Skin repairing composition based on NK cell activity regulation and preparation method thereof

Abstract

The invention relates to the technical field of skin repair. The invention aims to provide a skin repair composition based on NK cell activity regulation. The skin repair composition comprises the following raw materials in percentage by weight: 10-20% of an astragalus extract, 5-10% of paeoniflorin, 5-10% of a liquorice extract, 2-5% of sodium hyaluronate, 5-10% of oligopeptide, 2-3% of an emulsifier and the balance of deionized water. The radix astragali seu hedysari extract, paeoniflorin and liquorice root extract are taken as cores. Astragalus polysaccharide and paeoniflorin in the astragalus extract synergistically activate NK cells, promote the NK cells to secrete cell factors such as IFN-gamma and accelerate skin cell repair and regeneration; meanwhile, cycloastragenol contained in the radix astragali extract inhibits telomerase shortening, maintains the activity of skin stem cells and delays cell senescence. The radix glycyrrhizae extract serves as a synergistic transmitter, adjusts the inflammation microenvironment, activates an NF-kappa B signal channel, enhances the activation effect of the radix astragali extract and paeoniflorin on NK cells, promotes the synergistic effect of all the components, reduces scar formation and jointly improves the skin repair efficiency and quality.

Description

Technical Field

[0001] The present invention relates to the technical field of skin repair, and in particular to a skin repair composition based on NK cell activity regulation and a preparation method thereof. Background Art

[0002] As the largest organ in the human body, the skin plays a critical role in defending against external damage and maintaining a stable internal environment. However, repairing skin damage caused by factors such as trauma, burns, chronic ulcers, and skin aging remains a research challenge in both medicine and cosmetics. Traditional repair methods rely on exogenous growth factors, cell transplantation, or biological dressings, but these methods are associated with high costs, the risk of immune rejection, and poor storage stability. With advances in immunology and cell biology, immune cell-mediated tissue repair has become an emerging research direction. Natural killer (NK) cells, as a key component of innate immunity, not only possess anti-tumor and antiviral activities but also promote skin repair by secreting cytokines such as interferon-γ (IFN-γ) and granulocyte-macrophage colony-stimulating factor (GM-CSF), regulating fibroblast proliferation and extracellular matrix synthesis. Furthermore, telomere length is closely associated with cellular aging, and modulation of telomerase activity has been shown to prolong the lifespan of skin stem cells and maintain their repair capacity. However, synergistic skin repair strategies based on NK cell activity regulation and telomerase inhibition have not been reported. Summary of the Invention

[0003] The purpose of the present invention is to provide a skin repair composition based on NK cell activity regulation and a preparation method thereof. The skin repair composition activates the activity of NK cells, fully exerts the immune killing effect of NK cells, activates the body's self-healing and repair function, and cooperates with cycloastragenol to inhibit telomerase shortening and maintain healthy cell activity. Licorice extract can enhance synergy, regulate inflammation, promote collagen synthesis, reduce scars, and achieve efficient skin repair.

[0004] To achieve the above-mentioned purpose of the invention, the technical solution adopted by the present invention is a skin repair composition based on NK cell activity regulation, comprising the following raw materials in the following weight percentages: astragalus extract 10-20%, paeoniflorin 5-10%, licorice extract 5-10%, sodium hyaluronate 2-5%, oligopeptide 5-10%, emulsifier 2-3%, and the balance is deionized water.

[0005] Preferably, the purity of the paeoniflorin is ≥98%.

[0006] Preferably, the preparation method of the skin repair composition comprises the following steps:

[0007] S1. Raw material pretreatment

[0008] Astragalus extract: Select the extract with cycloastragenol content ≥10%, and pass it through an 80-mesh sieve to remove impurities;

[0009] Paeoniflorin: Use pharmaceutical grade raw materials with a purity of ≥98%, crush to 100 mesh powder for later use;

[0010] Licorice extract: Prepare a 10% pre-solution with 95% ethanol;

[0011] S2. Solution Preparation

[0012] Preparation of sodium hyaluronate solution: Take 3 times the amount of deionized water as sodium hyaluronate, slowly add sodium hyaluronate into 40℃ deionized water, and stir at a low speed of 80r / min for 1 hour;

[0013] Active ingredient premixing: Add 3 times the amount of astragalus extract to deionized water, stir at 150 rpm for 30 minutes, then add paeoniflorin, stir at 120 rpm for 20 minutes to obtain an astragalus extract and paeoniflorin pre-solution;

[0014] S3. Mixing and emulsification

[0015] Basic mixing: Add oligopeptides to sodium hyaluronate solution, stir at 60°C and 100 r / min for 10 minutes; then add astragalus extract and paeoniflorin pre-solution, and slowly drop the licorice extract pre-solution, then add the remaining deionized water, maintain 60°C, and stir at 120 r / min for 20 minutes to obtain the basic solution;

[0016] Emulsification molding: add 5g of emulsifier to the base solution, heat to 75℃, start the high-speed homogenizer, and process at 3000r / min for 15 minutes to form a uniform emulsion; then continue stirring at 80r / min at 50℃ for 30 minutes to promote system stability.

[0017] Preferably, the preparation method of the astragalus extract is as follows: take dried astragalus medicinal materials, grind them into coarse powder, add 70% ethanol solution at a material-liquid ratio of 1:10, and ultrasonically extract twice at 60°C, each time for 40 minutes, with an ultrasonic power of 300W; combine the extracts, and concentrate under reduced pressure until there is no alcohol taste, the concentration temperature is controlled at 50°C, and the vacuum degree is -0.06MPa; the concentrate is purified by a macroporous adsorption resin column, eluted with deionized water and 50% ethanol in sequence, and the 50% ethanol eluate is collected; the eluate is concentrated under reduced pressure again and then freeze-dried to obtain an extract rich in cycloastragenol and astragalus polysaccharides.

[0018] Preferably, the preparation method of the licorice extract is as follows: take dry and mold-free licorice raw materials, remove impurities and then grind them into coarse powder, add water at a solid-liquid ratio of 1:10, and stir and extract at 80°C for 2 hours; filter while hot, and extract the residue twice; combine the filtrate, and concentrate under reduced pressure at 60°C and -0.08MPa; purify the concentrate with macroporous adsorption resin, remove impurities with water and elute with 70% ethanol; the eluate is spray-dried or freeze-dried to obtain licorice extract powder.

[0019] The beneficial effects of the present invention are concentrated in the following aspects: the skin repair composition is based on astragalus extract, paeoniflorin, and licorice extract. Astragalus polysaccharide and paeoniflorin in the astragalus extract synergistically activate NK cells, prompting them to secrete cytokines such as IFN-γ, accelerating skin cell repair and regeneration. At the same time, cycloastragenol contained in the astragalus extract inhibits telomerase shortening, maintains skin stem cell activity, and delays cell aging. Licorice extract acts as a co-transmitter, regulating the inflammatory microenvironment and activating the NF-κB signaling pathway, enhancing the NK cell activation effects of astragalus extract and paeoniflorin, promoting synergistic effects among the components, reducing scar formation, and collectively improving the efficiency and quality of skin repair. DETAILED DESCRIPTION

[0020] The present invention is a skin repair composition based on NK cell activity regulation, comprising the following raw materials in the following weight percentages: 10-20% astragalus extract, 5-10% paeoniflorin, 5-10% licorice extract, 2-5% sodium hyaluronate, 5-10% oligopeptide, 2-3% emulsifier, and the balance being deionized water. Paeoniflorin is the main NK cell activity activator of the present invention, and its purity is ≥98%. The oligopeptide is one or more of oligopeptide-1, oligopeptide-3, oligopeptide-5, palmitoyl pentapeptide-4, and acetyl hexapeptide-8.

[0021] The preparation method of the astragalus extract comprises the following steps: taking dried astragalus medicinal materials, grinding them into coarse powder, adding 70% ethanol solution at a material-liquid ratio of 1:10, performing ultrasonic extraction twice at 60° C. for 40 minutes each time, and using an ultrasonic power of 300 W; combining the extracts, concentrating under reduced pressure until there is no alcohol taste, controlling the concentration temperature at 50° C. and the vacuum degree at -0.06 MPa; purifying the concentrate through a macroporous adsorption resin column, eluting with deionized water and 50% ethanol in sequence, and collecting the 50% ethanol eluate; concentrating the eluate under reduced pressure again, and then freeze-drying to obtain an extract rich in cycloastragenol and astragalus polysaccharide.

[0022] The preparation method of the licorice extract comprises the following steps: taking dried, mold-free licorice raw materials, removing impurities and then grinding them into coarse powder, adding water at a solid-liquid ratio of 1:10, and extracting with stirring at 80° C. for 2 hours; filtering while hot, and extracting the filter residue twice; combining the filtrates, and concentrating under reduced pressure at 60° C. and -0.08 MPa; purifying the concentrate with a macroporous adsorption resin, removing impurities with water, and eluting with 70% ethanol; and spray drying or freeze drying the eluate to obtain licorice extract powder.

[0023] The overall preparation method of the skin repair composition comprises the following steps:

[0024] S1. Raw material pretreatment

[0025] Astragalus extract: Select the extract with cycloastragenol content ≥10%, and pass it through an 80-mesh sieve to remove impurities;

[0026] Paeoniflorin: Use pharmaceutical grade raw materials with a purity of ≥98%, crush to 100 mesh powder for later use;

[0027] Licorice extract: Prepare a 10% pre-solution with 95% ethanol;

[0028] S2. Solution Preparation

[0029] Preparation of sodium hyaluronate solution: Take 3 times the amount of deionized water as sodium hyaluronate, slowly add sodium hyaluronate into 40℃ deionized water, and stir at a low speed of 80r / min for 1 hour;

[0030] Active ingredient premixing: Add 3 times the amount of astragalus extract to deionized water, stir at 150 rpm for 30 minutes, then add paeoniflorin, stir at 120 rpm for 20 minutes to obtain an astragalus extract and paeoniflorin pre-solution;

[0031] S3. Mixing and emulsification

[0032] Basic mixing: Add oligopeptides to sodium hyaluronate solution, stir at 60°C and 100 r / min for 10 minutes; then add astragalus extract and paeoniflorin pre-solution, and slowly drop the licorice extract pre-solution, then add the remaining deionized water, maintain 60°C, and stir at 120 r / min for 20 minutes to obtain the basic solution;

[0033] Emulsification molding: add 5g of emulsifier to the base solution, heat to 75℃, start the high-speed homogenizer, and process at 3000r / min for 15 minutes to form a uniform emulsion; then continue stirring at 80r / min at 50℃ for 30 minutes to promote system stability.

[0034] In this invention, astragalus extract is one of the core ingredients in the skin repair composition, and its benefits are manifested in multiple dimensions. From an immunomodulatory perspective, astragalus polysaccharides, rich in astragalus extract, can activate natural killer (NK) cells. NK cells, a crucial line of defense in the human immune system, significantly increase the secretion of cytokines such as interferon-γ (IFN-γ) and granulocyte-macrophage colony-stimulating factor (GM-CSF) upon activation. IFN-γ regulates immune responses in the skin microenvironment, inhibiting the overexpression of inflammatory factors and alleviating skin inflammation symptoms. GM-CSF, on the other hand, promotes the proliferation of fibroblasts and keratinocytes, accelerating the repair process of skin tissue. Furthermore, cycloastragenol, contained in astragalus extract, is an effective telomerase regulator.

[0035] Telomeres are protective structures at the ends of chromosomes. They shorten continuously with cell division. When they reach a critical length, cells enter a state of senescence or apoptosis. Cycloastragenol can specifically bind to telomerase, slowing the rate of telomere shortening and maintaining the proliferation and activity of skin stem cells, thereby extending cell lifespan and providing a continuous cell source for skin repair. Furthermore, astragalus extract has antioxidant properties. Its flavonoids, phenols, and other ingredients can scavenge free radicals within skin cells, reducing damage to cells caused by oxidative stress, protecting the integrity of cell membranes, and further promoting normal metabolism and repair of skin cells.

[0036] Paeoniflorin, the primary NK cell activator in this invention, plays a crucial role in NK cell activation during skin repair. By modulating intracellular signaling pathways, it enhances NK cells' ability to recognize and kill target cells, while also promoting the secretion of various bioactive substances. When acting on NK cells, paeoniflorin activates the phosphatidylinositol 3-kinase (PI3K) / protein kinase B (Akt) signaling pathway, promoting the activation of related transcription factors within NK cells. This in turn upregulates the gene expression of cytokines such as IFN-γ and tumor necrosis factor-α (TNF-α), increasing their secretion. These cytokines not only regulate immune cell function but also directly act on skin cells, stimulating fibroblasts to synthesize and secrete extracellular matrix components such as collagen and elastin, enhancing skin elasticity and toughness and promoting the structural reconstruction of damaged skin tissue. In addition, paeoniflorin exhibits anti-inflammatory properties. It can inhibit the activation of the nuclear factor κB (NF-κB) signaling pathway, reduce the production and release of inflammatory mediators such as interleukin-1β (IL-1β) and interleukin-6 (IL-6), alleviate skin inflammation, relieve symptoms such as redness, swelling, and pain, and create a relatively stable microenvironment for skin repair. At the same time, paeoniflorin can regulate the proliferation and differentiation of skin cells, promote the normal differentiation of keratinocytes, accelerate the repair and regeneration of the epidermis, and help restore the skin's barrier function.

[0037] Licorice extract plays a unique and critical role in skin repair compositions. The main active ingredients in licorice extract include glycyrrhizic acid and glycyrrhizin. Glycyrrhizic acid has potent anti-inflammatory and immunomodulatory properties. Its structure is similar to that of adrenal cortex hormones, competitively inhibiting the activity of phospholipase A2 in inflammatory cells, reducing the release of arachidonic acid, and thereby blocking the synthesis of inflammatory mediators such as prostaglandins and leukotrienes, thereby alleviating the inflammatory response at its source. Glycyrrhizic acid can also regulate the function of immune cells, inhibiting the overactivation of T lymphocytes, reducing the secretion of proinflammatory cytokines, and promoting the production of anti-inflammatory cytokines such as interleukin-10 (IL-10), thereby achieving a balanced immune microenvironment in the skin. Glycyrrhizin is a highly effective antioxidant that scavenges various free radicals, including superoxide anion radicals and hydroxyl radicals, inhibiting lipid peroxidation and protecting skin cells from oxidative damage. Licorice extract also has antibacterial properties. The flavonoids it contains inhibit common skin pathogens such as Staphylococcus aureus and Escherichia coli, reducing the risk of bacterial infection and preventing skin wounds from further damage or inflammation caused by bacterial invasion, thereby providing a favorable external environment for skin repair. Furthermore, licorice extract can also promote skin cell metabolism, accelerate cell renewal and repair, and enhance the skin's self-repair ability.

[0038] Astragalus extract and paeoniflorin, as NK cell activators, exhibit a significant synergistic effect in enhancing NK cell activity. Astragalus polysaccharides and paeoniflorin in astragalus extract jointly activate NK cells through different signaling pathways, prompting them to secrete more cytokines, exerting a synergistic effect in regulating immunity and promoting cell proliferation. Licorice extract, on the other hand, plays a key role as a co-transmitter. Its anti-inflammatory and antioxidant properties can reduce skin inflammation and oxidative damage, creating a favorable microenvironment for astragalus extract and paeoniflorin to exert their effects. Furthermore, the active ingredients in licorice extract modulate intracellular signaling pathways, enhancing the binding of astragalus extract and paeoniflorin to cell receptors and promoting intracellular signaling, further enhancing NK cell activation and skin repair efficiency. The three ingredients work synergistically across multiple levels, including immune regulation, cell activity maintenance, inflammation control, and antimicrobial protection, to promote the repair and regeneration of damaged skin tissue, improving skin health and repair quality.

[0039] The present invention will be further described below in conjunction with examples and experiments, but the examples do not limit the present invention in any form. Unless otherwise stated, the raw materials and reagents used in the examples of the present invention are conventionally purchased raw materials and reagents.

[0040] According to the ratios described in Table 1, the skin repair composition of each example was prepared.

[0041] Table 1

[0042] Case No. Example 1 Example 2 Comparative Example 1 Comparative Example 2 Comparative Example 3 Astragalus extract 20 10 30 0 20 Paeoniflorin 10 5 0 30 10 Licorice Extract 10 5 0 0 0 Sodium hyaluronate 5 2 5 5 5 oligopeptides 10 5 10 10 10 emulsifiers 3 2 3 3 3 Deionized water margin margin margin margin margin

[0043] 1. Experimental Process

[0044] 1. Experimental Subjects

[0045] A total of 140 12-month-old SD rats were selected. A 1 cm2 area on the back of the model rats was shaved as the experimental area. The experimental area was scalded with boiling water. After each scalding, the experimental area was observed for 3 minutes until redness, swelling, and blisters appeared on the skin of the model rats' backs. This was to establish a scald model rat.

[0046] The scald model mice were randomly divided into 7 groups, with 20 mice in each group.

[0047] 2. Experimental Methods

[0048] Seven groups of burn model mice were labeled and treated as follows for four consecutive weeks:

[0049] Blank control group: no treatment;

[0050] Normal saline group: Apply normal saline once in the morning and evening;

[0051] High-dose group: Apply the skin repair composition of Example 1 once in the morning and once in the evening;

[0052] Low-dose group: Apply the skin repair composition of Example 2 once in the morning and once in the evening;

[0053] Astragalus extract group: Apply the skin repair composition of control example 1 once in the morning and evening;

[0054] Paeoniflorin-only group: Apply the skin repair composition of control example 2 once in the morning and evening;

[0055] Astragalus extract and paeoniflorin group: Apply the skin repair composition of control example 3 once in the morning and once in the evening;

[0056] 2. Experimental Results

[0057] After the experiment, the model mice were killed and the back skin tissue was taken for examination;

[0058] Testing indicators and data

[0059] 1. NK cell activity: IFN-γ secretion was detected (ELISA method), as shown in Table 2;

[0060] Table 2

[0061] Group IFN-γ secretion (pg / ml) Blank control group 24.5±2.1 Normal saline group 46.5±3.2 High-dose group 178.6±4.2 Low-dose group 145.3±3.7 Astragalus extract group 124.5±4.3 Single paeoniflorin group 98.4±3.4 Astragalus extract and paeoniflorin group 130.4±4.6

[0062] 2. Telomerase activity: Telomerase activity was detected by TRAP method, as shown in Table 3;

[0063] Table 3

[0064] Group Telomerase activity (%), with the blank control group as 1 Blank control group 100 Normal saline group / High-dose group 230±11 Low-dose group 198±6 Astragalus extract group 298±13 Single paeoniflorin group 102±2 Astragalus extract and paeoniflorin group 210±7

[0065] 3. Inflammatory factor levels: Detection of IL-1β content (ELISA method), as shown in Table 4;

[0066] Table 4

[0067] Group IL-1β content (pg / ml) Blank control group 134.2±4.9 Normal saline group 118.6±4.7 High-dose group 21.4±3.3 Low-dose group 35.7±5.5 Astragalus extract group 98.6±5.2 Single paeoniflorin group 122.4±4.8 Astragalus extract and paeoniflorin group 142.4±8.6

[0068] 4. Cell proliferation ability: The proliferation rate of fibroblasts was detected by CCK-8 method, as shown in Table 5;

[0069] Table 5

[0070] Group Fibroblast proliferation rate (%), with the blank control group as 1 Blank control group 100 Normal saline group 98±11 High-dose group 215±4 Low-dose group 185±3 Astragalus extract group 178±6 Single paeoniflorin group 132±9 Astragalus extract and paeoniflorin group 164±4

[0071] 5. Antibacterial properties: The diameter of the inhibition zone against Staphylococcus aureus was detected by agar diffusion method; as shown in Table 6

[0072] Table 6

[0073] Group Antibacterial diameter (mm) Blank control group / Normal saline group 6.7±0.4 High-dose group 10.4±1.2 Low-dose group 9.8±0.8 Astragalus extract group 7.5±0.6 Single paeoniflorin group 7.7±0.4 Astragalus extract and paeoniflorin group 7.5±0.7

[0074] In summary, the skin repair composition of the present invention has excellent performance in activating NK cell activity, activating telomerase activity, inflammation control level, promoting cell proliferation and repair, antibacterial properties, etc., and it is particularly prominent that when the licorice extract is missing as a reconciling medium, when the astragalus extract and paeoniflorin group are used alone as NK cell activity activators, the inflammation level is even higher than that of the rubber blank control group and the normal saline group. The inventor speculates that this is related to the following reasons: the use of astragalus extract and paeoniflorin alone to activate NK cells leads to an increase in inflammation level, mainly due to three reasons. First, after NK cell activation, the immune response is enhanced, more cytokines are released, macrophages are recruited and activated, etc., which promotes the increase of inflammatory mediators; second, the activation of NK cells by the two disrupts the balance of the cytokine network, the pro-inflammatory cytokines are relatively increased, and the anti-inflammatory regulation is insufficient; third, the astragalus extract and paeoniflorin have too strong an effect on NK cell activation, causing excessive stress on the immune system, and immune cells release a large amount of inflammatory mediators, which ultimately leads to an increase in inflammation level.

Claims

1. A skin repair composition based on NK cell activity regulation, characterized in that: The raw materials include the following weight percentages: 10-20% astragalus extract, 5-10% paeoniflorin, 5-10% licorice extract, 2-5% sodium hyaluronate, 5-10% oligopeptide, 2-3% emulsifier, and the balance is deionized water.

2. The skin repair composition based on NK cell activity regulation according to claim 1, characterized in that: The purity of the paeoniflorin is ≥98%.

3. The method for preparing a skin repair composition based on NK cell activity regulation according to any one of claims 1-2, characterized in that: The following steps are involved: S1. Raw material pretreatment Astragalus extract: Select the extract with cycloastragenol content ≥10%, and pass it through an 80-mesh sieve to remove impurities; Paeoniflorin: Use pharmaceutical grade raw materials with a purity of ≥98%, crush to 100 mesh powder for later use; Licorice extract: Prepare a 10% pre-solution with 95% ethanol; S2. Solution Preparation Preparation of sodium hyaluronate solution: Take 3 times the amount of deionized water as sodium hyaluronate, slowly add sodium hyaluronate into 40℃ deionized water, and stir at a low speed of 80r / min for 1 hour; Active ingredient premixing: Add 3 times the amount of astragalus extract to deionized water, stir at 150 rpm for 30 minutes, then add paeoniflorin, stir at 120 rpm for 20 minutes to obtain an astragalus extract and paeoniflorin pre-solution; S3. Mixing and emulsification Basic mixing: Add oligopeptides to sodium hyaluronate solution, stir at 60°C and 100 r / min for 10 minutes; then add astragalus extract and paeoniflorin pre-solution, and slowly drop the licorice extract pre-solution, then add the remaining deionized water, maintain 60°C, and stir at 120 r / min for 20 minutes to obtain the basic solution; Emulsification molding: add 5g of emulsifier to the base solution, heat to 75℃, start the high-speed homogenizer, and process at 3000r / min for 15 minutes to form a uniform emulsion; Then, stirring was continued at 80 r / min at 50° C. for 30 minutes to promote system stability.

4. The method for preparing a skin repair composition based on NK cell activity regulation according to claim 3, characterized in that: The preparation method of the astragalus extract comprises the following steps: taking dried astragalus medicinal materials, grinding them into coarse powder, adding 70% ethanol solution at a material-liquid ratio of 1:10, performing ultrasonic extraction twice at 60° C. for 40 minutes each time, and using an ultrasonic power of 300 W; combining the extracts, concentrating under reduced pressure until there is no alcohol taste, controlling the concentration temperature at 50° C. and the vacuum degree at -0.06 MPa; purifying the concentrate through a macroporous adsorption resin column, eluting with deionized water and 50% ethanol in sequence, and collecting the 50% ethanol eluate; concentrating the eluate under reduced pressure again, and then freeze-drying to obtain an extract rich in cycloastragenol and astragalus polysaccharide.

5. The method for preparing a skin repair composition based on NK cell activity regulation according to claim 4, characterized in that: The preparation method of the licorice extract comprises the following steps: taking dried, mold-free licorice raw materials, removing impurities and then grinding them into coarse powder, adding water at a solid-liquid ratio of 1:10, and extracting with stirring at 80° C. for 2 hours; filtering while hot, and extracting the filter residue twice; combining the filtrates, and concentrating under reduced pressure at 60° C. and -0.08 MPa; purifying the concentrate with a macroporous adsorption resin, removing impurities with water, and eluting with 70% ethanol; and spray drying or freeze drying the eluate to obtain licorice extract powder.