Soothing and repairing complex applicable to sensitive skin as well as preparation method and application of soothing and repairing complex

Abstract

The invention relates to a soothing and repairing complex suitable for sensitive skin and a preparation method and application thereof, and belongs to the technical field of cosmetics. The soothing and repairing complex provided by the invention is prepared by coating the surface of microalgae extracellular vesicles with stachyose, astragalus polysaccharide and galactoglucan, the four raw material components cooperate with one another to play a role, and the soothing and repairing complex has the advantages that the soothing and repairing complex aims at a passive fragile state caused by skin immune system imbalance and low tolerance threshold; the saccharide-extracellular vesicle compound is used for simulating mild regulation on a skin immune system, so that the resistance of skin to external environment pressure is systematically improved on the premise of no stimulation risk, and the transformation from passive damage-repair to active defense-toughness is realized; the problems that sensitive skin is easy to stimulate and repeatedly red, the barrier function is weak and the like are fundamentally improved.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology, and relates to a soothing and repairing complex suitable for sensitive skin, its preparation method, and its application. Background Technology

[0002] Currently, skincare products for sensitive skin mainly focus on "passive repair." These products typically alleviate discomfort and aid repair after visible symptoms of irritation such as redness, burning, and barrier damage appear, by adding anti-inflammatory agents, moisturizing ingredients, and lipid supplements. While this approach provides immediate relief, it has a fundamental limitation: its mode of action is delayed and reactive, failing to enhance the skin's own resistance before the irritation occurs. Frequent exposure to complex stressors such as environmental pollution, seasonal changes, and psychological stress traps the skin in a cycle of "damage-repair-re-damage," making it difficult to break free from its sensitive and fragile state.

[0003] While some existing technologies propose concepts like "strengthening the skin barrier" or "enhancing skin immunity," their methods are mostly limited to supplementing physical barrier components or using broad-spectrum antioxidants, failing to delve into the core regulatory mechanisms of the skin's immune system. As an immune-active organ, the skin contains pattern recognition receptors carried by its keratinocytes, enabling it to recognize specific molecular patterns. How to utilize safe and gentle ligands to appropriately modulate these receptors, thereby regulating the skin's immune system's tolerance and enabling it to respond more precisely and stably to challenges, rather than simply inhibiting or overactivating it, remains a gap that current technologies have not effectively addressed. Furthermore, many bioactive ingredients with regulatory potential, such as extracellular vesicles, face problems such as poor stability in formulations, easy inactivation, and difficulty in effectively delivering them to targets, further limiting their practical application effectiveness.

[0004] It is worth noting that research in the field of nanodelivery systems has provided theoretical support for vesicle surface modification. For example, Tenchov et al. pointed out in their study in ACS Nano (2022) that extracellular vesicles can be regarded as "natural lipid nanoparticles," and their phospholipid bilayer structure is highly similar to that of artificial liposomes. Meanwhile, multiple studies have confirmed the feasibility and effectiveness of polysaccharide coating on liposomes. For instance, Wang et al. (Food Sci. Nutr., 2021) and Cong L et al. (Food Chem., 2023) reported on the improvement of liposome performance by polysaccharide modification. A recent study, "Influence of Auricularia cornea Polysaccharide Coating on the Stability and Antioxidant Activity of Liposomes Ginsenoside Rh2," also directly demonstrates that glycoside coating technology can significantly enhance the stability and activity of lipid vesicles, solving the problem of vesicle aggregation. However, transferring the aforementioned sugar-liposome encapsulation technology to microalgal extracellular vesicle systems still faces challenges. How to achieve stable encapsulation of various specific sugars (stachyose, astragalus polysaccharide, galactoglucan) on the surface of microalgal vesicles without damaging their biological activity remains a problem that existing technologies have not yet effectively solved.

[0005] This invention aims to overcome the aforementioned shortcomings of existing technologies and provide a novel skincare strategy. The primary technical problem this invention addresses is to shift from the passive, reactive nature of traditional soothing products to a daily-use solution that actively regulates skin's immune tolerance. Secondly, this invention addresses how to integrate various active ingredients with immunomodulatory and barrier-repairing functions, particularly easily deactivated bioactive components such as extracellular vesicles, into a stable, efficient, and synergistic delivery system. Ultimately, this invention strives not only to alleviate immediate skin sensitivity symptoms but also to fundamentally improve the skin's health baseline, reducing the intensity and frequency of its response to external stimuli, and achieving a transformation from "sensitive and fragile" to "resilient and stable." Summary of the Invention

[0006] The purpose of this invention is to provide a soothing and repairing complex suitable for sensitive skin, its preparation method, and its application. The soothing and repairing complex of this invention is prepared by coating the surface of extracellular vesicles of microalgae with stachyose, astragalus polysaccharide, and galactoglucan. The four raw materials work synergistically to target the passive fragility caused by imbalance of the skin's immune system and low tolerance threshold. Through the sugar-extracellular vesicle complex, it simulates the gentle regulation of the skin's immune system, systematically improving the skin's resistance to external environmental stresses (such as pollutants, seasonal changes, and emotional stress) without the risk of irritation. It achieves a transformation from "passive damage-repair" to "active defense-strengthening", fundamentally improving problems such as irritation, recurrent redness, and weak barrier function in sensitive skin.

[0007] The objective of this invention can be achieved through the following technical solutions: The first aspect of the present invention provides a soothing and repairing complex suitable for sensitive skin, the soothing and repairing complex comprising the following raw materials: stachyose, astragalus polysaccharide, galactoglucan and microalgal extracellular vesicles, wherein the weight ratio of stachyose, astragalus polysaccharide, galactoglucan and microalgal extracellular vesicles is (0.5-5):(0.05-3):(0.01-2):(0.001-1).

[0008] Preferably, the weight ratio of stachyose, astragalus polysaccharide, galactoglucan and microalgal extracellular vesicles is (1-2):(0.1-1):(0.05-0.5):(0.005-0.1).

[0009] More preferably, the weight ratio of stachyose, astragalus polysaccharide, galactoglucan and microalgal extracellular vesicles is (1-1.5):(0.5-0.8):(0.1-0.3):(0.01-0.05).

[0010] A second aspect of the present invention provides a method for preparing the soothing and repairing complex suitable for sensitive skin as described in the first aspect, comprising the following steps: S1. Reconstitute the lyophilized powder of microalgal extracellular vesicles to prepare a suspension of microalgal extracellular vesicles, and place it on ice for later use; dissolve stachyose, astragalus polysaccharide, and galactoglucan in ultrapure water to prepare a mixed sugar stock solution, and filter it through a 0.22μm filter membrane for sterilization. S2. Under magnetic stirring, the microalgal extracellular vesicle suspension is added dropwise at a uniform rate to the sterilized mixed sugar stock solution to obtain a mixed system. S3. Adjust the pH of the mixture to 5.8-6.2 using a pH adjuster, and continue the reaction incubation to obtain the reaction solution; S4. The reaction solution is purified and separated to obtain the soothing and repairing complex.

[0011] Preferably, step S1 specifically involves: adding lyophilized microalgal extracellular vesicle powder to PBS buffer pre-cooled to 4°C and with a pH of 7.2-7.4, vortexing at 800-1200 rpm for 30-60 s to prepare a uniform microalgal extracellular vesicle suspension with a concentration of 0.5-2 mg / mL, and placing it on ice for later use to maintain the biological activity of the extracellular vesicles; dissolving stachyose, astragalus polysaccharide, and galactoglucan in ultrapure water to prepare a mixed sugar stock solution with a concentration of 10-30 mg / mL, and filtering it through a 0.22 μm filter membrane for sterilization.

[0012] Preferably, the magnetic stirring conditions in step S2 are magnetic stirring at 25-37℃ and 200-400 rpm; the uniform dripping time is 15-25 min.

[0013] The magnetic stirring conditions in this step provide the physical energy needed to overcome the electrostatic repulsion barrier. During this process, high-molecular-weight astragalus polysaccharides and galactoglucan primarily serve as the structural framework, forming a hydrogen bond network with the phospholipid heads and surface proteins of the vesicle membrane through their abundant hydroxyl groups. Combined with van der Waals forces and the spatial entanglement of the polymer chains, this forms a stable "glyco-vesicle" complex. Meanwhile, small-molecule stachyose is interspersed between the polysaccharide chains or embedded in the hydration layer of the glyco-vesicle complex through hydrogen bonding, synergistically regulating the system's osmotic pressure and providing immunomodulatory activity. This coating process overcomes the potential electrostatic repulsion between the polysaccharides and the vesicle surface. The low-power-density stirring avoids the damage to vesicle integrity caused by high-intensity methods such as ultrasound, improves the aggregation of extracellular vesicles in water, and contributes to the uniform dispersion and stability of the vesicles.

[0014] Preferably, the pH adjuster in step S3 is selected from one or more of citric acid, sodium citrate, phosphoric acid, sodium dihydrogen phosphate, and sodium hydroxide; the reaction incubation is carried out at 25-37°C by standing or stirring at low speed (≤100 rpm) for 1-4 h.

[0015] The incubation time in this step is another key parameter affecting the integrity of the coating and the stability of the complex. During incubation, the carboxyl and phosphate groups on the vesicle membrane surface are in a partially protonated state, maintaining negative charge to keep the vesicles dispersed while providing sufficient hydrogen bonding sites. Polysaccharide chains spontaneously and orderly adsorb and entwine onto the surface of extracellular vesicles through hydrogen bonds and hydrophobic interactions, ultimately forming a relatively dense "glycan coat," completing the coating process. During incubation, the pH of the reaction system should be monitored and maintained within the range of 5.8-6.2, as the sugar molecules are most conformated at this pH, facilitating sufficient contact with the vesicles.

[0016] Preferably, the purification and separation in step S4 is performed using a size exclusion chromatography column. Specifically, the reaction solution is loaded into the size exclusion chromatography column, eluted with PBS buffer, and the first eluent peak is collected to obtain the soothing and repairing complex. The packing material of the size exclusion chromatography column is agarose gel medium with a particle size of 50-150 μm, and the column bed volume is 5-10 times the volume of the reaction solution. The elution flow rate of the PBS buffer is controlled at 1-5 mL / min. The collection of the first eluent peak specifically involves collecting the eluent with an elution volume in the range of 0.3-0.6 times the column bed volume.

[0017] This step involves purifying the reaction product after incubation to separate the formed complex and remove unbound free polysaccharide molecules.

[0018] Preferably, the purification and separation in step S4 is performed by ultrafiltration or tangential flow filtration.

[0019] The third aspect of the present invention provides the application of the soothing and repairing complex for sensitive skin described in the first aspect in skin care products, the skin care products including toners, creams, masks, serums, and sprays, and the amount of the soothing and repairing complex added is 0.5%-5% of the total weight of the skin care products.

[0020] In a fourth aspect, the present invention provides a serum comprising the following ingredients by weight percentage: 0.5%-5% of the soothing and repairing complex suitable for sensitive skin as described in the first aspect, 0.05%-0.3% of a thickener, 5%-10% of a moisturizer, 0.5%-1% of an antioxidant, and the balance being deionized water.

[0021] Preferably, the ingredients in the essence include at least one of the following (a)-(c): (a) The thickener is selected from at least one of xanthan gum, carbomer, hydroxyethyl acrylate / sodium acryloyldimethyl taurate copolymer, ammonium acryloyldimethyl taurate / VP copolymer, and sclerotium gum; (b) The moisturizer is selected from at least one of allantoin, betaine, β-glucan, trehalose, caprylyl glycol, dipropylene glycol, sodium hyaluronate, 1,3-butanediol, 1,3-propanediol, 1,2-hexanediol, 1,2-pentanediol, glycerin, D-panthenol, and ceramide; (c) The antioxidant is p-hydroxyacetophenone.

[0022] The beneficial effects of this invention are: This invention provides a soothing and repairing complex suitable for sensitive skin. The complex is prepared by encapsulation of stachyose, astragalus polysaccharide, galactoglucan, and microalgal extracellular vesicles. Stachyose, as a bioactive oligosaccharide, possesses both immunomodulatory and osmotic pressure balancing effects. It gently acts on skin immune receptors, inducing the expression of defensins and the anti-inflammatory factor IL-10, rather than causing inflammation, thereby effectively increasing the skin's tolerance threshold. Simultaneously, as a skin prebiotic, it optimizes the microecological balance, synergistically enhancing the skin's resistance to external stimuli from immune training to barrier repair, achieving a shift from passive repair to active defense. Astragalus polysaccharide has clear immunomodulatory activity, enhancing macrophage function and regulating the Th1 / Th2 balance. In this system, it synergistically amplifies immunomodulatory signals with stachyose and provides antioxidant support, helping to maintain cellular homeostasis during the regulatory process. Galactoglucan, derived from yeast cell walls, is a classic β-glucan. It acts as a mild pathogen-associated molecular pattern (PAMP) mimic, recognized by pattern recognition receptors (such as TLR2 / 4) on the surface of skin keratinocytes. This recognition does not trigger strong inflammation, but rather induces the production of low levels of defensins, antimicrobial peptides, and anti-inflammatory cytokines (such as IL-10), thereby modulating the efficiency of the skin's innate immune system response and helping to increase the skin's tolerance threshold to stimuli. Simultaneously, it forms a breathable protective film on the skin surface, physically blocking some irritants and helping to reduce the direct impact of external irritants on the skin. The microalgal extracellular vesicles carry abundant nucleic acid, protein, and lipid signaling molecules, which can be effectively taken up by skin cells, directly regulating intracellular signaling pathways, promoting the synthesis of barrier-related proteins (such as filaggrin and naphthalene), and accelerating barrier repair. More importantly, the microRNA and other components they carry also help regulate the cell's adaptive response to stress, consolidating the long-term effects of "active defense." The four raw material components work synergistically to target the passive vulnerability caused by imbalances in the skin's immune system and low tolerance threshold. Through the polysaccharide-extracellular vesicle complex, it simulates the gentle regulation of the skin's immune system, systematically enhancing the skin's resistance to external environmental stresses (such as pollutants, seasonal changes, and emotional stress) without the risk of irritation. This achieves a transformation from "passive damage-repair" to "active defense-strengthening," fundamentally improving problems such as irritation, recurrent redness, and weak barrier function in sensitive skin. In addition, the sugar-extracellular vesicle coating structure also has significant physical protection and synergistic effects. The sugar layer acts as a physical barrier, effectively improving the stability of extracellular vesicles in storage and formulation systems, preventing vesicle aggregation and fusion or leakage of contents, and ensuring the long-term retention of bioactive ingredients. At the same time, the hydrophilic sugar shell improves the surface properties of the vesicles, enhances their adhesion and permeability on the skin surface, promotes more effective delivery of active ingredients to target cells, and further improves the soothing and repair efficiency. Attached Figure Description

[0023] Figure 1 Transmission electron micrograph of the soothing and repairing complex in Example 1; Figure 2 The changes in the red area on the faces of subjects before and after application in Example 1 and Comparative Example 13 are shown. Detailed Implementation

[0024] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with embodiments, is provided below.

[0025] Stachyose was purchased from Xi'an Weisbo Biotechnology Co., Ltd., product number WSB-SST; Astragalus polysaccharide was purchased from Shaanxi Haochen Biotechnology Co., Ltd., and its trade name is Astragalus Polysaccharide. Galactoglucan was purchased from Chengdu Saidike Biotechnology Co., Ltd., and its product name is Furuike. ® M120; Microalgal extracellular vesicles were purchased from ALGAKTIV, a company whose trade name is ALGAKTIV. ® Exometics™ G.

[0026] Unless otherwise specified, the conditions in the examples shall be performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the components of the formulations in the examples are all commercially available products.

[0027] Unless otherwise specified, all other materials, reagents, etc. used in all embodiments and comparative examples of this invention are commercially available.

[0028] Table 1. Raw materials and their weight ratios for the composite. Stachyose Astragalus polysaccharides galactoglucan extracellular vesicles of microalgae Example 1 1.25 0.65 0.2 0.03 Example 2 1 0.5 0.1 0.01 Example 3 1.5 0.8 0.3 0.05 Example 4 1 0.1 0.05 0.005 Example 5 2 1 0.5 0.1 Example 6 0.5 0.05 0.01 0.001 Example 7 5 3 2 1 Example 8 1.25 0.65 0.2 0.03 Example 9 1.25 0.65 0.2 0.03 Note: The total weight of each raw material in each embodiment in Table 1 is equal.

[0029] The preparation method of the soothing and repairing complex in Examples 1-7 includes the following steps: S1. Add the lyophilized microalgal extracellular vesicle powder to PBS buffer pre-cooled to 4℃ and pH 7.4, and vortex at 1000 rpm for 40s to prepare a homogeneous microalgal extracellular vesicle suspension with a concentration of 1 mg / mL. Place it on ice for later use. Dissolve stachyose, astragalus polysaccharide, and galactoglucan in ultrapure water according to a preset ratio to prepare a mixed sugar stock solution with a concentration of 20 mg / mL. Filter the solution through a 0.22 μm filter membrane for sterilization. S2. Under magnetic stirring conditions of 28℃ and 300rpm, the microalgal extracellular vesicle suspension was added dropwise at a uniform rate to the sterilized mixed sugar stock solution for 20 minutes to obtain the mixed system. S3. Adjust the pH of the mixture to 6.0 using citric acid, and let the mixture continue to be incubated at 28°C for 2 hours to obtain the reaction solution; S4. Pack the chromatographic column with agarose gel medium with a particle size of 50-150 μm, and control the column bed volume to be 8 times the total volume of the reaction solution. Carefully load the reaction solution into the equilibrated size exclusion column, and perform isocratic elution with PBS buffer at a flow rate of 2.5 mL / min. Monitor the eluent in real time and collect the eluent with an eluent volume of 0.3-0.5 times the column bed volume to obtain the soothing and repairing complex.

[0030] Depend on Figure 1 As can be seen, the glycoconjugate of stachyose, galactoglucan and astragalus polysaccharide described in this invention encapsulates extracellular vesicles of microalgae, giving the vesicles a more robust structure, and the resulting higher surface charge reduces vesicle aggregation.

[0031] The preparation method of the soothing and repairing complex in Example 8 includes the following steps: S1. Add the lyophilized microalgal extracellular vesicle powder to PBS buffer pre-cooled to 4℃ and pH 7.4, and vortex at 800 rpm for 60 s to prepare a homogeneous microalgal extracellular vesicle suspension with a concentration of 1 mg / mL. Place it on ice for later use. Dissolve stachyose, astragalus polysaccharide, and galactoglucan in ultrapure water according to a preset ratio to prepare a mixed sugar stock solution with a concentration of 20 mg / mL. Filter the solution through a 0.22 μm filter membrane for sterilization. S2. Under magnetic stirring conditions of 25℃ and 400rpm, the microalgal extracellular vesicle suspension was added dropwise to the sterilized mixed sugar stock solution at a constant rate for 15 minutes to obtain the mixed system. S3. Adjust the pH of the mixture to 5.8 using citric acid, and let the mixture continue to incubate at 25°C for 4 hours to obtain the reaction solution; S4. Pack the chromatographic column with agarose gel medium with a particle size of 50-150μm, and control the column bed volume to be 5 times the total volume of the reaction solution. Carefully load the reaction solution into the equilibrated size exclusion column, and perform isocratic elution with PBS buffer at a flow rate of 1mL / min. Monitor the eluent in real time and collect the eluent with an eluent volume of 0.3-0.5 times the column bed volume to obtain the soothing and repairing complex.

[0032] The preparation method of the soothing and repairing complex in Example 9 includes the following steps: S1. Add the lyophilized microalgal extracellular vesicle powder to PBS buffer pre-cooled to 4℃ and pH 7.4, and vortex at 1200 rpm for 30 s to prepare a uniform microalgal extracellular vesicle suspension with a concentration of 1 mg / mL. Place it on ice for later use. Dissolve stachyose, astragalus polysaccharide, and galactoglucan in ultrapure water according to a preset ratio to prepare a mixed sugar stock solution with a concentration of 20 mg / mL. Filter the solution through a 0.22 μm filter membrane for sterilization. S2. Under magnetic stirring conditions of 37℃ and 200rpm, the microalgal extracellular vesicle suspension was added dropwise at a uniform rate to the sterilized mixed sugar stock solution for 25 minutes to obtain the mixed system. S3. Adjust the pH of the mixture to 6.2 using citric acid, and incubate the mixture at 37°C for 1 hour to obtain the reaction solution; S4. Pack the chromatographic column with agarose gel medium with a particle size of 50-150μm, and control the column bed volume to be 10 times the total volume of the reaction solution. Carefully load the reaction solution into the equilibrated size exclusion column, and perform isocratic elution with PBS buffer at a flow rate of 5mL / min. Monitor the eluent in real time and collect the eluent with an eluent volume of 0.3-0.5 times the column bed volume to obtain the soothing and repairing complex.

[0033] Comparative Example 1 Compared with Example 1, the difference is that stachyose was omitted in Comparative Example 1, and the missing amount was made up with astragalus polysaccharide and galactoglucan in a weight ratio of 0.65:0.2. Other conditions were the same as in Example 1.

[0034] Comparative Example 2 Compared with Example 1, the difference is that Astragalus polysaccharide was omitted in Comparative Example 2, and the missing amount was made up with stachyose and galactoglucan in a weight ratio of 1.25:0.2. Other conditions were the same as in Example 1.

[0035] Comparative Example 3 Compared with Example 1, the difference is that galactoglucan was omitted in Comparative Example 3, and the missing amount was made up with stachyose and astragalus polysaccharide in a weight ratio of 1.25:0.65. Other conditions were the same as in Example 1.

[0036] Comparative Example 4 Compared with Example 1, the difference is that stachyose was omitted in Comparative Example 4, and the missing amount was made up with fructooligosaccharide (product number D31251, supplier: Qingdao Haiweisen Biotechnology Co., Ltd.). Other conditions were the same as in Example 1.

[0037] Comparative Example 5 Compared with Example 1, the difference is that Astragalus polysaccharide was omitted in Comparative Example 5, and the missing amount was made up with Lycium barbarum polysaccharide (product number zb-6425, supplier: Xi'an Zebang Biotechnology Co., Ltd.). Other conditions were the same as in Example 1.

[0038] Comparative Example 6 The difference between Comparative Example 6 and Example 1 is that galactoglucan was omitted in Comparative Example 6, and the missing amount was replaced with β-glucan (trade name: Celecoxib). ® The supplier (Sichuan Hetai Xinguang Biotechnology Co., Ltd.) supplemented the information, and other conditions remained consistent with those in Example 1.

[0039] Comparative Example 7 The difference between Comparative Example 7 and Example 1 is that the microalgal extracellular vesicles in the complex were omitted, and the missing amount was replaced with Centella asiatica extracellular vesicles (product name: Turmeria Zen). ™ The supplier is Vytrus, and other conditions remain the same as in Example 1.

[0040] Comparative Example 8 Compared with Example 1, the difference is that the weight ratio of stachyose, astragalus polysaccharide, galactoglucan and microalgal extracellular vesicles in Comparative Example 8 is 0.2:0.65:1.25:0.03, while other conditions remain the same as in Example 1.

[0041] Comparative Example 9 Compared with Example 1, the difference is that the weight ratio of stachyose, astragalus polysaccharide, galactoglucan and microalgal extracellular vesicles in Comparative Example 9 is 0.03:0.65:0.2:1.25, while other conditions remain the same as in Example 1.

[0042] Comparative Example 10 Compared with Example 1, the difference is that the soothing and repairing complex of Comparative Example 10 does not use the preparation steps S1, S2, S3 and S4. Instead, it is simply a mixture of stachyose, astragalus polysaccharide, galactoglucan and microalgal extracellular vesicles in a weight ratio of 1.25:0.65:0.2:0.03.

[0043] Comparative Example 11 Compared with Example 1, the difference is that the magnetic stirring speed in step S2 of the preparation of the soothing and repairing complex in Comparative Example 11 is 1000 rpm, and other conditions are the same as in Example 1.

[0044] Comparative Example 12 The difference from Example 1 is that the pH value in step S3 of Comparative Example 12 was adjusted to 7.4, while other conditions remained the same as in Example 1.

[0045] Comparative Example 13 The difference from Example 1 is that the pH value in step S3 of Comparative Example 13 was adjusted to 5.0, while other conditions remained the same as in Example 1.

[0046] Comparative Example 14 The difference from Example 1 is that the static incubation time in step S3 of Comparative Example 14 is 30 minutes, while other conditions remain the same as in Example 1.

[0047] Comparative Example 15 The difference from Example 1 is that the static incubation temperature in step S3 of Comparative Example 15 is 40°C, while other conditions remain the same as in Example 1.

[0048] Comparative Example 16 Compared with Example 1, the difference is that step S4 size exclusion chromatography purification was not performed in Comparative Example 16, while other conditions remained the same as in Example 1.

[0049] Comparative Example 17 Compared with Example 1, the difference is that in Comparative Example 17, step S4 collects effluent with an elution volume in the range of 0.8-1.0 times the column bed volume, while other conditions remain the same as in Example 1.

[0050] Example of results: Detection of the effect of the complex on the expression of immune-related factors in skin keratinocytes Human beta-defensin-2 (hBD-2) is a key antimicrobial peptide expressed by keratinocytes after sensing immune stimuli such as pathogen-associated molecular patterns (PAMPs). Moderate upregulation of hBD-2 is a marker of the skin's innate immune system being "awakened" and initiating primary defense. Interleukin-10 (IL-10) is a key anti-inflammatory cytokine that can inhibit excessive inflammation and promote immune tolerance. By examining the regulatory effects of the composition on hBD-2 and IL-10 expression in keratinocytes, we can precisely assess whether it can gently regulate the skin's immune system without inducing harmful inflammation, thus simultaneously enhancing basic defense capabilities and establishing a tolerance balance—the core embodiment of the "active defense" concept.

[0051] Experimental methods: This study investigates the effects of the soothing and repairing complex for sensitive skin prepared in Examples 1-9 and Comparative Examples 1-17 on the expression of hBD-2 and IL-10 in human immortalized keratinocytes (HaCaT).

[0052] The cell line used was immortalized human keratinocytes (HaCaT, from Beina Biotechnology). The incubator conditions were: temperature 37±1℃, saturated humidity, and carbon dioxide concentration 5±1%. Cells were cultured and treated according to groupings, followed by testing, specifically the detection of hBD-2 and IL-10 levels. The testing methods are as follows: (1) Cell seeding: HaCaT cells in the logarithmic growth phase were digested and counted, and their cell density was adjusted to 2×10⁶ cells / year using DMEM high-glucose culture medium containing 10% v / v fetal bovine serum. 5 Cell suspension of cells / mL was seeded into 1 mL per well of a 12-well cell culture plate; the culture plate was pre-cultured in an incubator for 24 h to allow the cells to adhere completely and reach about 80% confluence. (2) Cell treatment: Discard the original culture medium in each well, wash gently once with sterile PBS, and divide the cells into the following blank control group, model group, and sample group; among them, the blank control group is added with DMEM complete culture medium (containing 1% v / v FBS) without any stimulants or samples; the model group is added with DMEM complete culture medium (containing 1% v / v FBS) containing 100 ng / mL LPS (lipopolysaccharide, Sigma-Aldrich); the sample group is added with DMEM complete culture medium (containing 1% v / v FBS) containing 100 ng / mL LPS and 1% v / v of the corresponding example or comparative sample; each group is set with at least 3 replicates, and the culture plate is put back into the incubator for continued culture; (3) Sample collection: 24 h after drug treatment, collect cell culture supernatant; transfer the supernatant to a sterile centrifuge tube, centrifuge at 4℃ and 1000 rpm for 10 min to remove any cells that may detach, take the supernatant, store at -80℃ for later use in ELISA detection; (4) Detection of hBD-2 and IL-10 levels: The detection was performed according to the experimental procedures of the Human β-defensin-2 (hBD-2) ELISA kit (Huamei Biotechnology, CSB-E13201h) and the Human Interleukin-10 (IL-10) ELISA kit (Jianglai Biotechnology, JL19246); 50 μL of standard or cell supernatant sample to be tested was accurately added to the standard wells and sample wells of the pre-coated plate, followed by 100 μL of... HRP-labeled detection antibody was sealed and incubated at 37°C for 60 min. After incubation, the liquid in the wells was discarded, and the samples were washed thoroughly 5 times with washing buffer and patted dry. 50 μL each of substrate A and B were added to each well sequentially, and the mixture was incubated at 37°C in the dark for 15 min. Finally, 50 μL of stop solution was added to each well to terminate the reaction. The absorbance (OD value) of each well was immediately measured at 450 nm using a microplate reader. The concentrations of hBD-2 and IL-10 in the samples were calculated based on the standard curve, and their relative promotion rates were calculated using the following formulas: The relative promotion rate of hBD-2 (%) = [(hBD-2 concentration in the sample group - hBD-2 concentration in the model group) / hBD-2 concentration in the model group] × 100%. The higher this value, the more the composition "modulates" and enhances the direct defense response of keratinocytes under the same immune challenge (LPS). The relative promotion rate of IL-10 (%) = [(IL-10 concentration in the sample group - IL-10 concentration in the model group) / IL-10 concentration in the model group] × 100%. The higher this value, the more effectively the composition can guide the immune response towards anti-inflammatory and tolerance-promoting directions, which is a key indicator for improving the tolerance threshold. The average of the calculation results is recorded in Table 2.

[0053] Table 2. Promotion rates of hBD-2 and IL-10 in each group of samples hBD-2 relative promotion rate / % IL-10 relative promotion rate / % Example 1 68.5 61.2 Example 2 65.2 57.1 Example 3 64.7 57.3 Example 4 60.6 53.3 Example 5 61.9 54.5 Example 6 59.1 51.8 Example 7 58.3 50.2 Example 8 67.1 58.7 Example 9 66.4 59.0 Comparative Example 1 20.4 13.1 Comparative Example 2 21.5 14.8 Comparative Example 3 19.4 14.1 Comparative Example 4 26.1 16.9 Comparative Example 5 30.9 22.8 Comparative Example 6 23.5 18.5 Comparative Example 7 21.3 15.3 Comparative Example 8 45.3 37.9 Comparative Example 9 44.7 36.4 Comparative Example 10 39.4 31.1 Comparative Example 11 33.2 25.8 Comparative Example 12 35.2 27.8 Comparative Example 13 11.3 4.7 Comparative Example 14 41.2 33.1 Comparative Example 15 42.1 34.7 Comparative Example 16 40.2 32.9 Comparative Example 17 14.2 6.8 As shown in Table 2, compared with Comparative Examples 1-17, the soothing and repairing complex provided in Examples 1-9 can synergistically and gently regulate skin immune cells, significantly increase the expression of human β-defensin-2 and interleukin-10, enhance basic defense capabilities and establish tolerance balance.

[0054] As can be seen from the comparison of Example 1 and Comparative Examples 1-7, the stachyose, astragalus polysaccharide, galactoglucan, and microalgal extracellular vesicles in the complex work synergistically to form an "immunoregulation-barrier repair" synergistic system. The absence or replacement of any component will lead to a significant decrease in the promoting effect of the complex on β-defensin-2 and interleukin-10. As can be seen from the comparison of Example 1 and Comparative Examples 8-17, the soothing and repairing complex of the present invention has its own characteristics in terms of the proportion of raw materials and preparation process. Any change in process parameters will result in its final immunomodulatory efficacy failing to reach the level of the examples.

[0055] Application Example 1-9 and Comparative Application Example 1-17 The soothing and repairing complexes of Examples 1-9 and Comparative Examples 1-17 were added to the serums at a concentration of 3 wt% to obtain the serums of Application Examples 1-9 and Comparative Application Examples 1-17, respectively. In addition, a serum without the soothing and repairing complex was prepared as a blank application example. The formula of the serums is shown in Table 3.

[0056] The preparation methods of the serums in Application Examples 1-9 and Comparative Application Examples 1-17 include the following steps: (1) Mix phase A with deionized water, heat to 85°C, and homogenize at 1300 rpm for 4 min to obtain pre-prepared component A, and keep it warm for later use; (2) Mix phase B and heat to 60°C to melt, to obtain pre-prepared component B; (3) Cool the pre-prepared component A to 60°C, add the pre-prepared component B while stirring at 250 rpm, and continue stirring for 10-15 min; continue cooling to 40°C, add the soothing and repairing complex and stir for 8 min, stir evenly, and discharge to obtain the essence.

[0057] The preparation method of the serum in the blank application example includes the following steps: (1) Mix phase A with deionized water, heat to 85°C, and homogenize at 1300 rpm for 4 min to obtain pre-prepared component A, and keep it warm for later use; (2) Mix phase B and heat to 60°C to melt, to obtain pre-prepared component B; (3) Cool the pre-prepared component A to 60°C, add the pre-prepared component B while stirring at 150 rpm, stir evenly, and discharge to obtain the essence.

[0058] Table 3. Serum Components and Dosage

[0059] Application example: Human efficacy evaluation for sensitive skin Experimental Methods: In order to accurately evaluate the efficacy of the soothing and repairing complex of the present invention in reducing the immediate subjective discomfort and objective inflammatory response of sensitive skin to chemical stimulation, this application test was designed in accordance with the relevant principles of human efficacy evaluation in the "Cosmetic Safety Technical Specifications (2015 Edition)".

[0060] Volunteer recruitment and grouping: 81 healthy Asian female volunteers aged 18 to 45 who self-assessed as having sensitive skin were recruited and divided into 27 groups of 3 people each. Inclusion criteria: Lactic acid stinging test (10% lactic acid applied to the nasolabial fold for 5 minutes) score ≥3; intact skin in the test area (both cheeks) without active lesions; no use of hormonal drugs or related cosmetic treatments on the face within 1 month prior to the test; signed informed consent form.

[0061] Test products: The serums prepared using Application Examples 1-9 or Comparative Application Examples 1-17 were used as test groups; the serums prepared using the blank application examples were used as blank groups. Test method: Apply 1g of the blank group to the nasolabial fold on one side of the volunteer's face and 1g of the test group to the other side. Allow the product to be absorbed. Each subject had a 10% lactic acid solution applied to their nasolabial folds (using 0.8cm diameter double-layered filter paper) for 5 minutes. After application, the solution was removed to assess facial sensitivity to lactic acid stimulation. A 4-point questionnaire was used to assess sensitivity scores: 0 - no sensation, 1 - slight stinging, 2 - noticeable stinging, 3 - severe stinging. The average lactic acid stinging score for each group of subjects was calculated. In addition, the a* value of the cheeks was measured using a CL440 skin colorimeter, and the transepidermal water loss (TEWL) value at the cheekbone was measured using a Tewameter®™ Hex transepidermal water loss meter. The lactic acid stinging score indicates the improvement in skin's defense and tolerance; the lower the score, the stronger the skin's defense and tolerance. The a* value indicates facial redness; a lower a* value indicates improved facial redness, and the complex has a soothing effect. The TEWL value indicates the improvement in the skin barrier; a lower TEWL value indicates enhanced barrier function. Follow-up Measurement (Day 14): After completing 14 days of product use, volunteers returned for a follow-up visit. Under the same environmental conditions, all measurement items were repeated, and the improvement in data for each group was calculated. The calculation formula is as follows: Lactic acid stinging score = scores from each volunteer / 3 TEWL value improvement rate, facial redness improvement rate (%) = [(mean before use - mean after use) / mean before use] × 100%; The average of the calculation results is recorded in Table 4.

[0062] Table 4 Results of Human Efficacy Evaluation Lactic acid stinging score / % TEWL value improvement rate / % Facial redness improvement rate / % Blank application example 3 2.2 4.2 Application Example 1 0.3 27.4 52.1 Application Example 2 0.7 22.6 45.5 Application Example 3 1.0 24.2 47.7 Application Example 4 0.7 21.2 40.4 Application Example 5 0.7 21.8 41.2 Application Example 6 1.0 19.2 35.6 Application Example 7 1.0 18.3 34.7 Application Example 8 0.3 26.7 50.8 Application Example 9 0.3 25.5 49.3 Comparative Application Example 1 2.3 3.7 9.3 Comparative Application Example 2 2.3 4.2 10.9 Comparative Application Example 3 2.3 3.3 9.5 Comparative Application Example 4 2.3 5.6 11.7 Comparative Application Example 5 2.3 6.0 13.3 Comparative Application Example 6 2.3 5.1 12.2 Comparative Application Example 7 2.3 4.7 11.5 Comparative Application Example 8 1.3 12.1 25.6 Comparative Application Example 9 1.3 13.6 24.6 Comparative Application Example 10 1.3 7.2 17.8 Comparative Application Example 11 1.7 6.2 14.6 Comparative Application Example 12 1.3 7.3 16.2 Comparative Application Example 13 3.0 2.1 2.3 Comparative Application Example 14 1.3 11.4 21.1 Comparative Application Example 15 1.3 11.7 22.3 Comparative Application Example 16 1.3 10.5 18.2 Comparative Application Example 17 2.7 3.1 5.3 As shown in Table 4, the soothing and repairing complex prepared in this invention can significantly improve the skin's defense capabilities and tolerance, reduce facial redness, and strengthen the skin barrier. It can be applied to people with sensitive skin and has good soothing and repairing effects. Regarding skin tolerance, the lactic acid stinging scores of subjects in Application Examples 1-9 were significantly lower than those in Comparative Application Examples 1-17, indicating that the complex of this invention can effectively improve the skin's tolerance threshold to external chemical stimuli. Regarding soothing and repair, the improvement rate of facial redness in subjects in Application Examples 1-9 was significantly higher, and the a* value decreased significantly, indicating that the complex has excellent immediate soothing and anti-inflammatory redness-reducing abilities. Regarding barrier function, the improvement rate of TEWL values ​​in subjects in Application Examples 1-9 was significantly better than that in Comparative Application Examples 1-17, indicating that the complex can promote the physical repair of the skin barrier structure and reduce moisture loss.

[0063] As shown in Application Example 1 and Comparative Application Examples 1-7, the stachyose, astragalus polysaccharide, galactoglucan, and microalgal extracellular vesicles in the complex also exhibit significant synergistic effects on human skin, forming an "immunomodulation-barrier repair" synergistic system. The absence or replacement of any component leads to a rebound in lactic acid stinging scores, a significant decrease in redness improvement rate, and a marked decline in barrier repair effect, proving that all four raw materials are indispensable. As shown in Application Example 1 and Comparative Application Examples 8-17, the soothing and repairing complex of this invention has unique characteristics in terms of raw material ratios and preparation processes, such as pH value, incubation time, and purification methods. Any change in process parameters will result in the final product's clinical efficacy failing to reach the level shown in the application examples, indicating that a specific encapsulation process is crucial to ensuring stable delivery and efficacy of the active ingredients.

[0064] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any indirect modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A soothing and repairing complex suitable for sensitive skin, characterized in that, The soothing and repairing complex comprises the following ingredients: stachyose, astragalus polysaccharide, galactoglucan, and microalgal extracellular vesicles, wherein the weight ratio of stachyose, astragalus polysaccharide, galactoglucan, and microalgal extracellular vesicles is (0.5-5):(0.05-3):(0.01-2):(0.001-1).

2. The soothing and repairing complex suitable for sensitive skin according to claim 1, characterized in that, The weight ratio of stachyose, astragalus polysaccharide, galactoglucan and microalgal extracellular vesicles is (1-2):(0.1-1):(0.05-0.5):(0.005-0.1).

3. The soothing and repairing complex suitable for sensitive skin according to claim 1, characterized in that, The weight ratio of stachyose, astragalus polysaccharide, galactoglucan and microalgal extracellular vesicles is (1-1.5):(0.5-0.8):(0.1-0.3):(0.01-0.05).

4. A method for preparing a soothing and repairing complex suitable for sensitive skin according to any one of claims 1-3, characterized in that, Includes the following steps: S1. Reconstitute the lyophilized powder of microalgal extracellular vesicles to prepare a suspension of microalgal extracellular vesicles, and place it on ice for later use; dissolve stachyose, astragalus polysaccharide, and galactoglucan in ultrapure water to prepare a mixed sugar stock solution, and filter it through a 0.22μm filter membrane for sterilization. S2. Under magnetic stirring, the microalgal extracellular vesicle suspension is added dropwise at a uniform rate to the sterilized mixed sugar stock solution to obtain a mixed system. S3. Adjust the pH of the mixture to 5.8-6.2 using a pH adjuster, and continue the reaction incubation to obtain the reaction solution; S4. The reaction solution is purified and separated to obtain the soothing and repairing complex.

5. The method for preparing the soothing and repairing complex suitable for sensitive skin according to claim 4, characterized in that, Step S1 is as follows: Add the lyophilized powder of microalgal extracellular vesicles to PBS buffer pre-cooled to 4°C and with a pH of 7.2-7.4, and vortex at 800-1200 rpm for 30-60 s to prepare a uniform microalgal extracellular vesicle suspension with a concentration of 0.5-2 mg / mL, and place it on ice for later use; dissolve stachyose, astragalus polysaccharide, and galactoglucan in ultrapure water to prepare a mixed sugar stock solution with a concentration of 10-30 mg / mL, and filter it through a 0.22 μm filter membrane for sterilization.

6. The method for preparing the soothing and repairing complex suitable for sensitive skin according to claim 4, characterized in that, The magnetic stirring conditions in step S2 are magnetic stirring at 25-37℃ and 200-400 rpm; the uniform dripping time is 15-25 min.

7. The method for preparing the soothing and repairing complex suitable for sensitive skin according to claim 4, characterized in that, The pH adjuster mentioned in step S3 is selected from one or more of citric acid, sodium citrate, phosphoric acid, sodium dihydrogen phosphate, and sodium hydroxide; the reaction incubation is carried out by standing or stirring at low speed at 25-37°C for 1-4 hours.

8. The method for preparing the soothing and repairing complex suitable for sensitive skin according to claim 4, characterized in that, The purification and separation described in step S4 is performed using a size exclusion chromatography column. Specifically, the reaction solution is loaded into the size exclusion chromatography column, eluted with PBS buffer, and the first eluent peak is collected to obtain the soothing and repairing complex. The packing material of the size exclusion chromatography column is agarose gel medium with a particle size of 50-150 μm, and the column bed volume is 5-10 times the volume of the reaction solution. The elution flow rate of the PBS buffer is controlled at 1-5 mL / min. The collection of the first eluent peak specifically involves collecting the eluent with an elution volume in the range of 0.3-0.6 times the column bed volume.

9. The application of the soothing and repairing complex suitable for sensitive skin according to any one of claims 1-8 in skincare products, characterized in that, The skincare products include toners, creams, masks, serums, and sprays, and the amount of the soothing and repairing complex added is 0.5%-5% of the total weight of the skincare products.

10. An essence, characterized in that, The serum comprises the following ingredients by weight percentage: 0.5%-5% of the soothing and repairing complex suitable for sensitive skin as described in any one of claims 1-8, 0.05%-0.3% of thickener, 5%-10% of moisturizer, 0.5%-1% of antioxidant, and the balance being deionized water.

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