Application of circium japonicum extract

Abstract

The invention discloses application of a circium japonicum extract. The circium japonicum extract is applied to preparation of products for preventing skin problems caused by cortisol unbalance. The invention also relates to application in preparation of products for relieving skin problems caused by cortisol imbalance. The circium japonicum extract can improve skin problems caused by excessive transformation of cortisol and increase the expression quantity of dermal collagen, skin barrier related protein and skin basement membrane related protein, so that the problems of skin barrier damage, skin sensitivity, wrinkle generation, skin elasticity reduction and the like caused by emotional pressure are effectively relieved.

Description

Technical Field

[0001] This application belongs to the field of cosmetic technology, and in particular relates to the use of a thistle extract in the preparation of products for preventing and / or alleviating skin problems caused by cortisol imbalance. Background Technology

[0002] Thistle is a plant belonging to the genus *Cirsium* in the family Asteraceae. It contains a variety of physiologically active components, such as flavonoids, including sauroprin and sauroprin, as well as lignins, triterpenoids, sterols, enols, volatile oils, acids, and glycosides. It has various pharmacological effects, including hemostasis, blood pressure reduction, antitumor, antidiabetic, antibacterial, anti-osteoporosis, liver protection, and diuresis.

[0003] Cortisol, also known as hydrocortisone, is a glucocorticoid released and secreted by the adrenal glands in response to activation of the HPA axis. It is considered a "stress hormone." When the body is under stress, cortisol can be activated locally in the skin. Sudden release of cortisol under stress can cause damage to the epidermal barrier, skin sensitivity and fragility, and skin aging.

[0004] There are currently no reports of using thistle extract to treat skin problems caused by cortisol imbalance. Summary of the Invention

[0005] This application provides the use of thistle extract in the preparation of products for preventing and / or alleviating skin problems caused by cortisol imbalance, which can improve skin problems caused by excessive cortisol conversion.

[0006] In a first aspect, this application provides a use of thistle extract, the use of which includes at least one of (1) to (2): (1) use in the preparation of a product for preventing skin problems caused by cortisol imbalance; (2) use in the preparation of a product for relieving skin problems caused by cortisol imbalance.

[0007] In any embodiment of this application, cortisol imbalance includes at least one of an increase in cortisol content in the skin and an increase in cortisol activity in the skin.

[0008] In any embodiment of this application, cortisol imbalance includes cortisol imbalance caused by stress.

[0009] In any embodiment of this application, skin problems include at least one of the following: skin barrier damage caused by long-term stress, skin aging, decreased skin moisturizing ability, decreased oil control ability, acne, skin inflammation, fatigued skin appearance, skin ulcers, skin petechiae, increased capillary fragility, and poor healing of skin wounds.

[0010] In any embodiment of this application, the thistle extract includes at least one of thistle glycerol extract, thistle water extract, thistle alcohol extract, and thistle ester extract.

[0011] In any embodiment of this application, the method for preparing the glycerol extract of thistle includes: mixing thistle powder with a glycerol extractant and performing extraction treatment, followed by post-treatment to obtain the glycerol extract of thistle.

[0012] In any embodiment of this application, in the step of mixing and extracting thistle powder with a glycerol extractant to obtain thistle glycerol extract, the mass concentration of the glycerol extractant is 35% to 65%.

[0013] In any embodiment of this application, the extraction temperature is maintained at 76°C to 83°C.

[0014] In any embodiment of this application, the extraction time is 1.8h to 2.8h.

[0015] In any embodiment of this application, the mass ratio of thistle powder to glycerol extractant is 1:(8~13).

[0016] In any embodiment of this application, the extractant for the thistle glycerol extract includes glycerol.

[0017] In any embodiment of this application, the product includes at least one of pharmaceuticals and cosmetics; and / or, the dosage form of the product includes at least one of creams, lotions, liquids, gels, sprays, aerosols, patches, and lyophilized agents.

[0018] In any embodiment of this application, the mass-volume concentration of thistle extract in the product is greater than or equal to 10 μg / mL.

[0019] In any embodiment of this application, the product further includes excipients, said excipients including at least one of fragrances, dyes, emulsifiers, stabilizers, lubricants, solvents, humectants, emollients, wetting agents, film-forming agents, UV absorbers, essential oils, vitamins, trace metals, anti-irritants, antimicrobial agents, antioxidants, chelating agents, preservatives, pH adjusters, skin conditioning agents, thickeners, and organosilicon compounds. This application provides the use of thistle extract, including its application in the preparation of products for preventing skin problems caused by cortisol imbalance; and its application in the preparation of products for alleviating skin problems caused by cortisol imbalance. The thistle extract of this application can improve skin problems caused by excessive cortisol conversion, increase the expression levels of dermal collagen, skin barrier-related proteins, and skin basement membrane-related proteins, thereby effectively alleviating problems such as skin barrier damage, skin sensitivity, wrinkles, and decreased skin elasticity caused by emotional stress. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments of this application will be briefly introduced below. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a schematic diagram showing the cytotoxicity results of different concentrations of thistle extract on human keratinocytes in Example 2 of this application.

[0022] Figure 2 This is a schematic diagram showing the effect of different concentrations of Cirsium japonicum extract on the inhibition of cortisol production in Example 3 of this application (wherein, "*" in the figure represents a significant difference between the sample treatment group and the corticosterone treatment group, ****P<0.0001, using one-way ANOVA analysis method).

[0023] Figure 3 This diagram illustrates the effects of different concentrations of *Cirsium japonicum* extract on the mRNA expression of keratin, filaggrin, and scutellarin in Example 4 of this application. (Figure A shows the effect of different concentrations of *Cirsium japonicum* extract on keratin 1 mRNA expression in Example 4; Figure B shows the effect of different concentrations of *Cirsium japonicum* extract on keratin 10 mRNA expression in Example 4; Figure C shows the effect of different concentrations of *Cirsium japonicum* extract on filaggrin FLG mRNA expression in Example 4; Figure D shows the effect of different concentrations of *Cirsium japonicum* extract on scutellarin LOR mRNA expression in Example 4; In the diagram, "#" indicates a significant difference between the corticosterone-treated group and the blank group, ##P<0.01, analyzed using Student's t-test; in the diagram, "*" indicates a significant difference between the sample treatment group (sample co-treated with corticosterone) and the corticosterone-treated group, *P<0.05, **P<0.01, ***P<0.001, analyzed using one-way ANOVA).

[0024] Figure 4This diagram illustrates the effects of different concentrations of *Cirsium japonicum* extract on the expression of skin basement membrane-related genes mRNA in Example 5 of this application. Figure A shows the effect of different concentrations of *Cirsium japonicum* extract on the expression of type IV collagen-IV mRNA in Example 5 of this application; Figure B shows the effect of different concentrations of *Cirsium japonicum* extract on the expression of type VII collagen-VII mRNA in Example 5 of this application; Figure C shows the effect of different concentrations of *Cirsium japonicum* extract on the expression of type XVII collagen-XVII mRNA in Example 5 of this application. In the diagram, "#" indicates a significant difference between the corticosterone-treated group and the control group (##P<0.01), analyzed using Student's t-test; "*" indicates a significant difference between the sample treatment group (sample co-treated with corticosterone) and the corticosterone-treated group (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001), analyzed using one-way ANOVA. (Analytical methods).

[0025] Figure 5 This application presents a schematic diagram illustrating the effects of different concentrations of *Cirsium japonicum* extract on the expression of skin collagen-related genes mRNA in Example 6 of this application (Figure A represents the effect of different concentrations of *Cirsium japonicum* extract on the expression of type I collagen-I mRNA in Example 6 of this application; Figure B represents the effect of different concentrations of *Cirsium japonicum* extract on the expression of type III collagen-III mRNA in Example 6 of this application; where, in the figure, "#" indicates a significant difference between the corticosterone-treated group and the blank group, ##P<0.01, analyzed using Student's t-test method; in the figure, "*" indicates a significant difference between the sample treatment group (sample co-treated with corticosterone) and the corticosterone-treated group, ***P<0.001, ****P<0.0001, analyzed using one-way ANOVA method). Detailed Implementation

[0026] To make the purpose, technical solution, and beneficial technical effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the implementation details described in this specification are merely for illustrative purposes and are not intended to limit the scope of this application.

[0027] For simplicity, this application only explicitly discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form a range not explicitly stated; and any lower limit can be combined with other lower limits to form a range not explicitly stated, just as any upper limit can be combined with any other upper limit to form a range not explicitly stated. Furthermore, although not explicitly stated, every point or individual value between the endpoints of the range is included within that range. Therefore, each point or individual value can be used as its own lower or upper limit and combined with any other point or individual value or with other lower or upper limits to form a range not explicitly stated.

[0028] It should be noted that the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0029] Unless otherwise stated, the values ​​of the parameters mentioned in this application can be measured using various measurement methods commonly used in the art (e.g., they can be tested according to the methods given in the embodiments of this application). Unless otherwise stated, the test temperature for all parameters mentioned in this application is 25°C and the test pressure is standard atmospheric pressure.

[0030] The foregoing description of this application is not intended to describe every disclosed implementation or method. Instead, the following description provides more specific examples of exemplary embodiments. Throughout the application, guidance is provided through a series of embodiments, which can be used in various combinations. The examples listed are representative only and should not be construed as exhaustive.

[0031] As a plant of the genus *Cirsium* in the family Asteraceae, *Cirsium japonicum* is rich in various flavonoids such as sauroprin and monaxioline, as well as active ingredients such as lignin, triterpenes, and sterols. It has a variety of pharmacological effects, including hemostasis, anti-inflammation, anti-tumor, antibacterial, and liver protection.

[0032] Cortisol, a stress hormone secreted by the adrenal glands, can weaken the immune system when excessively released under stress. Furthermore, in the skin, it can cause a range of skin problems, such as damaged skin barrier, dryness, sensitivity, oiliness, acne, aging, decreased elasticity, and wrinkles, by inhibiting barrier-related proteins and collagen, and accelerating sebum secretion. Currently, there are no reports on using thistle extract to treat skin problems caused by cortisol imbalance. Therefore, there is an urgent need to develop a use for thistle extract in the preparation of products for preventing and / or alleviating skin problems caused by cortisol imbalance, in order to overcome the shortcomings of existing technologies.

[0033] The first aspect of the embodiments of this application provides a use of thistle extract, the use including at least one of (1) to (2): (1) use in the preparation of a product for preventing skin problems caused by cortisol imbalance; (2) use in the preparation of a product for relieving skin problems caused by cortisol imbalance.

[0034] The thistle extract of this application can improve skin problems caused by excessive cortisol conversion, increase the expression of skin barrier-related proteins and skin basement membrane-related proteins, and promote collagen regeneration in the dermis, thereby effectively alleviating skin barrier damage, skin sensitivity, wrinkles and decreased skin elasticity caused by emotional stress.

[0035] In some embodiments, cortisol imbalance includes at least one of an increase in cortisol content in the skin and an increase in cortisol activity in the skin.

[0036] Preferably, cortisol imbalance includes at least one of increased activity of 11β-HSD1 in the skin and increased content of 11β-HSD1 in the skin.

[0037] In some implementations, cortisol imbalance includes cortisol imbalance caused by stress.

[0038] As an example, stress can be at least one of psychological, emotional, mental, and physical stress.

[0039] In some implementations, prevention and / or relief of skin problems caused by cortisol imbalance includes at least one of (1) to (9): (1) upregulating the expression level of skin barrier-related gene mRNA; (2) increasing the content of skin barrier-related protein; (3) enhancing the activity of skin barrier-related protein; (4) upregulating the expression level of skin basement membrane-related gene mRNA; (5) increasing the content of skin basement membrane-related protein; (6) enhancing the activity of skin basement membrane-related protein; (7) upregulating the expression level of dermal collagen-related gene mRNA; (8) increasing the content of dermal collagen; (9) enhancing the activity of dermal collagen.

[0040] Thistle extract can inhibit cortisol production and promote the expression of keratin or filaggrin in the skin, thereby effectively relieving skin barrier damage and skin sensitivity caused by emotional stress. At the same time, thistle extract can also improve the effect of stress on the skin basement membrane, help maintain the content and structure of collagen and elastin fibers in the skin basement membrane, enhance the skin's support and elasticity, and thus alleviate problems such as skin sagging and deepening wrinkles caused by long-term stress.

[0041] Optionally, prevention and / or relief of skin problems caused by cortisol imbalance may also include upregulating the expression of dermal extracellular matrix collagen-related gene mRNA, increasing the content of dermal extracellular matrix collagen, and enhancing the activity of dermal extracellular matrix collagen.

[0042] As an example, dermal extracellular matrix collagen includes at least one of collagen I and collagen III.

[0043] In some implementations, skin problems include at least one of the following: chronic stress-induced skin barrier damage, skin aging, skin sensitivity, pigmentation, decreased skin hydration and oil control, acne, skin inflammation, fatigued skin appearance, skin ulcers, petechiae, increased capillary fragility, and poor wound healing.

[0044] In some embodiments, the skin barrier-related gene mRNA includes at least one of keratin 1 mRNA, keratin 10 mRNA, filaggrin mRNA, and nautiline mRNA; and / or, the skin barrier-related protein includes at least one of keratin 1, keratin 10, filaggrin, and nautiline.

[0045] Thistle extract can improve the downregulation of the expression of the above-mentioned skin barrier-related genes caused by cortisol imbalance under stress, and has a significant repair effect on corticosterone-induced skin barrier damage, thereby effectively alleviating skin barrier dysfunction, skin sensitivity, and pigmentation caused by abnormal cortisol levels.

[0046] In some embodiments, the skin basement membrane-associated gene mRNA includes at least one of type IV collagen mRNA, type VII collagen mRNA, and type XVII collagen mRNA; and / or, the skin basement membrane-associated protein includes at least one of type IV collagen, type VII collagen, and type XVII collagen.

[0047] Thistle extract can improve the downregulation of the expression of the above-mentioned skin basement membrane-related genes caused by cortisol imbalance under stress, and has a significant repair effect on cortisol-induced skin basement membrane damage, thereby effectively alleviating skin basement membrane damage, skin sensitivity, and pigmentation caused by abnormal cortisol levels.

[0048] In some embodiments, the dermal collagen-related gene mRNA includes at least one of type I collagen mRNA and type III collagen mRNA; and / or, dermal collagen includes at least one of type I collagen and type III collagen.

[0049] Thistle extract can improve the downregulation of the expression of the above-mentioned skin collagen-related genes caused by cortisol imbalance under stress, and has a significant repair effect on cortisol-induced skin collagen loss, thereby effectively alleviating skin collagen loss caused by abnormal cortisol levels, and thus improving skin aging and wrinkles.

[0050] In some embodiments, the thistle extract includes at least one of thistle glycerol extract, thistle water extract, thistle alcohol extract, and thistle ester extract.

[0051] Optionally, the thistle extract can be obtained by at least one of solvent extraction, supercritical extraction or ultrasonic extraction. In some embodiments, the preparation method of thistle glycerol extract includes: mixing thistle powder with a glycerol extractant and performing extraction treatment, followed by post-treatment to obtain thistle glycerol extract.

[0052] In some embodiments, in the step of mixing and extracting thistle powder with a glycerol extractant to obtain thistle glycerol extract, the mass concentration of the glycerol extractant is 35% to 65%.

[0053] As an example, the mass concentration of glycerol extract can be 35%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, or 65%.

[0054] In some implementations, the extraction temperature is maintained at 76°C to 83°C.

[0055] As an example, the extraction temperature can be maintained at 76℃, 77℃, 78℃, 79℃, 80℃, 81℃, 82℃, or 83℃.

[0056] In some implementations, the extraction time is 1.8h to 2.8h.

[0057] As an example, the extraction time can be 1.8h, 1.9h, 2.0h, 2.1h, 2.2h, 2.3h, 2.4h, 2.5h, 2.6h, 2.7h, or 2.8h.

[0058] In some embodiments, the mass ratio of thistle powder to glycerol extract is 1:(8~13).

[0059] As an example, the mass ratio of thistle powder to glycerol extract can be 1:8, 1:8.5, 1:9, 1:9.5, 1:10, 1:10.5, 1:11, 1:11.5, 1:12, 1:12.5 or 1:13.

[0060] In some embodiments, the extractant for thistle glycerol extracts includes glycerol.

[0061] Optionally, the thistle extract can be obtained from one or more parts of the thistle flower, leaf, fruit or stem, which means that active extracts can be obtained from different parts of the thistle, which may contain different concentrations and types of active ingredients.

[0062] In some implementations, the product includes at least one of pharmaceuticals and cosmetics.

[0063] In some embodiments, the dosage form of the product includes at least one of creams, lotions, liquids, gels, sprays, aerosols, films, and lyophilized agents.

[0064] As an example, products can be formulated into various forms such as moisturizing toner (for gently hydrating the skin), nourishing toner (to replenish the skin's nutrients), nourishing cream (for deep nourishment), massage cream (for massage to promote absorption), serum (with high concentration of active ingredients), eye cream (for eye care), cleansing cream (for gentle cleansing), cleansing foam (for rich foam cleansing), cleansing water (for refreshing cleansing), face mask (for intensive care), spray (for convenient moisturizing), or powder (for easy carrying and use).

[0065] This diverse range of dosage forms allows the cosmetic compositions of the present invention to be better suited to different skin types and usage scenarios. For example, oily skin may prefer a refreshing lotion or spray, while dry skin may benefit from a nourishing cream or massage cream. Furthermore, the different dosage forms facilitate product packaging and use; for instance, powders are convenient for travel, while sprays can be used for large-area skin moisturizing.

[0066] In some embodiments, the mass-volume concentration of thistle extract in the product is greater than or equal to 10 μg / mL.

[0067] As an example, the mass-volume concentration of thistle extract in the product can be 10 μg / mL, 11 μg / mL, 12 μg / mL, 13 μg / mL, 14 μg / mL, 15 μg / mL, 16 μg / mL, 17 μg / mL, 18 μg / mL, 19 μg / mL, 20 μg / mL, 22 μg / mL, 24 μg / mL, 26 μg / mL, 28 μg / mL, 30 μg / mL, 32 μg / mL, 34 μg / mL, 36 μg / mL, 38 μg / mL, 40 μg / mL, 42 μg / mL, 44 μg / mL, 46 μg / mL. 48μg / mL, 50μg / mL, 52μg / mL, 54μg / mL, 56μg / mL, 58μg / mL, 60μg / mL, 62μg / mL, 64μg / mL, 66μg / mL, 68μg / mL, 70μg / mL, 72μg / mL, 74μg / mL, 76μg / mL, 78μg / mL, 80μg / mL, 82μg / mL, 84μg / mL, 86μg / mL, 88μg / mL, 90μg / mL, 92μg / mL, 94μg / mL, 96μg / mL, 98μg / mL or 100μg / mL.

[0068] In some embodiments, the product further includes excipients, such as fragrances, dyes, emulsifiers, stabilizers, lubricants, solvents, humectants, emollients, wetting agents, film-forming agents, UV absorbers, essential oils, vitamins, trace metals, anti-irritants, antimicrobial agents, antioxidants, chelating agents, preservatives, pH adjusters, skin conditioning agents, thickeners, and organosilicon compounds. These ingredients can work in conjunction with thistle extract to provide multiple functions, including improving skin condition, while ensuring product stability and user experience.

[0069] In some embodiments, the mass content of thistle extract in the product is 0.01% to 10%.

[0070] As an example, the mass content of thistle extract in the product can be 0.01%, 0.02%, 0.04%, 0.06%, 0.08%, 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.5%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, or 10%.

[0071] In this application, keratin 1 refers to Keratin 1.

[0072] In this application, keratin 10 refers to Keratin 10.

[0073] Keratin 1 and keratin 10 are the main intermediate filament proteins in keratinocytes. They form a complex network of cellular fibers in the cytoplasm, providing mechanical support for keratinocytes, maintaining cell morphology and structural integrity, and enabling the stratum corneum to withstand external mechanical pressure and friction, preventing skin damage. During keratinocyte differentiation, the expression levels of keratin 1 and keratin 10 change. Through interactions with other proteins, they regulate cell differentiation, promote the formation and maturation of the stratum corneum, and thus enhance the skin's barrier function.

[0074] In this application, filaggrin refers to filaggrin.

[0075] Filagrin (FLG) is an important molecule in the stratum corneum of human skin that connects keratin fibers. With the assistance of FLG monomers, keratin and fibers aggregate regularly, forming a solid physical barrier on the outermost layer of the epidermis. This barrier prevents moisture loss and the invasion of external allergens, further strengthening the skin's physical barrier function. Simultaneously, FLG can degrade into small amino acid molecules in the stratum corneum, possessing moisturizing properties and known as a "natural moisturizing factor," playing a crucial role in hydration and barrier integrity.

[0076] In this application, loricrin is referred to as loricrin.

[0077] Loricrin is a major component of the keratinized capsule. It cross-links with other proteins such as inner lining proteins and plaque proteins under the action of transglutaminase, forming a highly cross-linked, insoluble protein network. This network strengthens the structure of the keratinized capsule, making it more resilient and stable, thereby enhancing the mechanical strength and pressure resistance of keratinocytes. Furthermore, loricrin can interact with proteins such as inner lining proteins to tightly connect keratin fibers with other cellular structures of keratinocytes, forming a unified cytoskeletal network. This makes the stratum corneum structure more compact and orderly, improving the integrity of the skin barrier.

[0078] In this application, Type I collagen refers to Collagen Type I, abbreviated as I-Col. Type I collagen is the most abundant type of collagen in the human body, accounting for approximately 90% of the total collagen. It is mainly found in connective tissues such as skin, bones, tendons, and ligaments, providing structural support and strength for these tissues. In the skin, Type I collagen helps maintain skin thickness and elasticity, delays the appearance of wrinkles, and improves problems such as skin laxity and sagging. In the skeletal system, it enhances bone strength and stability, and promotes bone regeneration and repair.

[0079] In this application, type III collagen refers to Collagen Type III, abbreviated as III-Col. Type III collagen is mainly found in tissues such as infant skin, vascular endothelium, cartilage, and vitreous humor of the eye, and is also known as "infant collagen." In the skin, it is located between the epidermis and dermis, playing a supporting role for the epidermis. The main function of type III collagen is to maintain the elasticity and stability of tissues, especially in tissues such as cartilage, blood vessel walls, and the eyeball. During skin repair, type III collagen is a key component in the early stages of wound healing. It forms a network structure at the site of new wound formation, maintaining the rigidity and stability of newly formed tissue and promoting skin wound repair.

[0080] In this application, type IV collagen refers to Collagen Type IV, abbreviated as IV-Col. It is a major component of the basement membrane reticular structure and, after being synthesized intracellularly, directly participates in the formation of the extracellular matrix as procollagen. Under normal circumstances, the expression and renewal of type IV collagen genes help maintain the stability and function of the basement membrane. The effect of cortisol may interfere with this renewal process, preventing the basement membrane from repairing and rebuilding in a timely manner, further exacerbating the weakening of the basement membrane and reducing its supporting and protective function for cells.

[0081] In this application, type VII collagen refers to Collagen Type VII, abbreviated as VII-Col. It is primarily produced by basal keratinocytes and dermal fibroblasts, forming anchoring fibrils at the dermal-epidermal junction, firmly attaching the epidermal basement membrane to the extracellular matrix of the dermis. The stability of these two layers is crucial for firm and elastic skin. When type VII collagen gene expression decreases, the quantity and quality of anchoring fibers are affected, leading to a loosening of the connection between the epidermis and dermis, reducing the stability of the basement membrane, and making skin lesions such as blisters more likely to occur.

[0082] In this application, type XVII collagen refers to Collagen Type XVII, abbreviated as XVII-Col. It is a core component of the hemidesmosomes of the basement membrane, primarily located in hair follicle stem cells in the bulb of the hair follicle and epidermal stem cells at the junction of the epidermis and dermis. Its function is to ensure a tight connection between the epidermis and dermis through the basement membrane band, promoting epidermal turnover and stabilizing the basement membrane. Proteins encoded by type XVII collagen play a crucial role in maintaining the proliferation and differentiation of epidermal stem cells and the stability of the basement membrane. Reduced expression of type XVII collagen genes leads to impaired function of epidermal stem cells, affecting normal epidermal renewal and repair, and consequently causing the basement membrane to lose normal cellular support, becoming fragile and unstable.

[0083] Example The following embodiments describe the disclosure of this application in more detail. These embodiments are for illustrative purposes only, as various modifications and variations will be apparent to those skilled in the art within the scope of the disclosure of this application. Unless otherwise stated, all parts, percentages, and ratios reported in the following embodiments are based on mass, and all reagents used in the embodiments are commercially available or synthesized by conventional methods and can be used directly without further processing, and the instruments used in the embodiments are commercially available.

[0084] Example 1: Preparation of thistle extract First, fresh thistles were air-dried (naturally dried in a cool place) and then pulverized to obtain thistle powder. The thistle powder was mixed with a 60% glycerol aqueous solution and extracted. The extraction temperature was maintained at 80℃ for 2.5 hours. The mass ratio of thistle powder to extractant (60% glycerol aqueous solution) was 1:12 (in g / g). After extraction, the mixture was filtered, and the filtrate was collected to obtain the thistle glycerol extract.

[0085] Example 2 Cytotoxicity of thistle extract 1. Experimental Materials and Methods Human immortalized keratinocytes (Wuhan Pronosai Life Science Technology Co., Ltd., Product No.: CCL-0090, Specification: 1×10) were used. 6 The experiments were conducted using Cells / Vials. These cells were stored at 2 × 10⁶ cells per well. 4 Cells were seeded at a density of 1000 cells per well in 96-well plates. They were then incubated at 37°C with 5% CO2 for one day to allow the cells to stabilize.

[0086] A solvent control group (CTR) and a sample group were set up. For the sample group, the *Cirsium japonicum* extract prepared in Example 1 was diluted with culture medium to concentrations of 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL. These different concentrations of *Cirsium japonicum* extract were then added to wells containing keratinocytes and incubated with the cells for 24 hours. For the CTR group, an equal volume of complete culture medium as the sample group was added. After incubation, 10 μL of CCK-8 solution was added to each well, and the culture plate was incubated in an incubator for 1–4 hours. The absorbance at 450 nm was measured using a microplate reader.

[0087] 2. Calculation of cell viability and experimental results Cell viability = [(As-Ab) / (Ac-Ab)] × 100% As: Absorbance of experimental wells (containing cells, culture medium, CCK-8 solution, and thistle extract); Ac: Absorbance of control wells (containing cells, culture medium, and CCK-8 solution, but excluding thistle extract); Ab: Absorbance of blank wells (containing culture medium and CCK-8 solution, but excluding cells and thistle extract).

[0088] The experimental results will be listed in Figure 1 This figure visually illustrates the effect of different concentrations of thistle extract on the survival rate of keratinocytes, thus allowing us to determine whether thistle extract is cytotoxic and the degree of toxicity.

[0089] The experimental results are shown in Figure 1. The thistle extract of the present invention did not exhibit cytotoxicity at concentrations up to 100 μg / mL.

[0090] Example 3: Thistle extract inhibits cortisol production. 1. Experimental Materials and Methods 1.1 Cell Culture Human immortalized keratinocytes - HaCaT cells (Wuhan Pronosei Life Science Technology Co., Ltd., product number: CCL-0090, specification: 1×10⁻⁶) were used. 6 The experiments were conducted using Cells / Vials. These cells were stored at 5 × 10⁶ cells per well. 4 Cells were seeded at a density of 1,000 μL of culture medium into each well of a 24-well plate. The plates were then incubated at 37°C with 5% CO2 for one day to allow the cells to stabilize. These culture conditions mimic the growth environment of human skin cells, ensuring normal cell growth and physiological function.

[0091] 1.2 Stress stimuli and treatment After cell stabilization, 3 μM (µmol / L) of cortisone (a corticosteroid that can act as a stress stimulant) was added to the culture medium. Following cortisone treatment, the thistle extract prepared in Example 1 was diluted to concentrations of 2 μg / mL, 10 μg / mL, and 50 μg / mL, and added to the cortisone-treated cells, respectively. The cells were then allowed to react with the thistle extract for 24 hours. By setting up different concentrations of thistle extract treatment groups, its concentration-dependent effect can be studied, i.e., whether there are differences in the inhibitory effect on cortisol production at different concentrations.

[0092] 1.3 Cell treatment and cortisol content determination After the reaction was complete, the culture medium in the supernatant was collected and centrifuged at 12,000 rpm at 4°C for 10 minutes.

[0093] Finally, the intracellular cortisol level was determined using a human cortisol ELISA kit (manufacturer: Shanghai Yanqi Biotechnology Co., Ltd., catalog number YQ-58261K, specification 96T). ELISA is a commonly used biological detection technique with high sensitivity and specificity, capable of accurately measuring cortisol concentration.

[0094] 2. Experimental Results The experimental results are calculated as a percentage of cortisol production inhibition rate and listed in Table 1. The formula for calculating the cortisol production inhibition rate is: Cortisol production inhibition rate = [(Cortisol content in control group - Cortisol content in treatment group) / Cortisol content in control group] × 100%.

[0095] Table 1 clearly shows the inhibitory effect of different concentrations of thistle extract on cortisol production. If thistle extract can significantly reduce cortisol production, it demonstrates its potential to improve skin problems caused by cortisol imbalance under stress.

[0096] Table 1

[0097] As shown in Table 1 and Figure 2 As shown, the *Cirsium japonicum* extract of the present invention inhibits cortisol production in a concentration-dependent manner. At a low concentration of 2 μg / mL, the extract achieves an inhibition rate of 18% on cortisol production; when the concentration is increased to 50 μg / mL, the inhibitory effect is further enhanced, with a maximum inhibition rate of 38%.

[0098] Example 4: Thistle extract can improve the downregulation of skin barrier-related genes (Keratin1, Keratin10, Filaggrin, and Loricrin) caused by cortisol imbalance under stress. 1. Experimental materials: Please see Table 2 for specific experimental materials.

[0099] Table 2 Reagent Information

[0100] 2. Experimental Methods: 1) Cell seeding: Human immortalized keratinocytes-HaCaT cells (Wuhan Pronosei Life Science Technology Co., Ltd., catalog number: CCL-0090, specification: 1×10⁻⁶) 6 Cells / Vials) according to 2×10 5 Incubate overnight in a 5% CO2 incubator per cell / well; 2) Experimental groups: The solvent control group (CTR), corticosterone induction group, and sample group were set up separately, with 3 replicates. The experimental groups are shown in Table 3.

[0101] Table 3 Experimental Groups

[0102] 3) Sample feeding: When the cell seeding rate of the 6-well plate reaches 60-70%, discard the old culture medium. Add fresh culture medium to the solvent control group (CTR), add fresh culture medium containing 3 μM corticosterone to the corticosterone-induced group, and add liquid culture medium containing 3 μM corticosterone and the *Cirsium japonicum* extract prepared in Example 1 to the sample group. The final concentrations of the extract in the sample group are 2 μg / mL, 10 μg / mL, and 50 μg / mL, respectively. After sample feeding, place the 6-well plate in an incubator (37℃, 5% CO2) and incubate for 24 hours. 4) After the incubation period, discard the supernatant; wash twice with pre-cooled sterile PBS.

[0103] 5) RNA extraction (follow the RNA extraction kit procedure below) 6) Add 300 μL of lysis buffer to each well of the 6-well plate and blow it 5-10 times until the solid suspension dissolves and the solution becomes clear.

[0104] 7) Add an equal volume of binding solution to the lysis solution and mix by inverting 3-5 times.

[0105] 8) Transfer the mixture to a purification column, centrifuge at 12000 rcf for 30 s, and discard the liquid in the collection tube.

[0106] 9) Add 600 μL of washing solution I, centrifuge at 12000 rcf for 30 s, and discard the liquid in the collection tube.

[0107] 10) Add 600 μL of washing buffer II, centrifuge at 12000 rcf for 30 s, discard the liquid in the collection tube, and repeat this step.

[0108] 11) Centrifuge at the highest speed for 2 minutes to remove residual liquid.

[0109] 12) Place the RNA purification column in the RNA elution tube provided in the kit, add 30 μL of elution buffer, incubate at room temperature for 2-3 min, centrifuge at the highest speed for 30 s, and the resulting solution is the purified RNA.

[0110] 13) Add an appropriate amount of DEPC water to dissolve the mRNA, determine the concentration, and perform reverse transcription according to the instructions of the reverse transcription kit.

[0111] 14) Reverse transcription PCR System (20 μl): 5× PrimeScript Buffer (for Real Time, containing dNTP Mixture and Mg) 2+ 4µl, 2PrimeScript RT Enzyme Mix I 1µl, Random 6 mers 2µl, template (extracted mRNA as template) 2-10µl, ultrapure water to make up to 20µl.

[0112] Reaction conditions: Pre-denaturation 94℃, 5 min; 30 cycles: denaturation 94℃, 30 s, annealing 60℃, 30 s, extension 72℃, 30 s; post-extension 72℃, 10 min.

[0113] 3. Experimental Results like Figure 3 As shown, after treating HaCaT cells with 3 μM corticosterone for 24 hours, the mRNA expression levels of Keratin1, Keratin10, Filaggrin, and Loricrin were significantly downregulated; however, after co-incubation with the thistle extract prepared in Example 1, the expression of these barrier-related genes was significantly restored. These results indicate that the thistle extract has a repairing effect on corticosterone-induced skin barrier damage, and thus can improve skin barrier function damage caused by cortisol imbalance under stress.

[0114] Furthermore, as shown in Table 4, based on the analysis results of protein expression recovery rate, the thistle extract prepared in Example 1 increased the expression level of barrier-related proteins in a concentration-dependent manner, further confirming its significant potential to improve skin barrier function damage caused by cortisol imbalance under stress.

[0115] The formula for calculating the protein expression recovery rate is: Protein expression recovery rate = [(sample histone expression level - corticosterone-induced histone expression level) / corticosterone-induced histone expression level] × 100%.

[0116] Table 4

[0117] Example 5: Thistle extract can improve the downregulation of expression of genes related to skin basement membrane weakening (Collagen-Ⅳ, Collagen-Ⅶ and Collagen-ⅩⅦ) caused by cortisol imbalance under stress. 1. The experimental materials and methods are the same as those described in Example 4.

[0118] 2. Experimental Results like Figure 4As shown, after 24 hours of treatment with 3 μM corticosterone, the mRNA expression of Collagen-IV, Collagen-VII, and Collagen-XVII in HaCaT cells was significantly reduced; however, after co-treatment with the thistle extract prepared in Example 1, the transcription levels of these genes related to basement membrane structure showed a significant rebound, indicating that the extract has a repairing effect on corticosterone-induced skin basement membrane damage and can thus improve skin basement membrane damage caused by cortisol imbalance under stress.

[0119] The protein expression recovery rate results listed in Table 5 further show that this thistle extract can increase the expression of basement membrane-related proteins in a concentration-dependent manner, confirming its ability to improve basement membrane damage caused by cortisol imbalance under stress at the protein level. The formula for calculating the protein expression recovery rate is: Protein expression recovery rate = [(Sample histone expression level - Corticosterone-induced histone expression level) / Corticosterone-induced histone expression level] × 100%.

[0120] Table 5

[0121] Example 6: Thistle extract can improve the downregulation of skin collagen-related genes (Collagen Type I and Collagen Type III) caused by cortisol imbalance under stress. 1. Experimental Materials Please see Table 6 for specific experimental materials.

[0122] Table 6 Reagent Information

[0123] 2. Experimental Methods: 1) Cell seeding: Human dermal fibroblasts (ATCC, catalog number: PCS-201-012, specification: ≥ 5.0 x 10⁻⁶) 5 Cells / Vials) according to 2×10 5 Incubate overnight in a 5% CO2 incubator per cell / well; 2) Experimental groups: The solvent control group (CTR), corticosterone induction group, and sample group were set up separately, with 3 replicates. The experimental groups are shown in Table 7.

[0124] Table 7 Experimental Groups

[0125] 3) Sample feeding: When the cell seeding rate of the 6-well plate reaches 60-70%, discard the old culture medium. Add fresh culture medium to the solvent control group (CTR), add fresh culture medium containing 3 μM corticosterone to the corticosterone-induced group, and add liquid culture medium containing 3 μM corticosterone and the *Cirsium japonicum* extract prepared in Example 1 to the sample group. The final concentrations of the extract in the sample group are 2 μg / mL, 10 μg / mL, and 50 μg / mL, respectively. After sample feeding, place the 6-well plate in an incubator (37℃, 5% CO2) and incubate for 24 hours. 4) After the incubation period, discard the supernatant; wash twice with pre-cooled sterile PBS.

[0126] 5) RNA extraction (follow the RNA extraction kit procedure below) 6) Add 300 μL of lysis buffer to each well of the 6-well plate and blow it 5-10 times until the solid suspension dissolves and the solution becomes clear.

[0127] 7) Add an equal volume of binding solution to the lysis solution and mix by inverting 3-5 times.

[0128] 8) Transfer the mixture to a purification column, centrifuge at 12000 rcf for 30 s, and discard the liquid in the collection tube.

[0129] 9) Add 600 μL of washing solution I, centrifuge at 12000 rcf for 30 s, and discard the liquid in the collection tube.

[0130] 10) Add 600 μL of washing buffer II, centrifuge at 12000 rcf for 30 s, discard the liquid in the collection tube, and repeat this step.

[0131] 11) Centrifuge at the highest speed for 2 minutes to remove residual liquid.

[0132] 12) Place the RNA purification column in the RNA elution tube provided in the kit, add 30 μL of elution buffer, incubate at room temperature for 2-3 min, centrifuge at the highest speed for 30 s, and the resulting solution is the purified RNA.

[0133] 13) Add an appropriate amount of DEPC water to dissolve the mRNA, determine the concentration, and perform reverse transcription according to the instructions of the reverse transcription kit.

[0134] 14) Reverse transcription PCR System (20 μl): 4 µl of 5× PrimeScript Buffer (for Real Time, containing dNTP Mixture and Mg2+), 1 µl of 2 PrimeScript RT Enzyme Mix I, 2 µl of Random 6 mers, 2-10 µl of template (extracted mRNA as template), and ultrapure water to bring the system to 20 µl.

[0135] Reaction conditions: Pre-denaturation 94℃, 5 min; 30 cycles: denaturation 94℃, 30 s, annealing 60℃, 30 s, extension 72℃, 30 s; post-extension 72℃, 10 min.

[0136] 3. Experimental Results like Figure 5 As shown, after treating HDF-α cells with 3 μM corticosterone for 24 hours, the mRNA expression levels of Collagen Type I and Collagen Type III were significantly downregulated; however, after co-incubation with the thistle extract prepared in Example 1, the expression of these collagen-related genes was significantly restored. This result indicates that the thistle extract has a repairing effect on corticosterone-induced collagen loss in the skin, and thus can improve collagen loss caused by cortisol imbalance under stress, i.e., stress-induced skin aging and wrinkles.

[0137] Furthermore, as shown in Table 8, based on the analysis results of protein expression recovery rate, the *Cirsium japonicum* extract prepared in Example 1 increased the expression level of collagen-related protein mRNA in a concentration-dependent manner, further confirming its significant potential to improve skin collagen loss caused by cortisol imbalance under stress.

[0138] The formula for calculating the protein expression recovery rate is: Protein expression recovery rate = [(sample histone expression level - corticosterone-induced histone expression level) / corticosterone-induced histone expression level] × 100%.

[0139] Table 8

[0140] Furthermore, the applicant treated cells not induced by corticosterone-simulated stress (e.g., HaCaT cells) with the *Cirsium japonicum* extract prepared in Example 1 (final extract concentration 50 μg / mL). The results showed that the *Cirsium japonicum* extract prepared in Example 1 had no effect on increasing the expression of skin barrier-related genes (Keratin1, Keratin10, Filaggrin, and Loricrin), skin basement membrane weakening-related genes (Collagen-Ⅳ, Collagen-Ⅶ, and Collagen-ⅩⅦ), and skin collagen-related genes (Collagen Type Ⅰ and Collagen Type Ⅲ) in cells not induced by corticosterone (e.g., HaCaT cells), or the effect was insignificant. This further clarifies that the *Cirsium japonicum* extract has a selective and significant effect on skin barrier function damage, skin basement membrane weakening, and skin collagen loss caused by cortisol imbalance under stress. The *Cirsium japonicum* extract can selectively and effectively alleviate skin barrier damage, skin sensitivity, wrinkle formation, and decreased skin elasticity caused by emotional stress.

[0141] The above description is merely a specific implementation of this application. Those skilled in the art will clearly understand that, for the sake of convenience and brevity, the specific working process described above can be referred to the corresponding process in the foregoing method embodiments, and will not be repeated here. It should be understood that the protection scope of this application is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in this application, and these modifications or substitutions should all be covered within the protection scope of this application.

Claims

1. The use of a thistle extract, characterized in that, The use includes at least one of (1) to (2): (1) Use in the preparation of products for the prevention of skin problems caused by cortisol imbalance; (2) Use in the preparation of products that alleviate skin problems caused by cortisol imbalance.

2. The use according to claim 1, characterized in that, The cortisol imbalance includes at least one of an increase in cortisol content in the skin and an increase in cortisol activity in the skin.

3. The use according to claim 1, characterized in that, The cortisol imbalance includes cortisol imbalance caused by stress.

4. The use according to claim 1, characterized in that, The skin problems mentioned include at least one of the following: skin barrier damage caused by long-term stress, skin aging, decreased skin moisturizing ability, decreased oil control ability, acne, skin inflammation, fatigued skin appearance, skin ulcers, skin petechiae, increased capillary fragility, and poor skin wound healing.

5. The use according to claim 1, characterized in that, The thistle extract includes at least one of thistle glycerol extract, thistle water extract, thistle alcohol extract, and thistle ester extract.

6. The use according to claim 5, characterized in that, The preparation method of the thistle glycerol extract includes: Thistle powder was mixed with a glycerol extractant and extracted, and then post-processed to obtain thistle glycerol extract.

7. The use according to claim 6, characterized in that, In the step of mixing and extracting *Cirsium japonicum* powder with a glycerol extractant to obtain *Cirsium japonicum* glycerol extract, the mass concentration of the glycerol extractant is 35%~65%; and / or, The extraction temperature is maintained at 76℃~83℃; and / or, The extraction time is 1.8h to 2.8h; and / or, The mass ratio of the thistle powder to the glycerol extractant is 1:(8~13); and / or, The extractant for the thistle glycerol extract includes glycerol.

8. The use according to claim 1, characterized in that, The product includes at least one of pharmaceuticals and cosmetics; and / or, The dosage form of the product includes at least one of creams, lotions, liquids, gels, sprays, aerosols, films, and lyophilized agents.

9. The use according to claim 8, characterized in that, The mass-volume concentration of the thistle extract in the product is greater than or equal to 10 μg / mL.

10. The use according to claim 8, characterized in that, The product also includes excipients, which include at least one of the following: fragrances, dyes, emulsifiers, stabilizers, lubricants, solvents, humectants, softeners, wetting agents, film-forming agents, UV absorbers, essential oils, vitamins, trace metals, anti-irritants, antimicrobial agents, antioxidants, chelating agents, preservatives, pH adjusters, skin conditioning agents, thickeners, and organosilicon compounds.

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