A soothing repair composition and process for making the same

Abstract

This invention discloses a soothing and repairing composition and its preparation process, relating to the field of cosmetic technology. The composition comprises *Hemiberlesia lataniae* callus, *Oroxylum indicum* callus, *Pteris vittata* callus, *Rhodiola rosea* callus, sodium hyaluronate, trehalose, dipotassium glycyrrhizate, hydrogenated lecithin, polyglycerol-10 stearate, cetearyl oleate / sorbitan oleate, 1,2-pentanediol, xanthan gum, and deionized water. Through differentiated extraction and multi-component compounding processes, this invention enables the five active callus components to synergistically promote cell migration, inhibit inflammatory factors, scavenge free radicals, improve skin barrier function, and increase the hydration content of the stratum corneum. Furthermore, the composition exhibits good stability and high safety, making it suitable for preparing soothing and repairing skincare products.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, specifically to a soothing and repairing composition and its preparation process. Background Technology

[0002] With increasing environmental pollution, rising life stress, and improper skincare practices, the incidence of sensitive skin is increasing year by year. Sensitive skin is mainly characterized by decreased tolerance to external stimuli, easily experiencing subjective sensations such as burning, stinging, itching, and tightness, with or without objective signs such as erythema and desquamation. The pathogenesis of sensitive skin is complex, typically involving multiple factors such as impaired skin barrier function, abnormal nerve sensory function, excessive activation of inflammatory responses, and elevated levels of oxidative stress. Therefore, soothing and repairing products for sensitive skin need to simultaneously address multiple aspects, including barrier repair, anti-inflammation, anti-oxidation, and nerve soothing.

[0003] Currently, the active ingredients used in soothing and repairing cosmetics on the market mainly fall into the following categories: First, moisturizing and repairing ingredients, such as sodium hyaluronate, ceramides, and squalane, primarily improve barrier function by replenishing intercellular lipids or enhancing stratum corneum hydration; second, anti-inflammatory and soothing ingredients, such as centella asiatica extract, purslane extract, and dipotassium glycyrrhizate, mainly reduce skin inflammation by inhibiting inflammatory factors or blocking inflammatory signaling pathways; and third, antioxidant ingredients, such as vitamin C, vitamin E, and ferulic acid, reduce oxidative stress damage to skin cells by scavenging free radicals. While these ingredients can help alleviate the symptoms of sensitive skin to some extent, most products still use a simple combination of single or a few active ingredients, making it difficult to simultaneously cover multiple levels of action such as barrier repair, anti-inflammation, anti-oxidation, and cell repair.

[0004] In recent years, plant callus culture technology has received increasing attention in the field of cosmetic raw material development. Plant callus is a mass of thin-walled cells with meristematic capacity formed by dedifferentiation of explants under in vitro culture conditions. These cells have vigorous metabolism, capable of synthesizing and accumulating various secondary metabolites, and their active ingredients are often more diverse and abundant than those of the original plant tissue. However, existing callus raw materials used in cosmetics are mainly limited to a few plants such as ginseng, snow lotus, and dendrobium, and their extraction and combination methods are relatively simple, resulting in products with limited efficacy and difficulty in achieving comprehensive soothing and repairing effects targeting multiple points.

[0005] Therefore, developing a soothing and repairing composition that can work simultaneously from multiple dimensions such as barrier repair, anti-inflammation, anti-oxidation and cell regeneration is of great practical significance. Summary of the Invention

[0006] To address the above problems, the present invention provides a soothing and repairing composition and its preparation process.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] This invention provides a soothing and repairing composition, comprising, by weight, the following components: 8-16 parts of *Ceratophyllum demersum* callus, 6-12 parts of *Oroxylum indicum* callus, 8-15 parts of *Pteris vittata* callus, 5-12 parts of *Rhodiola rosea* callus, 3-8 parts of sodium hyaluronate, 2-5 parts of trehalose, 0.5-2 parts of dipotassium glycyrrhizate, 2-5 parts of hydrogenated lecithin, 2-4 parts of polyglycerol-10 stearate, 1-3 parts of cetearyl oleate / sorbitan oleate (Olivem 1000), 3-8 parts of 1,2-pentanediol, 0.1-0.5 parts of xanthan gum, and deionized water to 100 parts; the pH is adjusted to 5.2-6.2 with a citrate-sodium citrate buffer solution.

[0009] Furthermore, the sodium hyaluronate is a compound of high molecular weight sodium hyaluronate (800,000-1,500,000 Da) and low molecular weight sodium hyaluronate (30,000-80,000 Da) in a mass ratio of 1:1.5-2.5.

[0010] Furthermore, the soothing and repairing composition is used in the preparation of skin care products for soothing, repairing the skin barrier, anti-inflammatory, or antioxidant purposes.

[0011] The present invention also provides a preparation process for the soothing and repairing composition, comprising the following steps:

[0012] S1: Five active components were prepared respectively: anti-inflammatory active component A1 of red thick shell callus, moisturizing barrier component A2 of red thick shell-fern root polysaccharide, antioxidant and soothing component A3 of wood butterfly callus, regeneration and repair component A4 of fern root callus, and anti-inflammatory and healing-promoting component A5 of rhodiola rosea callus.

[0013] S2: Deionized water accounting for 60%-80% of the total formula is designated as aqueous phase B1, deionized water accounting for 15%-30% of the total formula is designated as aqueous phase B2, and deionized water accounting for 5%-10% of the total formula is designated as aqueous phase B3. The sum of B1, B2, and B3 is 100 parts.

[0014] S3: Dissolve sodium hyaluronate, trehalose and dipotassium glycyrrhizate in aqueous phase B1 under stirring at a temperature not exceeding 40°C to obtain an auxiliary component solution;

[0015] S4: Hydrogenated lecithin, polyglycerol-10 stearate and cetearyl oleate / sorbitan oleate are heated and melted in a water bath at 65-75℃, and then 1,2-pentanediol is added and stirred evenly to obtain an oil phase; the oil phase is slowly added to the auxiliary component solution under high-speed homogenization conditions, homogenized for 3-5 min to form an emulsion matrix, and then cooled to 40-45℃.

[0016] S5: Mix A1, A2, A3, A4, and A5, and then dissolve or disperse them in the aqueous phase B2 at 30-45℃ to obtain a dispersion of the active component;

[0017] S6: Add the dispersion of the active component to the emulsion matrix and stir until homogeneous to obtain mixture A; then disperse xanthan gum in aqueous phase B3 and add it to mixture A, stirring until fully swollen to obtain mixture B;

[0018] S7: Adjust the pH of mixture B to 5.2-6.2 with citric acid-sodium citrate buffer solution; continue stirring at low speed for 15-25 minutes to homogenize the system, fill and seal, and store at 2-8℃ away from light to obtain the final product.

[0019] Further, in step S1, the preparation method of the anti-inflammatory active component A1 of the red thick-shelled callus is as follows: 70% of the total amount of red thick-shelled callus is added to a 50% volume fraction of ethanol aqueous solution at a material-to-liquid ratio of 1:5-1:10, and a compound enzyme system is added. The compound enzyme system is composed of cellulase and snail enzyme in a mass ratio of 2:1. The amount of enzyme added is 0.5%-1.5% of the wet weight of the callus. The pH is adjusted to 4.5-5.5, and enzymatic extraction is carried out at 35-45℃ for 6-10 hours. The enzyme is then inactivated at 85-95℃ for 15-30 minutes. The supernatant is collected by centrifugation, concentrated under reduced pressure, and extracted 2-3 times with an equal volume of ethyl acetate. The ethyl acetate phases are combined and evaporated to dryness under reduced pressure at 40-50℃ to obtain the final product.

[0020] Further, in step S1, the preparation method of the red thick-shelled callus-fern callus combined polysaccharide moisturizing barrier component A2 is as follows: 30% of the total amount of red thick-shelled callus and 60% of the total amount of fern callus are mixed, deionized water is added at a material-liquid ratio of 1:5-1:10, the pH is adjusted to 5.0-6.0, cellulase is added, the amount of enzyme added is 0.3%-0.8% of the wet weight of the mixture, and it is extracted at 20-30℃ for 6-12 hours. The supernatant is collected by centrifugation, concentrated under reduced pressure, and freeze-dried into powder to obtain the product.

[0021] Further, in step S1, the preparation method of the antioxidant and soothing component A3 of the Oroxylum indicum callus is as follows: add Oroxylum indicum callus to a 70% volume fraction of ethanol aqueous solution at a material-to-liquid ratio of 1:6-1:12, adjust the pH to 4.0-5.0, stir and extract at 40-50℃ for 8-14 hours, collect the supernatant by centrifugation, concentrate under reduced pressure until there is no alcohol odor, extract with petroleum ether and ethyl acetate in sequence, collect the ethyl acetate phase, evaporate to dryness under reduced pressure at 40-55℃ to obtain the product.

[0022] Further, in step S1, the preparation method of the *Pteris vittata* callus regeneration and repair component A4 is as follows: 40% of the total amount of *Pteris vittata* callus is added to deionized water at a material-to-liquid ratio of 1:8-1:15, and extracted twice by reflux at 80-95℃ for 1.5-2 hours each time. The extracts are combined, cooled, and concentrated under reduced pressure. The solution is then passed through a macroporous adsorption resin column and eluted sequentially with deionized water, 30% (v / v) ethanol, 60% (v / v) ethanol, and 95% (v / v) ethanol. The 30% ethanol and 60% ethanol eluates are collected, combined, and concentrated under reduced pressure at 40-55℃ to dryness to obtain the final product.

[0023] Further, in step S1, the preparation method of the anti-inflammatory and healing-promoting component A5 of Rhodiola rosea callus is as follows: Rhodiola rosea callus is added to a 60% volume fraction of ethanol aqueous solution at a material-to-liquid ratio of 1:5-1:12, the pH is adjusted to 5.0-6.0, and the mixture is stirred and extracted at 30-45℃ for 6-10 hours. The supernatant is collected by centrifugation, concentrated under reduced pressure until there is no alcohol odor, dispersed with deionized water, extracted successively with petroleum ether and ethyl acetate, the ethyl acetate phase is collected, and evaporated to dryness under reduced pressure at 40-55℃ to obtain the final product.

[0024] Furthermore, the callus tissues of *Sedum morganianum*, *Oroxylum indicum*, *Pteris vittata*, and *Sedum aizoon* were obtained by the following methods: Sterile explants of each plant were inoculated into an induction medium, which was MS basal medium supplemented with 2.5 mg / L 2,4-D, 0.45 mg / L 6-BA, 30 g / L sucrose, and 7.0 g / L agar, pH 5.8, and cultured at 24±1℃ in the dark for 25 days to obtain callus tissue. The callus tissue was then transferred to a liquid suspension proliferation medium, which was MS basal medium supplemented with 1.8 mg / L NAA, 0.4 mg / L KT, and 25 g / L sucrose, pH 5.8, and cultured at 130 rpm and 24±1℃ under weak light for 18 days. After filtration, callus cell clumps were collected, washed three times with deionized water, and centrifuged at 4000 rpm for 15 min to obtain the final product.

[0025] This invention employs a differentiated extraction process, targeting the differences in the physicochemical properties of various active ingredients. By controlling the extraction temperature, solvent polarity, and enzymatic hydrolysis conditions, components with different activity spectra are enriched stepwise from the callus culture system. Specifically: A1 uses *Pteris vittata* callus as raw material, and performs enzymatic extraction with cellulase and snail enzyme at 35-45℃ and 50% ethanol to fully rupture the cell walls, dissolving small molecule anti-inflammatory components such as coumarins and xanthones, followed by extraction and enrichment with ethyl acetate; A2 mixes *Pteris vittata* and *Pteris multifida* callus, and adds cellulase to a pure water system at 20-30℃ for extraction assistance. The low-temperature conditions maximize the preservation of the original structure and degree of polymerization of polysaccharides, and the polysaccharide powder is obtained by freeze-drying; A3 uses *Oroxylum indicum* callus... Using tissue as raw material, extracting at 40-50℃ and 70% ethanol, followed by sequential extraction with petroleum ether and ethyl acetate, yielded an antioxidant component rich in flavonoids such as oroxylin and oleoresin. A4, using *Pteris vittata* callus as raw material, was extracted under reflux at 80-95℃, followed by gradient elution with macroporous adsorption resin to enrich flavonoid-saponin active components. A5, using *Rhodiola rosea* callus as raw material, was extracted at 30-45℃ and 60% ethanol, followed by ethyl acetate extraction, yielding an anti-inflammatory and healing-promoting component rich in flavonols such as kaempferol and quercetin. These five components were prepared separately using their respective processes and then compounded in a multi-component formulation according to the specified proportions.

[0026] Due to differences in chemical composition and molecular weight distribution, the five active components complement and synergize at different levels and pathways of action in the skin: A2, rich in polysaccharide macromolecules, can form a hydrophilic protective film on the skin surface, sealing damaged barriers and reducing transepidermal water loss, primarily acting on the stratum corneum; A3, rich in flavonoid antioxidants, can scavenge free radicals and reduce oxidative stress damage, primarily acting on the epidermis; A4 contains flavonoids and saponins, which can promote keratinocyte proliferation and migration, primarily acting on the epidermis and epidermal-dermal junction; A1, rich in coumarin and xanthones, can inhibit the NF-κB inflammatory pathway and reduce IL-6 and TNF-α secretion, primarily acting on the dermis; A5, rich in flavonols and mucin, can regulate inflammatory mediators and promote tissue repair, primarily acting on the dermis. The five components work synergistically from four dimensions: moisturizing barrier, anti-oxidation, anti-inflammation, and cell repair, achieving a multi-target synergistic effect of "1+1+1+1+1>5".

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] (1) The soothing and repairing composition of the present invention can significantly promote the migration of keratinocytes and the closure of scratches, and has good cell repair and healing effects.

[0029] (2) The soothing and repairing composition of the present invention has excellent anti-inflammatory activity and can significantly inhibit the secretion of LPS-induced IL-6 and TNF-α inflammatory factors.

[0030] (3) The soothing and repairing composition provided by the present invention has good antioxidant capacity and can effectively remove DPPH free radicals.

[0031] (4) The soothing and repairing composition of the present invention can effectively improve the condition of sensitive skin, reduce transepidermal water loss, increase the moisture content of the stratum corneum, reduce skin erythema, and relieve subjective discomfort such as redness, tightness and stinging on human skin.

[0032] (5) In the soothing and repairing composition of the present invention, there is a significant synergistic effect among the five active components derived from callus tissue, and each component is indispensable for achieving the best efficacy.

[0033] (6) The soothing and repairing composition of the present invention has good stability and safety in use, and is non-irritating to the skin.

[0034] This invention achieves a multi-dimensional synergistic soothing and repairing effect, encompassing moisturizing barrier, anti-oxidation, anti-inflammation, and cell repair, by selecting various unique plant callus tissues and employing differentiated extraction processes. This overcomes the shortcomings of existing products with only one dimension of efficacy. Detailed Implementation

[0035] To make the objectives and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0036] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0037] Example 1: Preparation of callus tissue

[0038] Callus cell clusters of *Thick-shelled Red*, *Oroxylum indicum*, *Fernroot*, and *Sedum aizoon* were prepared using the following unified method:

[0039] Sterile explants from various plants (young leaves from *Echinochloa crus-galli*, stem segments from *Oroxylum indicum*, leaves from *Pteris vittata*, and leaves from *Rhodiola rosea*) were inoculated into callus induction medium. This induction medium was MS basal medium supplemented with 2.5 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.45 mg / L 6-benzylaminopurine (6-BA), 30 g / L sucrose, and 7.0 g / L agar, pH 5.8. The MS basal medium formula (per liter) is as follows: NH4NO3 1650mg, KNO3 1900mg, CaCl2·2H2O 440mg, MgSO4·7H2O 370mg, KH2PO4 170mg, KI 0.83mg, H3BO3 6.2mg, MnSO4·4H2O 22.3mg, ZnSO4·7H2O 8.6mg, Na2MoO4·2H2O 0.25mg, CuSO4·5H2O 0.025mg, CoCl2·6H2O 0.025mg, FeSO4·7H2O 27.8mg, Na2EDTA·2H2O 37.3mg, inositol 100mg, glycine 2mg, thiamine hydrochloride 0.1mg, pyridoxine hydrochloride 0.5mg, and nicotinic acid 0.5mg. Callus tissue was obtained by induction culture at 24±1℃ in the dark for 25 days. The callus tissue was then transferred to liquid suspension proliferation medium, which was MS basal medium supplemented with 1.8 mg / L naphthaleneacetic acid (NAA), 0.4 mg / L kinetin (KT) and 25 g / L sucrose, pH 5.8. The medium was cultured in suspension at 130 rpm and 24±1℃ under weak light for 18 days. After the culture was completed, the callus tissue cell clumps were collected by filtration through a 300-mesh filter, washed three times with deionized water, and centrifuged at 4000 rpm for 15 min. The precipitate was then collected for later use.

[0040] Example 2

[0041] This embodiment provides a soothing and repairing composition, which, by weight, consists of the following components: 8 parts of *Ceratophyllum demersum* callus, 6 parts of *Oroxylum indicum* callus, 8 parts of *Pteris vittata* callus, 5 parts of *Rhodiola rosea* callus, 3 parts of sodium hyaluronate (a mixture of high molecular weight sodium hyaluronate 800,000 Da and low molecular weight sodium hyaluronate 30,000 Da in a mass ratio of 1:1.5), 2 parts of trehalose, 0.5 parts of dipotassium glycyrrhizate, 2 parts of hydrogenated lecithin, 2 parts of polyglycerol-10 stearate, 1 part of cetearyl oleate / sorbitan oleate, 3 parts of 1,2-pentanediol, 0.1 parts of xanthan gum, and deionized water to a total of 100 parts.

[0042] Its preparation process is as follows:

[0043] S1: Prepare five active components respectively:

[0044] (1) Anti-inflammatory active component A1 of red thick shell callus: 70% of the total amount of red thick shell callus was added to 50% volume fraction of ethanol aqueous solution at a material-to-liquid ratio of 1:5, and a compound enzyme system (cellulase and snail enzyme were mixed at a mass ratio of 2:1) was added. The amount of enzyme added was 0.5% of the wet weight of the callus. The pH was adjusted to 4.5, and the callus was enzymatically extracted at 35℃ for 10h. The enzyme was then inactivated at 85℃ for 30min. The supernatant was collected by centrifugation, concentrated under reduced pressure, and extracted three times with an equal volume of ethyl acetate. The ethyl acetate phases were combined and evaporated to dryness under reduced pressure at 40℃ to obtain the final product.

[0045] (2) Red thick shell-fern callus combined polysaccharide moisturizing barrier component A2: Mix 30% of the total amount of red thick shell callus and 60% of the total amount of fern callus, add deionized water at a material-liquid ratio of 1:5, adjust the pH to 5.0, add cellulase at a rate of 0.3% of the wet weight of the mixture, extract at 20℃ for 12h, collect the supernatant by centrifugation, concentrate under reduced pressure and freeze dry into powder to obtain the product.

[0046] (3) Antioxidant and soothing component A3 of Oroxylum indicum callus: Oroxylum indicum callus was added to 70% volume fraction of ethanol aqueous solution at a material-to-liquid ratio of 1:6, the pH was adjusted to 4.0, and the mixture was stirred and extracted at 40℃ for 14h. The supernatant was collected by centrifugation, concentrated under reduced pressure until there was no alcohol odor, and then extracted with petroleum ether and ethyl acetate in sequence. The ethyl acetate phase was collected and evaporated to dryness under reduced pressure at 40℃ to obtain the final product.

[0047] (4) Fern callus regeneration and repair component A4: 40% of the total amount of fern callus in the formula was added to deionized water at a material-to-liquid ratio of 1:8. The mixture was refluxed at 80°C for 2 hours each time. The extracts were combined, cooled, and concentrated under reduced pressure. The mixture was then passed through a macroporous adsorption resin column and eluted sequentially with deionized water, 30% ethanol, 60% ethanol, and 95% ethanol. The eluted portions of 30% ethanol and 60% ethanol were collected, combined, and concentrated under reduced pressure at 40°C to dryness to obtain the final product.

[0048] (5) Anti-inflammatory and healing-promoting component A5 of Rhodiola rosea callus: Rhodiola rosea callus was added to 60% volume fraction of ethanol aqueous solution at a material-to-liquid ratio of 1:5, the pH was adjusted to 5.0, and the mixture was extracted at 30℃ with stirring for 10h. The supernatant was collected by centrifugation, concentrated under reduced pressure until there was no alcohol odor, and then dispersed with deionized water. The mixture was extracted with petroleum ether and ethyl acetate in sequence, the ethyl acetate phase was collected, and the mixture was evaporated to dryness under reduced pressure at 40℃ to obtain the final product.

[0049] S2: Deionized water accounting for 60% of the total formula amount is designated as aqueous phase B1, deionized water accounting for 30% of the total formula amount is designated as aqueous phase B2, and deionized water accounting for 10% of the total formula amount is designated as aqueous phase B3. The sum of B1, B2, and B3 is 100 parts.

[0050] S3: Dissolve sodium hyaluronate, trehalose, and dipotassium glycyrrhizate in aqueous phase B1 under stirring at a temperature not exceeding 40°C to obtain an auxiliary component solution.

[0051] S4: Hydrogenated lecithin, polyglycerol-10 stearate and cetearyl oleate / sorbitan oleate were heated and melted in a water bath at 65°C, and then 1,2-pentanediol was added and stirred evenly to obtain an oil phase; the oil phase was slowly added to the auxiliary component solution under high-speed homogenization conditions, homogenized for 3 min to form an emulsion matrix, and then cooled to 40°C.

[0052] S5: Mix A1, A2, A3, A4, and A5, and then dissolve or disperse them in the aqueous phase B2 at 30°C to obtain a dispersion of the active component.

[0053] S6: Add the dispersion of the active component to the emulsion matrix and stir until homogeneous to obtain mixture A; then disperse xanthan gum in aqueous phase B3 and add it to mixture A, stirring until fully swollen to obtain mixture B.

[0054] S7: Adjust the pH of mixture B to 5.2 with citric acid-sodium citrate buffer solution; continue stirring at low speed for 15 minutes to homogenize the system, fill and seal, and store at 2°C in the dark to obtain the final product.

[0055] Example 3

[0056] This embodiment provides a soothing and repairing composition, which, by weight, consists of the following components: 12 parts of *Ceratophyllum demersum* callus, 9 parts of *Oroxylum indicum* callus, 12 parts of *Pteris vittata* callus, 8 parts of *Rhodiola rosea* callus, 5 parts of sodium hyaluronate (a mixture of high molecular weight sodium hyaluronate 1.2 million Da and low molecular weight sodium hyaluronate 50,000 Da in a mass ratio of 1:2), 4 parts of trehalose, 1 part of dipotassium glycyrrhizate, 3 parts of hydrogenated lecithin, 3 parts of polyglycerol-10 stearate, 2 parts of cetearyl oleate / sorbitan oleate, 5 parts of 1,2-pentanediol, 0.3 parts of xanthan gum, and deionized water to a total of 100 parts.

[0057] Its preparation process is as follows:

[0058] S1: Prepare five active components respectively:

[0059] (1) Anti-inflammatory active component A1 of red thick shell callus: 70% of the total amount of red thick shell callus was added to 50% volume fraction of ethanol aqueous solution at a material-to-liquid ratio of 1:8, and a compound enzyme system (cellulase and snail enzyme were mixed at a mass ratio of 2:1) was added. The amount of enzyme added was 1.0% of the wet weight of the callus. The pH was adjusted to 5.0, and the callus was enzymatically extracted at 40℃ for 8 hours. The enzyme was then inactivated at 90℃ for 20 minutes. The supernatant was collected by centrifugation, concentrated under reduced pressure, and extracted three times with an equal volume of ethyl acetate. The ethyl acetate phases were combined and evaporated to dryness under reduced pressure at 45℃ to obtain the final product.

[0060] (2) Red thick shell-fern callus combined polysaccharide moisturizing barrier component A2: Mix 30% of the total amount of red thick shell callus and 60% of the total amount of fern callus, add deionized water at a material-liquid ratio of 1:8, adjust the pH to 5.5, add cellulase at a rate of 0.5% of the wet weight of the mixture, extract at 25℃ for 9 hours, collect the supernatant by centrifugation, concentrate under reduced pressure and freeze dry into powder to obtain the product.

[0061] (3) Antioxidant and soothing component A3 of Oroxylum indicum callus: Oroxylum indicum callus was added to 70% volume fraction of ethanol aqueous solution at a material-to-liquid ratio of 1:9, the pH was adjusted to 4.5, and the mixture was stirred and extracted at 45℃ for 12h. The supernatant was collected by centrifugation, concentrated under reduced pressure until there was no alcohol odor, and then extracted with petroleum ether and ethyl acetate in sequence. The ethyl acetate phase was collected and evaporated to dryness under reduced pressure at 50℃ to obtain the final product.

[0062] (4) Fern callus regeneration and repair component A4: 40% of the total amount of fern callus in the formula was added to deionized water at a material-to-liquid ratio of 1:12. The mixture was refluxed at 90°C twice for 1.8 hours each time. The extracts were combined, cooled, and concentrated under reduced pressure. The mixture was then passed through a macroporous adsorption resin column and eluted sequentially with deionized water, 30% ethanol, 60% ethanol, and 95% ethanol. The eluted portions of 30% ethanol and 60% ethanol were collected, combined, and concentrated under reduced pressure at 50°C to dryness to obtain the final product.

[0063] (5) Anti-inflammatory and healing-promoting component A5 of Rhodiola rosea callus: Rhodiola rosea callus was added to 60% volume fraction of ethanol aqueous solution at a material-to-liquid ratio of 1:8, the pH was adjusted to 5.5, and the mixture was stirred and extracted at 40℃ for 8 hours. The supernatant was collected by centrifugation, concentrated under reduced pressure until there was no alcohol odor, and then dispersed with deionized water. The mixture was extracted with petroleum ether and ethyl acetate in sequence, the ethyl acetate phase was collected, and the mixture was evaporated to dryness under reduced pressure at 50℃ to obtain the final product.

[0064] S2: Deionized water accounting for 70% of the total formula amount is designated as aqueous phase B1, deionized water accounting for 23% of the total formula amount is designated as aqueous phase B2, and deionized water accounting for 7% of the total formula amount is designated as aqueous phase B3. The sum of B1, B2, and B3 is 100 parts.

[0065] S3: Dissolve sodium hyaluronate, trehalose, and dipotassium glycyrrhizate in aqueous phase B1 under stirring at a temperature not exceeding 40°C to obtain an auxiliary component solution.

[0066] S4: Hydrogenated lecithin, polyglycerol-10 stearate and cetearyl oleate / sorbitan oleate were heated and melted in a 70°C water bath, and then 1,2-pentanediol was added and stirred evenly to obtain an oil phase; the oil phase was slowly added to the auxiliary component solution under high-speed homogenization conditions, homogenized for 4 min to form an emulsion matrix, and then cooled to 42°C.

[0067] S5: Mix A1, A2, A3, A4, and A5, and then dissolve or disperse them in the aqueous phase B2 at 40°C to obtain a dispersion of the active component.

[0068] S6: Add the dispersion of the active component to the emulsion matrix and stir until homogeneous to obtain mixture A; then disperse xanthan gum in aqueous phase B3 and add it to mixture A, stirring until fully swollen to obtain mixture B.

[0069] S7: Adjust the pH of mixture B to 5.8 with citric acid-sodium citrate buffer solution; continue stirring at low speed for 20 minutes to homogenize the system, fill and seal, and store at 5°C away from light to obtain the final product.

[0070] Example 4

[0071] This embodiment provides a soothing and repairing composition, which, by weight, consists of the following components: 16 parts of *Ceratophyllum demersum* callus, 12 parts of *Oroxylum indicum* callus, 15 parts of *Pteris vittata* callus, 12 parts of *Rhodiola rosea* callus, 8 parts of sodium hyaluronate (a mixture of high molecular weight sodium hyaluronate 1.5 million Da and low molecular weight sodium hyaluronate 80,000 Da in a mass ratio of 1:2.5), 5 parts of trehalose, 2 parts of dipotassium glycyrrhizate, 5 parts of hydrogenated lecithin, 4 parts of polyglycerol-10 stearate, 3 parts of cetearyl oleate / sorbitan oleate, 8 parts of 1,2-pentanediol, 0.5 parts of xanthan gum, and deionized water to a total of 100 parts.

[0072] Its preparation process is as follows:

[0073] S1: Prepare five active components respectively:

[0074] (1) Anti-inflammatory active component A1 of red thick shell callus: 70% of the total amount of red thick shell callus was added to 50% volume fraction of ethanol aqueous solution at a material-to-liquid ratio of 1:10, and a compound enzyme system (cellulase and snail enzyme were mixed at a mass ratio of 2:1) was added. The amount of enzyme added was 1.5% of the wet weight of the callus. The pH was adjusted to 5.5, and the callus was enzymatically extracted at 45℃ for 6 hours. The enzyme was then inactivated at 95℃ for 15 minutes. The supernatant was collected by centrifugation, concentrated under reduced pressure, and extracted twice with an equal volume of ethyl acetate. The ethyl acetate phases were combined and evaporated to dryness under reduced pressure at 50℃ to obtain the final product.

[0075] (2) Red thick shell-fern callus combined polysaccharide moisturizing barrier component A2: Mix 30% of the total amount of red thick shell callus and 60% of the total amount of fern callus, add deionized water at a material-liquid ratio of 1:10, adjust the pH to 6.0, add cellulase at a rate of 0.8% of the wet weight of the mixture, extract at 30℃ for 6 hours, collect the supernatant by centrifugation, concentrate under reduced pressure and freeze dry into powder to obtain the product.

[0076] (3) Antioxidant and soothing component A3 of Oroxylum indicum callus: Oroxylum indicum callus was added to 70% volume fraction of ethanol aqueous solution at a material-to-liquid ratio of 1:12, the pH was adjusted to 5.0, and the mixture was stirred and extracted at 50℃ for 8 hours. The supernatant was collected by centrifugation, concentrated under reduced pressure until there was no alcohol odor, and then extracted with petroleum ether and ethyl acetate in sequence. The ethyl acetate phase was collected and evaporated to dryness under reduced pressure at 55℃ to obtain the final product.

[0077] (4) Fern callus regeneration and repair component A4: 40% of the total amount of fern callus in the formula was added to deionized water at a material-to-liquid ratio of 1:15. The mixture was refluxed at 95°C twice for 1.5 hours each time. The extracts were combined, cooled, and concentrated under reduced pressure. The mixture was then passed through a macroporous adsorption resin column and eluted sequentially with deionized water, 30% ethanol, 60% ethanol, and 95% ethanol. The elution fractions of 30% ethanol and 60% ethanol were collected, combined, and concentrated under reduced pressure at 55°C to dryness to obtain the final product.

[0078] (5) Anti-inflammatory and healing-promoting component A5 of Rhodiola rosea callus: Rhodiola rosea callus was added to 60% volume fraction of ethanol aqueous solution at a material-to-liquid ratio of 1:12, the pH was adjusted to 6.0, and the mixture was stirred and extracted at 45℃ for 6 hours. The supernatant was collected by centrifugation, concentrated under reduced pressure until there was no alcohol odor, and then dispersed with deionized water. The mixture was extracted with petroleum ether and ethyl acetate in sequence, the ethyl acetate phase was collected, and the mixture was evaporated to dryness under reduced pressure at 55℃ to obtain the final product.

[0079] S2: Deionized water accounting for 80% of the total formula amount is designated as aqueous phase B1, deionized water accounting for 15% of the total formula amount is designated as aqueous phase B2, and deionized water accounting for 5% of the total formula amount is designated as aqueous phase B3. The sum of B1, B2, and B3 is 100 parts.

[0080] S3: Dissolve sodium hyaluronate, trehalose, and dipotassium glycyrrhizate in aqueous phase B1 under stirring at a temperature not exceeding 40°C to obtain an auxiliary component solution.

[0081] S4: Hydrogenated lecithin, polyglycerol-10 stearate and cetearyl oleate / sorbitan oleate were heated and melted in a 75°C water bath, and then 1,2-pentanediol was added and stirred evenly to obtain an oil phase; the oil phase was slowly added to the auxiliary component solution under high-speed homogenization conditions, homogenized for 5 min to form an emulsion matrix, and then cooled to 45°C.

[0082] S5: Mix A1, A2, A3, A4, and A5, and then dissolve or disperse them in the aqueous phase B2 at 45°C to obtain a dispersion of the active component.

[0083] S6: Add the dispersion of the active component to the emulsion matrix and stir until homogeneous to obtain mixture A; then disperse xanthan gum in aqueous phase B3 and add it to mixture A, stirring until fully swollen to obtain mixture B.

[0084] S7: Adjust the pH of mixture B to 6.2 with citric acid-sodium citrate buffer solution; continue stirring at low speed for 25 minutes to homogenize the system, fill and seal, and store at 8°C in the dark to obtain the final product.

[0085] Comparative Example 1: A1 only

[0086] The difference from Example 3 is that only A1 is used as the core active component, and the amount of A1 used is the same as in Example 3. A2, A3, A4, and A5 are not used. The remaining components, amounts, and preparation processes are the same as in Example 3.

[0087] Comparative Example 2 (A2 only)

[0088] The difference from Example 3 is that only A2 is used as the core active component, and the amount used is the same as that of A1 in Example 3. A1, A3, A4, and A5 are not used. The remaining components, amounts, and preparation processes are the same as in Example 3.

[0089] Comparative Example 3 (A3 only)

[0090] The difference from Example 3 is that only A3 is used as the core active component, and the amount used is the same as that of A1 in Example 3. A1, A2, A4, and A5 are not used. The remaining components, amounts, and preparation processes are the same as in Example 3.

[0091] Comparative Example 4 (A4 only)

[0092] The difference from Example 3 is that only A4 is used as the core active component, and the amount used is the same as that of A1 in Example 3. A1, A2, A3, and A5 are not used. The remaining components, amounts, and preparation processes are the same as in Example 3.

[0093] Comparative Example 5 (A5 only)

[0094] The difference from Example 3 is that only A5 is used as the core active component, and the amount used is the same as that of A1 in Example 3. A1, A2, A3, and A4 are not used. The remaining components, amounts, and preparation processes are the same as in Example 3.

[0095] Comparative Example 6: Missing A5

[0096] The difference from Example 3 is that only A1, A2, A3, and A4 are used as the core active components, and A5 is not used. The remaining components, amounts, and preparation processes are the same as in Example 3.

[0097] Comparative Example 7: Missing A4

[0098] The difference from Example 3 is that only A1, A2, A3, and A5 are used as the core active components, and A4 is not used. The remaining components, amounts, and preparation processes are the same as in Example 3.

[0099] Comparative Example 8: Missing A3

[0100] The difference from Example 3 is that only A1, A2, A4, and A5 are used as the core active components, while A3 is not used. The remaining components, amounts, and preparation processes are the same as in Example 3.

[0101] Comparative Example 9: Missing A2

[0102] The difference from Example 3 is that only A1, A3, A4, and A5 are used as the core active components, and A2 is not used. The remaining components, dosages, and preparation processes are the same as in Example 3.

[0103] Comparative Example 10: Missing A1

[0104] The difference from Example 3 is that only A2, A3, A4, and A5 are used as the core active components, and A1 is not used. The remaining components, dosages, and preparation processes are the same as in Example 3.

[0105] Comparative Example 11: Blank Control

[0106] No core active ingredients (A1-A5) or auxiliary active ingredients (sodium hyaluronate, trehalose, dipotassium glycyrrhizate) are used. Only the same base emulsion matrix as in Example 3 (i.e., hydrogenated lecithin, polyglycerol-10 stearate, cetearyl oleate and sorbitan oleate, 1,2-pentanediol, xanthan gum, B1, B2, and B3) are used. Preparation process: The oil phase is added to B1 to form the emulsion matrix; then B2 is added to the emulsion matrix, followed by xanthan gum dispersed in B3. Subsequent steps are the same as in Example 3.

[0107] Experimental Example 1: In vitro cell scratch healing experiment

[0108] Human immortalized keratinocytes (HaCaT) were used for cell culture. The complete culture medium used was DMEM high-glucose medium (containing 4.5 g / L D-glucose, 4 mM L-glutamine, and 1 mM sodium pyruvate) supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin. Cells were seeded in 6-well plates and cultured in the above complete medium until confluence reached approximately 95%. A straight line was drawn in the center of each well using a 200 μL pipette tip, and the cells were washed with PBS to remove suspension cells.

[0109] Preparation of test samples: The compositions of Examples 2-4 and Comparative Examples 1-11 were diluted with serum-free DMEM high-glucose basal medium (with the same composition as the above-mentioned DMEM high-glucose medium, but without the addition of FBS and antibiotics) to make the total active ingredient content of the composition in the final test concentration 100 μg / mL. A negative control group was set up: only an equal volume of serum-free DMEM high-glucose medium (without any composition) was added.

[0110] Add 2 mL of culture medium containing the corresponding sample to each well, with 3 replicates per group. Incubate at 37℃ in a 5% CO2 incubator for 48 h. Take photos under an inverted microscope (200×) at 0 h and 48 h, and measure the scratch area using ImageJ software. Calculate the scratch healing rate (%) = (initial scratch area - 48 h scratch area) / initial scratch area × 100%. Each sample has 3 replicates, and the results are averaged.

[0111] Table 1. Effect of each sample on the scratch healing rate of HaCaT cells (mean±SD, n=3)

[0112]

[0113] Table 1 shows that, on the one hand, the scratch healing rate of the negative control group (serum-free culture medium only) was 31.2%, and the healing rate of the blank emulsion matrix (comparative example 11) was 33.0%, with no significant difference between the two (p>0.05), indicating that the blank emulsion matrix itself has no promoting effect on keratinocyte migration. The scratch healing rates of Examples 2-4 were 78.9%, 86.0%, and 83.5%, respectively, all significantly higher than those of the negative control group and Comparative example 11 (p<0.01), indicating that the composition of the present invention can effectively promote keratinocyte migration and wound healing. On the other hand, the healing rates of Examples 2-4 were significantly higher than those of the single-component comparative examples 1-5 (46.2%-53.7%), and also significantly higher than those of comparative examples 6-10 lacking a core component (59.6%-67.0%), p<0.01. Among them, Example 3 showed the best effect, with a healing rate of 86.0%. This indicates that the combination of the five active components of the present invention can produce a cell migration promoting effect far superior to that of a single component or a component lacking a component.

[0114] In addition, a weighted expected value comparison method was used to analyze the synergistic effect:

[0115] (1) First, calculate the “synergistic value” of each single component relative to the negative control (i.e., the scratch healing rate minus the healing rate of the negative control). The healing rate of the negative control was 31.2%. The synergistic values ​​of each single component compared to examples 1-5 were: A1 (46.2-31.2=15.0), A2 (49.8-31.2=18.6), A3 (48.5-31.2=17.3), A4 (51.3-31.2=20.1), and A5 (53.7-31.2=22.5).

[0116] (2) The mass fractions of each active component in the formulation of Example 2 were weighted: based on a total of 41 parts of active ingredients, the mass fractions of A1, A2, A3, A4 and A5 were A1 (12×70% / 41≈0.205), A2 ((12×30%+12×60%) / 41≈0.263), A3 (9 / 41≈0.220), A4 (12×40% / 41≈0.117), and A5 (8 / 41≈0.195).

[0117] The weighted expected synergy value = 15.0 × 0.205 + 18.6 × 0.263 + 17.3 × 0.220 + 20.1 × 0.117 + 22.5 × 0.195 = 3.08 + 4.89 + 3.81 + 2.35 + 4.39 = 18.52 (%).

[0118] The expected scratch healing rate is calculated as follows: negative control 31.2% + 18.52% = 49.72%.

[0119] In Example 3, the measured healing rate was 86.0%, and the measured synergistic effect was 86.0-31.2=54.8%. The measured synergistic effect of 54.8% is much greater than the weighted expected synergistic effect of 18.52%, exceeding it by 36.28%. This result indicates that the five active components derived from callus tissue have a significant synergistic effect in promoting keratinocyte migration, and their combined effect far exceeds the weighted sum of the individual effects of each component.

[0120] The above cell scratch healing experiments show that the compositions of Examples 2-4 of this invention can significantly promote the migration and scratch closure of HaCaT keratinocytes, with Example 3 showing the best effect. Synergistic effect analysis confirmed that there is a significant synergistic effect among the five active components (A1-A5).

[0121] Experiment Example 2: In vitro anti-inflammatory experiment (detection of inflammatory factors in LPS-induced HaCaT cells)

[0122] HaCaT cells were seeded in 24-well plates and cultured in complete medium (DMEM high glucose + 10% FBS + antibiotics) until approximately 80% confluence. The original medium was discarded and replaced with DMEM high glucose medium containing 0.5% FBS (i.e., basal medium + 0.5% FBS) for 12 hours of starvation. Then, lipopolysaccharide (LPS) was added to a final concentration of 1 μg / mL to stimulate the cells. Simultaneously, samples from Examples 2-4 and Comparative Examples 1-11, diluted to 100 μg / mL in DMEM high glucose medium containing 0.5% FBS, were added, respectively. A model control group (LPS only, no sample) and a negative control group (no LPS, no sample, only DMEM high glucose medium containing 0.5% FBS) were set up. Each group had three replicates, and the cells were incubated at 37°C in a 5% CO2 incubator for 24 hours. The cell supernatant was collected, and the levels of IL-6 and TNF-α were measured according to the ELISA kit (Shanghai Enzyme-Link Biotechnology Co., Ltd.) instructions. Inflammatory factor inhibition rate (%) = (content in model control group - content in sample group) / content in model control group × 100%.

[0123] Table 2. Inhibition rates of IL-6 and TNF-α by each sample (mean±SD, n=3)

[0124]

[0125] Note: The negative control group (no LPS stimulation) had an IL-6 level of 28 pg / mL and an TNF-α level of 15 pg / mL; the model control group (LPS only) had an IL-6 secretion level of 1245 pg / mL and an TNF-α secretion level of 890 pg / mL (measured values). The inhibition rate of the model control group was defined as 0%. The inhibition rate of the negative control group was not calculated and is indicated by "-" because it did not receive LPS stimulation and was not included in the inhibition rate calculation.

[0126] The results in Table 2 show that the blank emulsion matrix (Comparative Example 11) had an inhibition rate of less than 4% against both inflammatory factors, indicating no substantial anti-inflammatory effect. Examples 2-4 showed inhibition rates of 72.5%-81.4% against IL-6 and 69.8%-78.1% against TNF-α, both significantly higher than those of the single-component comparative examples 1-5 (IL-6 inhibition rate 24.6%-51.0%, TNF-α inhibition rate 21.0%-47.6%), and also significantly higher than those of comparative examples 6-10 lacking any core component (IL-6 inhibition rate 52.4%-62.5%, TNF-α inhibition rate 49.7%-59.2%), p<0.01.

[0127] In addition, a synergistic effect analysis was conducted. First, the inhibition rate of each individual component (comparative examples 1-5) relative to the model control group was calculated (since the inhibition rate of the model control group was 0, the inhibition rate of each comparative example was directly used as the synergistic value). The weighted expected value was calculated in the same way as in Experiment 1, with the mass fraction of each component remaining unchanged.

[0128] The weighted expected IL-6 inhibition rate = 43.2×0.205 + 24.6×0.263 + 38.0×0.220 + 34.8×0.117 + 51.0×0.195 = 8.86 + 6.47 + 8.36 + 4.07 + 9.95 = 37.71 (%). The measured IL-6 inhibition rate in Example 3 was 81.4%, significantly higher than the weighted expected value of 37.71%, exceeding it by 43.69%. p<0.001.

[0129] Similarly, the weighted expected inhibition rate of TNF-α = 40.5×0.205 + 21.0×0.263 + 35.7×0.220 + 32.4×0.117 + 47.6×0.195 = 8.30 + 5.52 + 7.85 + 3.79 + 9.28 = 34.74 (%). The measured inhibition rate of TNF-α in Example 3 was 78.1%, which is much higher than the weighted expected value of 34.74%, exceeding it by 43.36%. p<0.001. This indicates that the five active components also have a significant synergistic effect in anti-inflammatory aspects.

[0130] The LPS-induced HaCaT cell inflammation model experiments above showed that the compositions of Examples 2-4 of this invention can significantly inhibit the secretion of inflammatory factors IL-6 and TNF-α, with Example 3 showing the best effect. Synergistic effect analysis confirmed that the five active components have a significant synergistic effect in anti-inflammation, and the combined anti-inflammatory effect far exceeds the weighted expected value.

[0131] Experimental Example 3: Antioxidant Test (DPPH Free Radical Scavenging Rate)

[0132] The compositions of Examples 2-4 and Comparative Examples 1-11 were diluted with deionized water to 1 mg / mL (based on total active matter). 2 mL of the sample solution was mixed with 2 mL of 0.1 mM DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) ethanol solution and reacted in the dark for 30 min. Deionized water was used as a control instead of the sample solution, and anhydrous ethanol was used as the background sample instead of the DPPH solution. The absorbance was measured at 517 nm. DPPH scavenging rate (%) = [1 - (A... 样品 -A 本底 ) / A 对照 ×100%. Each sample was measured 3 times, and the average value was taken.

[0133] Table 3. DPPH radical scavenging rate of each sample (mean±SD, n=3)

[0134]

[0135] The results in Table 3 show that the DPPH scavenging rate of the blank emulsion matrix (Comparative Example 11) was only 6.2%, indicating no substantial antioxidant activity. The DPPH scavenging rates of Examples 2-4 were 74.8%-83.2%, significantly higher than those of Comparative Examples 1-5 (30.8%-64.0%) for each single component, and also significantly higher than those of Comparative Examples 6-10 (57.0%-71.8%) lacking a core component, p<0.01.

[0136] Using the same method as Experiment 1, weighted expected values ​​were calculated with Example 3 as a representative. The synergistic values ​​of each single component's DPPH scavenging rate minus the blank matrix (6.2%) were as follows: A1 (37.4-6.2=31.2), A2 (30.8-6.2=24.6), A3 (64.0-6.2=57.8), A4 (43.6-6.2=37.4), and A5 (54.5-6.2=48.3).

[0137] Weighted expected synergy = 31.2×0.205 + 24.6×0.263 + 57.8×0.220 + 37.4×0.117 + 48.3×0.195 = 6.40 + 6.47 + 12.72 + 4.38 + 9.42 = 39.39 (%).

[0138] Expected clearance rate = 6.2% + 39.39% = 45.59%.

[0139] Example 3 showed a measured clearance rate of 83.2% and a measured synergistic effect of 83.2% - 6.2% = 77.0%, which far exceeded the expected synergistic effect of 39.39% by 37.61%, indicating that there is also a significant synergistic effect in terms of anti-oxidation.

[0140] The DPPH free radical scavenging experiments above show that the compositions of Examples 2-4 of this invention have good antioxidant capacity, with Example 3 showing the best effect. Synergistic effect analysis confirms that the five active components have a significant synergistic effect in antioxidant activity.

[0141] Experiment Example 4: Short-term efficacy test on human skin

[0142] Eighty-four healthy female volunteers, aged 25-50 years (mean age 36.5 years), who met the diagnostic criteria for sensitive skin (screening based on a 10% lactic acid stinging test with a stinging score ≥3), were recruited. All participants had no serious systemic diseases, no history of cosmetic allergies, and no skin diseases at the test sites. The participants were randomly divided into 14 groups of 6, corresponding to Examples 2-4 and Comparative Examples 1-11, respectively.

[0143] Subjects applied approximately 0.5g of the corresponding sample to the inside of their left forearm and face after cleansing in the morning and evening, for 28 consecutive days. No other skincare products were used during the testing period.

[0144] The following tests were conducted on day 0 (D0) before use, day 14 (D14) after use, and day 28 (D28):

[0145] (1) Transepidermal water loss (TEWL): Measured using a Tewameter® TM300 transepidermal water loss meter (Courage+ Khazaka, Germany), unit g / h / m 2 A decrease in TEWL value indicates improved skin barrier function.

[0146] (2) Stratum corneum moisture content: Measured using a Corneometer® CM825 (Courage + Khazaka) stratum corneum moisture meter, in units of au. Increased moisture content indicates a moisturizing effect.

[0147] (3) Skin erythema index (a* value): Measured using a Mexameter® MX18 (Courage + Khazaka) skin colorimeter. A lower a* value indicates a reduction in erythema.

[0148] (4) Subjective discomfort rating: The 0-10 point visual analog scale (VAS) was used, and the subjects rated facial redness, tightness and stinging / burning sensation respectively. 0 points indicated no discomfort and 10 points indicated the most severe discomfort.

[0149] Calculate the difference between D28 and D0 (Δ=D28-D0), a negative value indicates improvement; for easier and more intuitive comparison, this paper uses the improvement value = D0 value - D28 value (positive value indicates improvement, the larger the value, the more obvious the improvement).

[0150] All tests were conducted in a constant temperature and humidity environment (22±1℃, 50±5% RH), and each part was measured 3 times and the average value was taken.

[0151] Table 4 Results of human efficacy trials (mean±SD, n=6)

[0152]

[0153] Table 4 shows that the blank emulsion matrix (Comparative Example 11) showed very little improvement in all indicators (TEWL improvement of only 1.2, stratum corneum moisture content improvement of 2.8), indicating that the base emulsion matrix itself has limited effect on improving sensitive skin. Examples 2-4 showed significant improvement in all tested indicators, significantly higher than Comparative Examples 1-5 (each single component) and Comparative Examples 6-10 (lacking any core component), with Example 3 showing the best effect: TEWL improvement of 8.8 g / h / m. 2 The stratum corneum moisture content improved by 25.1 au, the erythema index improved by 6.1, and the VAS scores for redness, tightness, and stinging / burning sensation improved by 5.6, 5.2, and 5.4 points, respectively. These data indicate that the composition of the present invention can effectively improve the barrier function, moisturizing ability, erythema, and subjective discomfort of sensitive skin, and the combined effect of the five active ingredients is far superior to that of the individual proportions.

[0154] Taking the TEWL improvement value as an example, the weighted expected value method was used to analyze the synergistic effect. The TEWL improvement value of the blank emulsion matrix (Comparative Example 11) was 1.2. The synergistic values ​​of the TEWL improvement values ​​of each single component Comparative Examples 1-5 after subtracting the blank were: A1 (3.6-1.2=2.4), A2 (5.0-1.2=3.8), A3 (3.2-1.2=2.0), A4 (3.4-1.2=2.2), and A5 (3.9-1.2=2.7).

[0155] Weighted expected synergy = 2.4×0.205 + 3.8×0.263 + 2.0×0.220 + 2.2×0.117 + 2.7×0.195 = 0.49 + 1.00 + 0.44 + 0.26 + 0.53 = 2.72.

[0156] Expected TEWL improvement = 1.2 + 2.72 = 3.92.

[0157] In Example 3, the measured TEWL improvement value was 8.8, and the measured synergistic value was 8.8-1.2=7.6, which is much greater than the expected synergistic value of 2.72 and exceeds 4.88, indicating that the five active ingredients have a significant synergistic effect in improving skin barrier function.

[0158] The 28-day human trial results showed that the compositions of Examples 2-4 of this invention can effectively improve transepidermal water loss, stratum corneum moisture content, erythema index, and subjective discomfort in sensitive skin, with Example 3 showing the best effect. Synergistic effect analysis further confirmed that the five active ingredients have a significant synergistic effect in improving skin barrier function.

[0159] Experiment Example 5: Stability Test

[0160] The compositions of Examples 2-4 were respectively filled into 30 mL PET plastic bottles and placed in a constant temperature and humidity chamber at 40℃±2℃ and 75%RH±5% for accelerated storage for 4 weeks. Samples were taken at 0, 2, and 4 weeks for the following tests:

[0161] (1) Appearance observation: Observe with the naked eye whether there is layering, emulsion breaking, discoloration, or odor.

[0162] (2) pH value measurement: pH value is measured directly using a pH meter.

[0163] (3) Centrifugation stability: Take 10 mL of sample into a centrifuge tube, centrifuge at 4000 rpm for 30 minutes, observe whether it separates into layers, and measure the height of the precipitate layer (if present).

[0164] Each sample was repeated 3 times.

[0165] Table 5 Stability Test Results

[0166]

[0167] Table 5 shows that after 4 weeks of accelerated storage at 40°C, all samples from the examples maintained a uniform appearance, without any stratification, demulsification, discoloration, or off-odor; the pH value variation was within ±0.1, and the pH value remained stable; after centrifugation, there was no stratification or precipitation, and the precipitation rate was 0. This indicates that the compositions of the present invention have good physicochemical stability and can meet the shelf-life requirements of conventional cosmetics.

[0168] Experiment Example 6: Skin Irritation / Safety Test

[0169] Following the "Skin Occlusive Patch Test" method in the *Cosmetic Safety Technical Specifications* (2015 edition), 30 healthy volunteers aged 22-48 years who met the diagnostic criteria for sensitive skin (different from the subjects in the human efficacy test) were recruited. Approximately 0.02g of the composition sample from Example 3 was placed in the patch test chamber, with deionized water as a blank control. The patch was applied to normal skin on the back of the subjects and removed after 24 hours. Skin reactions were observed at 0.5h, 24h, and 48h after removal. The results showed that none of the 30 subjects experienced any skin irritation reactions such as erythema or edema at any of the observation time points (skin reaction grade was 0), with a positive reaction rate of 0%. The blank control also showed no reaction. This indicates that the composition of Example 3 of this invention is non-irritating to sensitive skin and has good skin safety and tolerability. Combined with the fact that no adverse reactions were reported in the human efficacy test, it can be considered that the composition of this invention has high safety for use.

[0170] In summary, this invention prepares a soothing and repairing composition by differentially extracting and compounding callus tissues from four plants: *Hypericum rubrum*, *Oroxylum indicum*, *Pteris vittata*, and *Rhodiola rosea*. The experiments above demonstrate that this composition significantly promotes keratinocyte migration, inhibits the secretion of inflammatory factors, scavenges free radicals, improves the barrier function of sensitive skin, increases stratum corneum moisture content, reduces erythema, and alleviates subjective discomfort. Furthermore, there is a significant synergistic effect among the five active components. Simultaneously, the composition exhibits good stability, is non-irritating to the skin, and has high safety for use.

[0171] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A soothing and repairing composition, characterized in that: By weight, it consists of the following components: 8-16 parts of red thick-shelled callus, 6-12 parts of wood butterfly callus, 8-15 parts of fern root callus, 5-12 parts of Rhodiola rosea callus, 3-8 parts of sodium hyaluronate, 2-5 parts of trehalose, 0.5-2 parts of dipotassium glycyrrhizate, 2-5 parts of hydrogenated lecithin, 2-4 parts of polyglycerol-10 stearate, 1-3 parts of cetearyl oleate / sorbitan oleate, 3-8 parts of 1,2-pentanediol, 0.1-0.5 parts of xanthan gum, and deionized water to 100 parts.

2. The soothing and repairing composition according to claim 1, characterized in that: The sodium hyaluronate is a compound of high molecular weight sodium hyaluronate (800,000-1,500,000 Da) and low molecular weight sodium hyaluronate (30,000-80,000 Da) in a mass ratio of 1:1.5-2.

5.

3. The soothing and repairing composition according to claim 1, characterized in that: The soothing and repairing composition is used in the preparation of skin care products for soothing, repairing the skin barrier, anti-inflammatory or antioxidant purposes.

4. The preparation process of the soothing and repairing composition according to any one of claims 1-3, characterized in that: Includes the following steps: S1: Five active components were prepared respectively: anti-inflammatory active component A1 of red thick shell callus, moisturizing barrier component A2 of red thick shell-fern root polysaccharide, antioxidant and soothing component A3 of wood butterfly callus, regeneration and repair component A4 of fern root callus, and anti-inflammatory and healing-promoting component A5 of rhodiola rosea callus. S2: Deionized water accounting for 60%-80% of the total formula is designated as aqueous phase B1, deionized water accounting for 15%-30% of the total formula is designated as aqueous phase B2, and deionized water accounting for 5%-10% of the total formula is designated as aqueous phase B3. The sum of B1, B2, and B3 is 100 parts. S3: Dissolve sodium hyaluronate, trehalose and dipotassium glycyrrhizate in aqueous phase B1 under stirring at a temperature not exceeding 40°C to obtain an auxiliary component solution; S4: Hydrogenated lecithin, polyglycerol-10 stearate and cetearyl oleate / sorbitan oleate are heated and melted in a water bath at 65-75℃, and then 1,2-pentanediol is added and stirred evenly to obtain an oil phase; the oil phase is slowly added to the auxiliary component solution under high-speed homogenization conditions, homogenized for 3-5 min to form an emulsion matrix, and then cooled to 40-45℃. S5: Mix A1, A2, A3, A4, and A5, and then dissolve or disperse them in the aqueous phase B2 at 30-45℃ to obtain a dispersion of the active component; S6: Add the dispersion of the active component to the emulsion matrix and stir until homogeneous to obtain mixture A; then disperse xanthan gum in aqueous phase B3 and add it to mixture A, stirring until fully swollen to obtain mixture B; S7: Adjust the pH of mixture B to 5.2-6.2 with citric acid-sodium citrate buffer solution; continue stirring at low speed for 15-25 minutes to homogenize the system, fill and seal, and store at 2-8℃ away from light to obtain the final product.

5. The preparation process according to claim 4, characterized in that: In step S1, the preparation method of the anti-inflammatory active component A1 of the red thick-shelled callus is as follows: 70% of the total amount of red thick-shelled callus is added to a 50% volume fraction of ethanol aqueous solution at a material-to-liquid ratio of 1:5-1:10, and a compound enzyme system is added. The compound enzyme system is composed of cellulase and snail enzyme in a mass ratio of 2:

1. The amount of enzyme added is 0.5%-1.5% of the wet weight of the callus. The pH is adjusted to 4.5-5.5, and enzymatic extraction is carried out at 35-45℃ for 6-10 hours. The enzyme is then inactivated at 85-95℃ for 15-30 minutes. The supernatant is collected by centrifugation, concentrated under reduced pressure, and extracted 2-3 times with an equal volume of ethyl acetate. The ethyl acetate phases are combined and evaporated to dryness under reduced pressure at 40-50℃ to obtain the final product.

6. The preparation process according to claim 4, characterized in that: In step S1, the preparation method of the red thick shell-fern callus combined polysaccharide moisturizing barrier component A2 is as follows: 30% of the total amount of red thick shell callus and 60% of the total amount of fern callus are mixed, and deionized water is added at a material-liquid ratio of 1:5-1:

10. The pH is adjusted to 5.0-6.0, and cellulase is added at a concentration of 0.3%-0.8% of the wet weight of the mixture. The mixture is extracted at 20-30℃ for 6-12 hours, and the supernatant is collected by centrifugation. After concentration under reduced pressure, the mixture is freeze-dried into powder to obtain the final product.

7. The preparation process according to claim 4, characterized in that: In step S1, the preparation method of the antioxidant and soothing component A3 of the Oroxylum indicum callus is as follows: add Oroxylum indicum callus to a 70% volume fraction of ethanol aqueous solution at a material-to-liquid ratio of 1:6-1:12, adjust the pH to 4.0-5.0, stir and extract at 40-50℃ for 8-14 hours, collect the supernatant by centrifugation, concentrate under reduced pressure until there is no alcohol odor, extract with petroleum ether and ethyl acetate in sequence, collect the ethyl acetate phase, evaporate to dryness under reduced pressure at 40-55℃ to obtain the product.

8. The preparation process according to claim 4, characterized in that: In step S1, the preparation method of the *Pteris vittata* callus regeneration and repair component A4 is as follows: 40% of the total amount of *Pteris vittata* callus is added to deionized water at a material-to-liquid ratio of 1:8-1:15, and extracted twice by reflux at 80-95℃ for 1.5-2 hours each time. The extracts are combined, cooled, and concentrated under reduced pressure. The solution is then passed through a macroporous adsorption resin column and eluted sequentially with deionized water, 30% ethanol, 60% ethanol, and 95% ethanol. The 30% ethanol and 60% ethanol eluates are collected, combined, and concentrated under reduced pressure at 40-55℃ to dryness to obtain the final product.

9. The preparation process according to claim 4, characterized in that: In step S1, the preparation method of the anti-inflammatory and healing-promoting component A5 of Rhodiola rosea callus is as follows: Rhodiola rosea callus is added to a 60% volume fraction of ethanol aqueous solution at a material-to-liquid ratio of 1:5-1:12, the pH is adjusted to 5.0-6.0, and the mixture is extracted by stirring at 30-45℃ for 6-10 hours. The supernatant is collected by centrifugation, concentrated under reduced pressure until there is no alcohol odor, dispersed with deionized water, and extracted successively with petroleum ether and ethyl acetate. The ethyl acetate phase is collected and evaporated to dryness under reduced pressure at 40-55℃ to obtain the final product.

10. The preparation process according to claim 4, characterized in that: The callus tissues of *Sedum morganianum*, *Oroxylum indicum*, *Pteris vittata*, and *Sedum aizoon* were obtained by the following methods: Sterile explants from each plant were inoculated into an induction medium consisting of MS basal medium supplemented with 2.5 mg / L 2,4-D, 0.45 mg / L 6-BA, 30 g / L sucrose, and 7.0 g / L agar, pH 5.8, and cultured at 24±1℃ in the dark for 25 days to obtain callus tissue. The callus tissue was then transferred to a liquid suspension proliferation medium consisting of MS basal medium supplemented with 1.8 mg / L NAA, 0.4 mg / L KT, and 25 g / L sucrose, pH 5.8, and cultured at 130 rpm and 24±1℃ under low light for 18 days. After filtration, callus cell clumps were collected, washed three times with deionized water, and centrifuged at 4000 rpm for 15 min to obtain the final product.

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