains NAD + It was confirmed that the oxidative stress caused by H2O2 was reduced by approximately 6.63% compared to alone (Comparative Example 1).

[0137]

[0138] Experimental Example 5: Cell regeneration effect

[0139] A scratch assay was performed to evaluate the cell regeneration-promoting effect of the NAD liposome complex.

[0140] Specifically, 24 hours prior to the experiment, fibroblast cells (HS68) were cultured in a 24-well culture plate. After 24 hours, the culture medium was removed, and after UV irradiation, some of the cells attached to the bottom were scraped off using a scraper. After 24 hours, the culture medium containing the experimental sample was treated, and the area of ​​regenerated cells was calculated.

[0141] As a result, as shown in FIGS. 12 and 13, the NAD liposome complex of the present invention (Example 2) was found to increase cell regeneration after UV irradiation by approximately 52.83% compared to the group that was not treated after UV irradiation.

[0142]

[0143] To confirm the cell regeneration effect under NAD+ depletion conditions, the expression levels of cell regeneration-related gene markers were analyzed using RT-qPCR.

[0144] Specifically, RNA was extracted after 48 hours of treatment with a culture medium containing experimental substances in fibroblasts. cDNA was synthesized from the extracted RNA, and RT-qPCR was performed.

[0145] As a result, as shown in Fig. 14, the NAD liposome complex of the present invention (Example 2) contains NAD +Compared to the single (comparative example 1), it was confirmed that COL3A1 increased by 2.86%, FGF2 by 4.30%, TFTB1 by 12.62%, and PDGFB by 91.3%.

[0146]

[0147] Intracellular fluorescence intensity and expression were measured using fluorescence microscopy and flow cytometry.

[0148] Specifically, the Ki67 marker was labeled using immunohistochemistry (IHC). After treating cells with a culture medium containing experimental substances for 48 hours, cells were fixed and treated with a Ki67 detection primary antibody and a fluorescent molecule-conjugated secondary antibody. The average fluorescence intensity and fluorescence positivity distribution within the cells were measured using a flow cytometer and are shown in Figures 15 to 17.

[0149] As a result, as shown in FIG. 15, FIG. 16 and FIG. 17, the NAD liposome complex of the present invention (Experimental Example 2) contains NAD + Compared to the single (comparative example 1), the fluorescence intensity and marker-expressing cell distribution for Ki67, a cell growth marker, were found to increase by 130.87% and 122.89%, respectively.

[0150]

[0151] To analyze the recovery of fibrillin expression, fluorescence imaging and fluorescence intensity were measured according to the presence or absence of NAD liposome complex treatment after UV irradiation of fibrillin.

[0152] Specifically, fibrillin markers were labeled using immunohistochemistry (IHC). After treating cells with a culture medium containing experimental substances for 48 hours, cells were fixed and treated with a fibrillin-detecting primary antibody and a fluorescent molecule-conjugated secondary antibody.

[0153] As a result, as shown in FIGS. 18 and 19, the NAD liposome complex of the present invention (Experimental Example 2) contains NAD + It was confirmed that the expression of fibrillin, which had been reduced after UV irradiation compared to single (Comparative Example 1), was restored by approximately 38.33%.

[0154]

[0155] After applying the test substance to the artificial skin for 3 days, the artificial skin was sectioned and stained using the Masson trichrome staining technique, and the area of ​​generated collagen was calculated.

[0156] As a result, as shown in FIGS. 20 and 21, the NAD liposome complex of the present invention (Experimental Example 2) was found to increase the amount of collagen produced in the skin dermis by 193% compared to the existing untreated complex.

[0157]

[0158] Experimental Example 6: Skin Safety

[0159] The skin safety of the liposome complex of the present invention was evaluated.

[0160] Base formulation: NAD liposome-containing essence; Skin irritation index: 0.100.15

[0161]

[0162] To assess the skin stability of cosmetic compositions containing the liposome complex of the present invention, skin irritation indices were compared. As a result, as shown in Table 3, the essence containing the NAD liposome complex of the present invention exhibited a skin irritation index of 0.15, which is lower than 0.2, demonstrating low irritation, indicating a level of stability suitable for use on skin.

[0163]

[0164] In summary of the examples and experimental examples of the present invention, a cosmetic composition including an NAD liposome complex manufactured by the manufacturing method of the present invention has solved the existing problem of aggregation, thereby ensuring formulation stability, and has been confirmed to be effective in preventing skin aging by increasing skin permeability in the form of an NAD liposome complex.

[0165]

[0166] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. Hydrogenated lecithin, fatty acids, and nicotinamide adenine dinucleotide (NAD) + ), NAD liposome complex.

2. In paragraph 1, The above NAD liposome complex is an NAD liposome complex in which the nicotinamide adenine dinucleotide is associated with the surface of the liposome through ionic bonding.

3. In paragraph 1, An NAD liposome complex having a surface charge of -60 to 5 mV.

4. In paragraph 1, An NAD liposome complex wherein the fatty acid has a monovalent, divalent or trivalent anionization degree.

5. In paragraph 1, An NAD liposome complex comprising the fatty acid in an amount of 0.001 to 1 wt% relative to the total weight of the NAD liposome complex.

6. In paragraph 1, An NAD liposome complex, wherein the nicotinamide adenine dinucleotide is contained in an amount of 0.01 to 30 wt% relative to the total weight of the liposome complex.

7. In paragraph 1, An NAD liposome complex wherein the nicotinamide adenine dinucleotide is loaded on the surface or core of the liposome, and the content ratio of the core: nicotinamide adenine dinucleotide loaded on the surface is 50:50 to 0:

100.

8. In paragraph 1, An NAD liposome complex, wherein the hydrogenated lecithin is contained in an amount of 0.005 to 5 wt% relative to the total weight of the NAD liposome complex.

9. In paragraph 1, An NAD liposome complex wherein the nicotinamide adenine dinucleotide is loaded in a weight ratio of 1:0.5 to 200 with respect to the hydrogenated lecithin.

10. In paragraph 1, An NAD liposome complex having an average diameter of 50 nm to 500 nm.

11. In paragraph 1, An NAD liposome complex further comprising a solvent in the above NAD liposome complex.

12. In paragraph 11, The above solvent is an NAD liposome complex containing dipropylene glycol.

13. In paragraph 1, An NAD liposome complex further comprising a nonionic surfactant in the above NAD liposome complex.

14. In paragraph 13, The above nonionic surfactant is an NAD liposome complex comprising sorbitan oleate.

15. A cosmetic composition comprising the NAD liposome complex of any one of claims 1 to 14.

16. A method for preparing an NAD liposome complex according to any one of claims 1 to 14, A step of forming a liposome with a solution containing a lipid, an anionic fatty acid, and a solvent; forming a mixture comprising nicotinamide adenine dinucleotide and a solvent; and A method for producing an NAD liposome complex, comprising the step of combining the above liposome and the above mixture.

Images