Anti-aging composition, preparation method and application thereof, cosmetics and skin care products

Abstract

The invention discloses an anti-aging composition which comprises DNA (deoxyribonucleic acid) sodium, enzyme-digested hyaluronic acid and madecassoside, and the mass ratio of the DNA sodium to the enzyme-digested hyaluronic acid to the madecassoside is (0.01-1): (0.01-1): (0.01-0.5). The invention also provides a preparation method and application of the anti-aging composition, cosmetics and skin care products. The anti-aging composition acts on epidermal cells and dermal cells at the same time, collagen synthesis and hyaluronic acid synthesis can be improved, the effects of barrier repairing and skin filling are achieved, and the purpose of multi-dimensional anti-aging is achieved.

Description

Technical Field

[0001] The invention belongs to the field of skin care products, and in particular relates to an anti-aging substance and a preparation method, application, cosmetics and skin care products thereof. Background Art

[0002] Skin aging is a degeneration of skin structure and a decline in function, which becomes more obvious with age. Skin aging is a direct manifestation of human aging and is the result of the interaction between the external environment and genetic material. It manifests itself as abnormalities in skin structure, function and appearance, such as sagging, wrinkles, abnormal pigmentation and other problems. Although the skin aging process cannot be avoided, it can be delayed through appropriate maintenance methods, which is also the fundamental reason why anti-aging skin care products are enduring and continue to be popular.

[0003] The skin consists of three parts: the epidermis, the dermis, and the subcutaneous tissue. The epidermis is mainly composed of keratinocytes, while the dermis is mainly composed of fibroblasts and endothelial cells. Different layers of the skin have different forms of aging. When the skin begins to age, the renewal of epidermal cells slows down, the epidermis becomes rough and pigmented, and is accompanied by problems such as poor barrier function and reduced skin thickness. When the dermis ages, the proliferation rate of dermal fibroblasts slows down, the ability to synthesize collagen and elastin in the extracellular matrix ECM decreases, and the activity of matrix metalloproteinases gradually increases with age, resulting in a decrease in the collagen content in the ECM. In addition, similar situations occur with macromolecules such as hyaluronic acid, leading to problems such as sagging skin and wrinkles.

[0004] The technical problems existing in the prior art are as follows:

[0005] 1. Not paying attention to the impact of the epidermis on the development of skin aging: Generally speaking, skin aging is mainly related to the dermis, collagen, extracellular matrix, etc. In particular, anti-aging by promoting collagen synthesis has basically become the preferred method in the industry. However, most researchers ignore the impact of the epidermis in the process of skin aging. The aging of the epidermis will lead to slower cell proliferation and migration, increase the transepidermal water loss of the skin, and make it more susceptible to damage, accompanied by dehydration, wrinkles, fine lines and other problems, which will further accelerate the formation of skin wrinkles. There are few reports on research on delaying skin aging by promoting the proliferation and migration of keratinocytes.

[0006] 2. Not acting on the epidermis and dermis at the same time: Many anti-aging products focus on collagen, free radicals, antioxidants and other dimensions, and a small number of patents use fibroblasts as efficacy targets. Keratinocytes are mainly reflected in inventions related to barrier repair, and there are few related reports in anti-aging inventions. Reports that use fibroblasts and keratinocytes as anti-aging targets at the same time are even rarer. Summary of the invention

[0007] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the above background technology and provide an anti-aging composition with many advantages such as good anti-aging effect, simple ingredients, high safety, good compatibility, and a wide range of applications, as well as its preparation method, application, cosmetics and skin care products.

[0008] In order to solve the above technical problems, the technical solution proposed by the present invention is:

[0009] An anti-aging composition comprises sodium DNA, enzyme-cleaved hyaluronic acid and madecassoside, wherein the mass ratio of the sodium DNA, enzyme-cleaved hyaluronic acid and madecassoside is (0.01-1): (0.01-1): (0.01-0.5).

[0010] In the above anti-aging composition, preferably, the mass ratio of the sodium DNA, enzyme-cleaved hyaluronic acid and madecassoside is (0.3-1): (0.2-1): (0.1-0.2).

[0011] In the above anti-aging composition, preferably, the mass ratio of the sodium DNA, enzyme-cleaved hyaluronic acid and madecassoside is 0.5:0.2:0.2.

[0012] In the above anti-aging composition, preferably, it also includes disodium EDTA and arginine, the mass ratio of the disodium EDTA to the total mass of the sodium DNA, enzyme-cleaved hyaluronic acid and madecassoside is 1:(1-25), and the mass ratio of the arginine to the total mass of the sodium DNA, enzyme-cleaved hyaluronic acid and madecassoside is 1:(1-25).

[0013] As a general technical concept, the present invention also provides a method for preparing the above-mentioned anti-aging composition, comprising the following steps:

[0014] (1) crush fresh salmon testis, disperse in deionized water, add arginine to adjust the pH value of the solution to 9-11, heat in a water bath and stir, and let stand to cool after the reaction;

[0015] (2) adding disodium EDTA to the reaction solution to adjust the pH value to 6-8, then adding a sufficient amount of saturated sodium chloride aqueous solution to react, centrifuging, and taking the supernatant; adding water to dilute the supernatant, and when the precipitate no longer increases, centrifuging, taking the precipitate, and obtaining the intermediate product 1;

[0016] (3) adding water to dissolve the intermediate product 1, stirring to dissolve, adding arginine to adjust the pH value to 9-11, and homogenizing using a high-pressure homogenizer to obtain an intermediate product 2;

[0017] (4) using a membrane filtration method, intercepting and obtaining filtrates with a molecular weight greater than 1 million, a molecular weight between 100,000 and 100, and a molecular weight less than 100,000, which are filtrate 1, filtrate 2, and filtrate 3, respectively; concentrating and freeze-drying them to obtain product 1, product 2, and product 3, respectively; and mixing product 1, product 2, and product 3 to obtain sodium DNA;

[0018] (5) Dissolving the sodium DNA, enzymatically hydrolyzed hyaluronic acid and madecassoside in water to obtain an anti-aging composition.

[0019] In the above preparation method, preferably, in the step (4), when the product 1, product 2 and product 3 are mixed, the mass ratio of product 1, product 2 and product 3 is (5-7): (2-3): (1-2). When selecting DNA sodium, the present invention limits the molecular weight of DNA sodium, and uses a large molecular weight, a medium molecular weight and a small molecular weight to mix in a mass ratio of (5-7): (2-3): (1-2), which can improve the effect of DNA sodium.

[0020] As a general technical concept, the present invention also provides an application of the above anti-aging composition in skin care products.

[0021] As a general technical concept, the present invention also provides a skin care product, the components of which include the above-mentioned anti-aging composition.

[0022] As a general technical concept, the present invention also provides an application of the above anti-aging composition in cosmetics.

[0023] As a general technical concept, the present invention also provides a cosmetic, the components of which include the above-mentioned anti-aging composition.

[0024] The skin is the largest organ in the human body and the first barrier that isolates the human body from the outside world. The skin is mainly divided into three parts: the epidermis, dermis and subcutaneous tissue. The epidermis can be further divided into the basal layer, the spinous layer, the granular layer, the transparent layer and the stratum corneum, which are mainly composed of keratinocytes. The dermis is mainly composed of the extracellular matrix (ECM) secreted by fibroblasts, including connective tissue composed of glycosaminoglycans, proteoglycans, structural proteins (collagen and elastin) and some special macromolecules (fibronectin, hyaluronic acid) that provide the skin with strong mechanical resistance and elasticity.

[0025] Skin aging can be divided into two types: endogenous aging and exogenous aging. Endogenous aging refers to aging caused by the body's internal factors (mainly genetic factors), and its obvious characteristics are the appearance of wrinkles and sagging skin, which is difficult to avoid. Endogenous aging only accounts for a part of aging, and a large part comes from exogenous aging. The most important influencing factors are photoaging, oxidative stress, particulate matter, environmental and emotional stress, etc. It manifests as roughness of exposed skin parts, deepening and thickening of wrinkles, structural abnormalities, pigmentation, vascular dilation, and epidermal keratinization dyskeratosis.

[0026] The main aging epidermal cells in the epidermis are flat, the life cycle of the spinous cells in the epidermis is shortened, and the granular layer gradually shrinks. The number of cell layers decreases, the epidermis becomes thinner, the tightness of the junction between the epidermis and the dermis decreases, the epidermis bulges, and the dermal papilla decreases. The aging of the epidermis will lead to a slowdown in cell proliferation, a decrease in the epidermal renewal rate, and epidermal atrophy and delayed wound healing. It leads to a decrease in the formation rate of neutral lipids in the stratum corneum barrier, an increase in transepidermal water loss, and the formation of dry, wrinkled, and fine lines on the skin.

[0027] Changes in the dermis structure are the main cause of skin aging. The dermis becomes thinner with aging, especially after the age of 40, the collagen content of women decreases significantly with age. When the dermis ages, the number of fibroblasts decreases, the collagen content gradually decreases, the spatial density of collagen bundles decreases, and the collagen becomes fragmented and lacks elasticity. The reduction of collagen makes the skin more vulnerable to damage, resulting in wrinkles, sagging, depressions and other problems.

[0028] The present invention starts with keratinocytes and fibroblasts, and realizes multi-dimensional anti-aging by promoting epidermal cell proliferation, migration, and dermal proliferation and filling ECM. The present invention selects the organic combination synergistic effect of DNA sodium, enzymatic hyaluronic acid and madecassoside, which can not only promote the proliferation of fibroblasts, increase hyaluronic acid synthesis, but also promote the proliferation and migration of keratinocytes. The composition acts on epidermal cells and dermal cells at the same time, achieves the effects of barrier repair and skin filling, and achieves the purpose of multi-dimensional anti-aging. In addition, in a more preferred scheme, the composition of the present invention also contains a small amount of arginine and disodium EDTA, and the addition of arginine can nourish the skin, provide a nutritional environment for the synergistic effect of the above-mentioned multiple substances, and the use of disodium EDTA can complex heavy metal ions and improve the cell growth environment. By adding arginine and disodium EDTA at the same time, the two are used at the same time to provide support for synergy, which can promote the synergistic effect of DNA sodium, enzymatic hyaluronic acid and madecassoside.

[0029] The mechanism of action and target of each active ingredient are analyzed as follows:

[0030] 1. DNA Sodium

[0031] DNA sodium is a DNA fragment of specific specifications extracted and optimized from salmon germ cells. Salmon DNA is 98% similar to human DNA. The DNA fragment extracted from salmon germ cells is most similar to human DNA and has a significant effect on skin regeneration and repair. DNA sodium mainly performs precise skin regeneration and repair by identifying human signals based on two pathways. Path one is to activate the A2a adenosine receptors on the cell surface, promote the release of cytokines and cell regeneration, and repair damaged cells; Path two is to restore cell proliferation and growth by promoting DNA synthesis or repair. Its main effects are: 1. Promote tissue regeneration and wound healing; 2. Anti-inflammatory acne and lighten scars and acne marks; 3. Anti-wrinkle, firming and anti-aging; 4. Protect cells from ultraviolet damage.

[0032] The DNA sodium of the present invention is not a purchased product. In order to better promote the synergistic effect of DNA sodium, enzymatically hydrolyzed hyaluronic acid and madecassoside, DNA sodium is prepared by the following method:

[0033] Take fresh salmon testis and crush it to a suitable size, and evenly disperse it in deionized water at a ratio of 1:10-1:20. Add arginine to the solution and adjust the pH value of the solution to between 9-11, preferably 10.0. Place the solution in a 60-75℃ water bath for heating and stirring, react for 10-15min and then let it stand to cool. After cooling to room temperature, measure the pH value of the supernatant, and use disodium EDTA to adjust the pH value to 6.0-8.0, preferably 7.0. Add enough saturated sodium chloride aqueous solution to adjust the sodium chloride concentration in the mother liquor to 2mol / L, and centrifuge to take the supernatant. Add deionized water to a sodium chloride concentration of 0.14mol / L, and when the precipitate no longer increases, centrifuge and take the precipitate. After re-dissolving the precipitate with deionized water, adjust the solid content to 0.1%-10%, use arginine to adjust the pH value of the solution to between 9.0-11.0, use a high-pressure homogenizer for processing, and the homogenization pressure is 200-400Bar, and cycle three times. After homogenization, membrane filtration is used for separation. The membrane molecular weight can be divided into 1 million Da and 100,000 Da. Finally, three separated liquids are obtained, namely, molecular weight greater than 1 million Da (high molecular weight), molecular weight 100,000-1 million Da (medium molecular weight), and molecular weight less than 100,000 Da (low molecular weight). After the filtrate is freeze-dried in vacuum, it is mixed in a mass ratio of (5-7): (2-3): (1-2) to obtain DNA sodium solid.

[0034] The DNA sodium of the present invention is a homemade product, which is cheaper than buying finished products, and the DNA sodium of the present invention is homemade according to the synergistic effect of the present invention, and the product performance is more matched with the needs of the present invention. The specific advantages are as follows: In the preparation process of the DNA sodium of the present invention, chemical reagents such as strong acids and strong bases are not used, but the useful components arginine and disodium EDTA are used to adjust the pH value, which eliminates the introduction of irritants from the source. In the preparation process of the DNA sodium of the present invention, based on the absence of the need to add additional additives, only water, sodium chloride, arginine and disodium EDTA are added throughout the process. Even if there are additives remaining in the DNA sodium product, the residual arginine and disodium EDTA are useful ingredients in the composition of the present invention, and there is no need to worry about the safety of the product. In addition, the present invention focuses on the optimization of the molecular weight of DNA sodium. The effect of large molecular weight DNA sodium is mainly moisturizing, while small and medium molecular weight DNA sodium is more conducive to skin penetration and skin target binding. Organically combining large molecular weight, medium molecular weight and small molecular weight DNA sodium can not only achieve the effect of moisturizing and moisturizing, but also further promote the transdermal absorption of DNA sodium, exert its biological activity, and improve the efficacy and effect of DNA sodium raw materials.

[0035] 2. Enzymatic cleavage of hyaluronic acid

[0036] Enzyme-cleaved hyaluronic acid is a hyaluronic acid with extremely low molecular weight (≤5000Da) prepared by biological enzyme-cleavage technology using conventional macromolecular hyaluronic acid as substrate. Due to its extremely low molecular weight, it is endowed with the characteristics of rapid penetration of the epidermis to reach the dermis. In addition, due to the preparation of biological enzyme-cleavage technology, small molecule hyaluronic acid still has extremely high biological activity, so it has the effects of small molecule hyaluronic acid such as scavenging free radicals, reducing the activity of inflammatory factors, inhibiting inflammatory reactions, repairing damaged cells, and resisting skin inflammation and sensitivity caused by various stimuli. The enzyme-cleaved hyaluronic acid aqueous solution is configured to a suitable concentration and is validated in a hyaluronic acid experimental model. It can be seen from the results that the amount of hyaluronic acid generated in fibroblasts increases with the increase in the amount of ultra-small molecular weight hyaluronic acid added, showing concentration-dependent characteristics. The above results show that the prepared ultra-small molecular weight hyaluronic acid can effectively promote the generation of hyaluronic acid in fibroblasts, and the effect gradually increases with the increase in concentration.

[0037] 3. Madecassoside

[0038] Centella asiatica (L.) Urb. is a perennial herbaceous plant of the genus Centella in the family Apiaceae. The stems of Centella asiatica are creeping and slender; the leaves are round, kidney-shaped or horseshoe-shaped, with serrated edges, glabrous on both sides, and a few soft hairs on the back veins; the petioles are glabrous, and the leaf sheaths at the bottom are transparent and membranous; the flowers of Centella asiatica are clustered in umbrellas, purple-red or milky white; the fruits are flat on both sides, spherical, and hairy on the surface. The flowering and fruiting period is from April to October. The whole plant of Centella asiatica is used as medicine, which tastes bitter and pungent, and is cold in nature. It has the effects of clearing away heat and dampness, detoxifying and reducing swelling, relieving jaundice and relieving stranguria, and promoting blood circulation and relieving pain. It is clinically used for damp-heat jaundice, carbuncle swelling and poison, traumatic injury, poisoning diarrhea, sore throat, liver and gallstones and other diseases.

[0039] Madecassoside (MC) is one of the main components of triterpenoid saponins in Centella asiatica extract, with multiple chemical groups such as hydroxyl and steroid ring. Studies have shown that MC has a wide range of in vitro and in vivo pharmacological activities, and is effective in reducing excessive proliferation, protecting cells, regulating inflammation, and rebuilding and repairing the extracellular matrix. Matrix metalloproteinases (MMPs) are one of the most important proteases that cause matrix component destruction and are also indispensable proteases in the process of cell apoptosis. In the serum cytokine experiment, compared with the model group, the addition of madecassoside can significantly reduce the expression of serum MMP-3 and MMP-13, which is statistically significant. MMP-3 can activate MMP-1 and other family members. Therefore, madecassoside can also inhibit the activity of MMP-1 and other MMPs to a certain extent, thereby reducing the loss of collagen in the skin matrix.

[0040] Compared with the prior art, the advantages of the present invention are:

[0041] The anti-aging composition of the present invention starts from the epidermis and dermis, and realizes multi-dimensional anti-aging by promoting the proliferation of keratinocytes and fibroblasts and filling the ECM layer. The organic combination of DNA sodium, enzymatic hyaluronic acid and madecassoside can not only promote the proliferation of fibroblasts, increase the synthesis of hyaluronic acid, but also promote the proliferation and migration of keratinocytes. The composition acts on epidermal cells and dermal cells at the same time, can improve collagen synthesis and hyaluronic acid synthesis, realize the effects of barrier repair and skin filling, and achieve the purpose of multi-dimensional anti-aging.

[0042] In addition, the composition is composed of active polysaccharides, ribonucleotides and plant extracts, and has many advantages such as simple ingredients, high safety, good compatibility and a wide range of applications, and is bound to have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0044] Figure 1 Graph showing the effect of the anti-aging compositions in Examples and Comparative Examples on the migration rate of cells in HaCaT.

[0045] Figure 2 It is a graph showing the effect of the anti-aging composition in the examples and comparative examples on the proliferation rate of HFF-1 cells.

[0046] Figure 3 This is a graph showing the effect of the anti-aging compositions in the examples and comparative examples on the growth rate of type III collagen in HFF-1 cells. DETAILED DESCRIPTION

[0047] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and meticulously below in conjunction with the accompanying drawings and preferred embodiments of the present invention, but the protection scope of the present invention is not limited to the following specific embodiments.

[0048] Unless otherwise defined, all the professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0049] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0050] Embodiment 1:

[0051] An anti-aging composition comprises sodium DNA, enzyme-cleaved hyaluronic acid and madecassoside, wherein the mass ratios of sodium DNA, enzyme-cleaved hyaluronic acid and madecassoside are shown in Table 1 below.

[0052] DNA sodium is a homemade product, and its preparation method is as follows: fresh salmon testis is crushed to a suitable size and evenly dispersed in deionized water at a ratio of 1:15. Arginine is added to the solution to adjust the pH value of the solution to 10.0. The solution is placed in a 70°C water bath for heating and stirring, and the reaction is allowed to stand and cool after 15 minutes. After cooling to room temperature, the pH value of the supernatant is measured, and the pH value is adjusted to 7.0 using disodium EDTA. Then add a sufficient amount of saturated sodium chloride aqueous solution to adjust the sodium chloride concentration in the mother liquor to 2 mol / L, and centrifuge to obtain the supernatant. Deionized water is added to a sodium chloride concentration of 0.14 mol / L. When the precipitate no longer increases, centrifuge and obtain the precipitate. After the precipitate is redissolved with deionized water, the solid content is adjusted to 5%, and the pH value of the solution is adjusted to 10 using arginine. It is treated with a high-pressure homogenizer with a homogenization pressure of 300 Bar and cycled three times. After homogenization, membrane filtration was used for separation. The membrane molecular weight can be divided into 1 million Da and 100,000 Da. Finally, three parts of separation liquid were obtained, namely, molecular weight greater than 1 million Da (high molecular weight), molecular weight 100,000-1 million Da (medium molecular weight), and molecular weight less than 100,000 Da (low molecular weight). After vacuum freeze drying, the filtrate was mixed at a mass ratio of 6:2:1.5 to obtain DNA sodium solid.

[0053] When preparing the anti-aging composition of this embodiment, sodium DNA, enzyme-cut hyaluronic acid, madecassoside, arginine and disodium EDTA are accurately weighed and measured, and then dissolved in deionized water and stirred evenly to obtain the anti-aging composition.

[0054] Table 1: Composition ratios in Examples and Comparative Examples (unit: g)

[0055]

[0056] Embodiment 2-6:

[0057] The anti-aging compositions in Examples 2-6 are different from those in Example 1 mainly in the different ratios of ingredients. The preparation methods of DNA sodium and the compositions remain the same. For specific ratios of ingredients, see Table 1 above.

[0058] Comparative Examples 1-6:

[0059] The anti-aging compositions in Comparative Examples 1-6 are different from those in Example 1 mainly in that the ratio of ingredients is different, and the preparation methods of DNA sodium and the composition remain the same. For specific ratios of ingredients, see Table 1 above.

[0060] The effects of the compositions with anti-aging effects in Examples 1-6 and Comparative Examples 1-6 were verified as follows:

[0061] (1) Keratinocyte scratch repair

[0062] 1) Test principle

[0063] The wound healing method is a simple method to measure cell migration and repair ability, similar to the in vitro wound healing model. On a monolayer of adherent cells cultured in a culture dish or plate in vitro, use a slot or a gun tip to scratch the central area of ​​cell growth, remove the cells in the central part, and then continue to culture the cells until the time set in the experiment. Take out the cell culture plate and observe the growth and migration ability of the surrounding cells. The experiment usually requires setting up a normal control group and an experimental group. The experimental group is a group that has been treated with certain factors or drugs, exogenous genes, etc. The difference in the ability of cells in different groups to repair the scratch area can be used to determine the effect of external factors (drugs, raw materials, etc.) on cell migration.

[0064] 2) Testing process

[0065] 1. Cell inoculation: Take cells in the logarithmic phase, digest the cells with 0.25% trypsin, use DMEM medium containing 15% FBS as the culture medium, and use sterile tweezers to place the bottom of the 2-well scratch plug on a 6-well plate. Adjust the density of the keratinocyte suspension to 4.0×10 4 70 μL / well was inoculated into a 6-well plate and cultured in a 37°C, 5% CO2 incubator for 24 h.

[0066] 2. Sample test: After the plug is removed, the supernatant is discarded, and the complete medium containing no serum is used as the diluent. According to the sample test concentration table, different concentrations of sample group working solutions (i.e., the anti-aging compositions of Examples 1-6 and Comparative Examples 1-6) are prepared, 2 mL / well, and added to the wells. After culturing in a 37°C, 5% CO2 incubator for 24 hours, the cell morphology is observed under a microscope and photographed. The blank control group is added with an equal amount of serum-free medium, and the positive control group is added with an equal amount of EGF solution with a concentration of 0.05 ng / mL.

[0067] 3. Image processing: Use Image J to analyze the fluorescence intensity and measure the area of ​​the cell scratch gap.

[0068] 3) Test results:

[0069] Calculation formula: W% = (W0 – Wt) × 100%;

[0070] Where W represents the cell migration rate in HaCaT;

[0071] Wt represents the scratch gap area of ​​the sample group after the HaCaT cell test;

[0072] W0 represents the scratch gap area of ​​the blank control group before the HaCaT cell test.

[0073] The data are sorted to obtain the following Table 2 and Figure 1 shown.

[0074] Table 2: Cell migration rate in HaCaT

[0075]

[0076] It can be seen from the chart that the anti-aging composition provided by the present invention promotes the proliferation and migration of HaCaT cells. By comparing the HaCaT cell migration rates in the comparative example and the embodiment, it can be seen that the components of the composition of the present invention have a synergistic effect. When some components are missing, the efficacy of the composition is significantly reduced. In particular, the anti-aging composition in Example 5, the components cooperate synergistically, and the effect is the best. In Example 6, arginine and disodium EDTA are not added, and the effect is inferior to that of Example 5.

[0077] (2) Fibroblast proliferation

[0078] 1) Test background

[0079] MTT is mainly used to determine the toxicity of drugs and raw materials to cells cultured in vitro, as well as for determining cell proliferation and cell activity. In the present invention, MTT is used to detect the proliferation effect of the anti-aging composition on fibroblasts (HFF-1) to explore and verify the optimal concentration and toxicological performance of the cell test.

[0080] 2) Test methods

[0081] 1. Cell inoculation: Adjust the density of fibroblast (HFF-1) suspension to 1.5×10 4 After that, the cells were inoculated into 96-well plates at 100 uL / well and cultured in a 37°C, 5% CO2 incubator for 24 h.

[0082] 2. Experimental grouping: set up a blank control group and a sample group.

[0083] 3. Dilution and addition of samples: Prepare the sample group working solution with complete culture medium as diluent, add 100 μL / well to 96-well plate, add the same amount of complete culture medium to the blank control group, and continue to culture in a 37°C, 5% CO2 incubator for 48 hours.

[0084] 4. Detection: Add 10uL MTT solution (5mg / ml, i.e. 0.5% MTT) to each well and continue culturing for 4h. Measure the absorbance of each well at OD490nm using an enzyme-linked immunosorbent assay (ELISA).

[0085] 5. Retest: Centrifuge, carefully remove the supernatant, add 100uL dimethyl sulfoxide to each well, place on a shaker and shake at low speed for 10 minutes to fully dissolve the crystals. Measure the absorbance of each well at OD490nm using an enzyme-linked immunosorbent assay.

[0086] 3) Test results

[0087] Calculation formula: P% = (Px-Pc) / Pc×100%;

[0088] Where P represents the proliferation rate of HFF-1 cells;

[0089] Px represents the average OD value of the sample group after the HFF-1 cell test;

[0090] Pc represents the average OD value of the blank control group after HFF-1 cell experiment.

[0091] The data are sorted to obtain the following Table 3 and Figure 2 shown.

[0092] Table 3: HFF-1 cell proliferation rate

[0093]

[0094] It can be seen from the chart that the anti-aging composition provided by the present invention promotes the proliferation of HFF-1 cells to a certain extent. By comparing the proliferation rate of fibroblasts in the comparative example and the example, it can be seen that the components of the composition of the present invention have a synergistic effect. When some components are missing, the efficacy of the composition is significantly reduced. In particular, the anti-aging composition in Example 5 has the best effect because the components cooperate synergistically. In Example 6, arginine and disodium EDTA are not added, and the effect is inferior to that of Example 5.

[0095] (3) Type III collagen

[0096] 1) Test principle:

[0097] Type III collagen is one of the many types of collagen in the human body. It is a homotrimer or a protein composed of three identical peptide chains (monomers), each of which is called the α1 chain of type III collagen. At the same time, type III collagen is an essential structural component of many organs and an important regulator of the diameter of type I and type II collagen fibrils. At the same time, type III collagen can also work with new cells and tissues to repair trauma and reshape collagen. Therefore, by testing type III collagen in an aging model, we can help evaluate the anti-aging and repair efficacy of the product.

[0098] 2) Testing process:

[0099] 1. After adjusting the density of the human immortalized fibroblast HFF-1 suspension to 7000 cells / well, inoculate it in a 96-well plate, 100 μL / well, and place it in a 37°C, 5% CO2 incubator for 24 hours. The cells are divided into a blank control group, a model group, and a sample group. The model group and the sample group are dehydrated, 50uL PBS is added, and placed in a UV irradiation instrument, irradiated vertically at a height of 3cm for 1min, PBS is removed, complete culture medium is added, and placed in an incubator for 24 hours; after repeating the operation for 5 days, the sample group is added with the sample to be tested (i.e., the anti-aging composition of Examples 1-6 and Comparative Examples 1-6), 100uL / well, and the model group is added with an equal amount of culture medium. The blank control group is not subjected to UV irradiation, and the other conditions remain unchanged.

[0100] 2. Immunofluorescence test

[0101] A. Sample collection: Aspirate the culture medium and wash the cells three times with PBS;

[0102] B. Cell fixation: Aspirate and discard PBS, add 4% paraformaldehyde fixative to fix the cells, and leave at room temperature for 20 minutes;

[0103] C. Washing: Wash 5 times with PBS to remove residual paraformaldehyde;

[0104] D. Blocking: Use blocking solution 5% BSA (or 5% FBS blocking solution prepared in PBS), 100uL / well, incubate at room temperature for 1 hour;

[0105] E. Primary antibody: Dilute the fluorescent primary antibody at a ratio of 1:50 using immunofluorescence diluent in a dark place. Remove the plate from the solution without washing, add the primary antibody diluent, and incubate overnight at 4°C in a dark place.

[0106] F. Washing: Rewarm for 30 minutes, remove the fluorescent primary antibody in the dark, and wash the cells 3 times with washing solution, 5 minutes each time;

[0107] G. Collect pictures: Keep PBS in each well and observe and take pictures under an inverted fluorescence microscope.

[0108] 3. Image processing: Use ImageJ to analyze the fluorescence results by averaging the fluorescence intensity.

[0109] 4. Statistical analysis: GraphPad Prism 8.0 software was used for data statistical analysis and graphs were drawn. The measured data were expressed as x±s. The differences between the groups were analyzed by one-way ANOVA. P < 0.05 was considered statistically significant.

[0110] 3) Test results:

[0111] Calculation formula: C% = (Cx-Cc) / Cc×100%;

[0112] Where C represents the growth rate of type III collagen in HFF-1 cells;

[0113] Cx represents the mean fluorescence intensity (MFI) of the sample group after the HFF-1 cell test;

[0114] Cc represents the mean fluorescence intensity (MFI) of the model group after HFF-1 cell experiment.

[0115] The data are sorted to obtain the following Table 4 and Figure 3 shown.

[0116] Table 4: Growth rate of type III collagen in HFF-1 cells

[0117]

[0118] It can be seen from the chart that the anti-aging composition provided by the present invention promotes the production of type III collagen in HFF-1 cells. By comparing the growth rate of type III collagen in HFF-1 cells in the comparative example and the example, it can be seen that the components of the composition of the present invention have a synergistic effect. When some components are missing, the efficacy of the composition is significantly reduced. In particular, the anti-aging composition in Example 5 has the best effect because the components cooperate synergistically. In Example 6, arginine and disodium EDTA are not added, and the effect is inferior to that of Example 5.

[0119] It can be seen from the above test results that the anti-aging effects of the anti-aging compositions in Examples 1-6 are significant and significantly better than those in Comparative Examples 1-6, and can be widely used in skin care products and cosmetics. When used, they can be added to skin care products and cosmetics in the form of aqueous solutions. Other components of skin care products and cosmetics can be existing conventional components, which are not limited in the present invention.

Claims

1. An anti-aging composition, characterized in that: The invention comprises DNA sodium, enzyme-cut hyaluronic acid and madecassoside, wherein the mass ratio of the DNA sodium, enzyme-cut hyaluronic acid and madecassoside is (0.01-1): (0.01-1): (0.01-0.5).

2. The anti-aging composition according to claim 1, characterized in that: The mass ratio of the DNA sodium, enzyme-cut hyaluronic acid and madecassoside is (0.3-1): (0.2-1): (0.1-0.2).

3. The anti-aging composition according to claim 2, characterized in that: The mass ratio of the DNA sodium, enzyme-cut hyaluronic acid and madecassoside is 0.5:0.2:0.

2.

4. The anti-aging composition according to any one of claims 1 to 3, characterized in that: It also includes disodium EDTA and arginine, the mass ratio of the disodium EDTA to the total mass of the sodium DNA, enzyme-cleaved hyaluronic acid and madecassoside is 1:(1-25), and the mass ratio of the arginine to the total mass of the sodium DNA, enzyme-cleaved hyaluronic acid and madecassoside is 1:(1-25).

5. A method for preparing the anti-aging composition according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) crush fresh salmon testis, disperse in deionized water, add arginine to adjust the pH value of the solution to 9-11, heat in a water bath and stir, and let stand to cool after the reaction; (2) adding disodium EDTA to the reaction solution to adjust the pH value to 6-8, then adding a sufficient amount of saturated sodium chloride aqueous solution to react, centrifuging, and taking the supernatant; adding water to dilute the supernatant, and when the precipitate no longer increases, centrifuging, taking the precipitate, and obtaining the intermediate product 1; (3) adding water to dissolve the intermediate product 1, stirring to dissolve, adding arginine to adjust the pH value to 9-11, and homogenizing using a high-pressure homogenizer to obtain an intermediate product 2; (4) using a membrane filtration method, intercepting and obtaining filtrates with a molecular weight greater than 1 million, a molecular weight between 100,000 and 100, and a molecular weight less than 100,000, which are filtrate 1, filtrate 2, and filtrate 3, respectively; concentrating and freeze-drying them to obtain product 1, product 2, and product 3, respectively; and mixing product 1, product 2, and product 3 to obtain sodium DNA; (5) Dissolving the sodium DNA, enzymatically hydrolyzed hyaluronic acid and madecassoside in water to obtain an anti-aging composition.

6. The preparation method according to claim 5, characterized in that: In the step (4), when the product 1, product 2 and product 3 are mixed, the mass ratio of product 1, product 2 and product 3 is (5-7): (2-3): (1-2).

7. Use of the anti-aging composition according to any one of claims 1 to 4 or the anti-aging composition prepared by the preparation method according to claim 5 or 6 in skin care products.

8. A skin care product, characterized in that: Its components include the anti-aging composition described in any one of claims 1 to 4 or the anti-aging composition prepared by the preparation method described in claim 5 or 6.

9. Use of the anti-aging composition according to any one of claims 1 to 4 or the anti-aging composition prepared by the preparation method according to claim 5 or 6 in cosmetics.

10. A cosmetic, characterized in that: Its components include the anti-aging composition described in any one of claims 1 to 4 or the anti-aging composition prepared by the preparation method described in claim 5 or 6.

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