Plant extract compositions, their preparation methods and applications

Abstract

This application discloses a plant extract composition, its preparation method, and its application. The plant extract composition is prepared by mixing trumpet creeper alcohol extract, thistle glycerol extract, and cardamom alcohol extract in a mass ratio of 100:(28~305):(28~305). The combination of these three extracts in the composition exhibits a synergistic effect, effectively protecting the skin from UV damage while simultaneously soothing and repairing it, achieving both core benefits and overcoming the limitations of traditional single-herb extraction methods that cannot fundamentally address skin allergies and fragility. Furthermore, the use of alcohol and glycerol extraction methods, compared to the irritating organic solvents used in traditional processes, is more suitable for people with sensitive skin, broadening the product's applicability.

Description

Technical Field

[0001] This application belongs to the field of cosmetic technology, and in particular relates to a plant extract composition, its preparation method and application. Background Technology

[0002] Trumpet creeper flowers are the dried flowers of the American trumpet creeper, a plant in the Bignoniaceae family. According to the pharmacopoeia, they can be used for rashes, itchy skin, acne, and other ailments. There are also records of its use in treating skin diseases in medical works throughout history. Thistle is the dried aerial part of the thistle plant in the Asteraceae family. The pharmacopoeia clearly states that it can be used for external bleeding, carbuncles, and boils. Red cardamom is the dried mature fruit of the Alpinia galanga plant in the Zingiberaceae family. Studies have confirmed that it has antibacterial and anti-inflammatory pharmacological activities.

[0003] However, current traditional extraction processes for these natural plants have significant technical limitations. The extraction and purification processes mostly use organic solvents such as water, ethanol, methanol, and ethyl acetate, which are highly irritating to the skin, especially exacerbating skin problems in people with sensitive skin. Furthermore, the single mechanism of action of individual herbs is insufficient to comprehensively address complex issues such as skin allergies and fragility, resulting in limited efficacy. Currently, there are no reports of using a combination of extracts from trumpet vine (Campsis grandiflora), thistle (Cirsium japonicum), and cardamom (Amomum villosum) to achieve protection against UV damage and immediate soothing and repair of the skin. Therefore, there is an urgent need to develop a novel compound extract to overcome the shortcomings of existing technologies. Summary of the Invention

[0004] This application provides a plant extract composition, its preparation method, and its application, which can achieve the effects of protecting the skin from ultraviolet light damage and providing immediate soothing and repair to the skin.

[0005] In a first aspect, this application provides a plant extract composition comprising: 100 parts of trumpet creeper alcohol extract; 28 to 305 parts of thistle glycerin extract; and 28 to 305 parts of cardamom alcohol extract; wherein the extractant of the trumpet creeper alcohol extract comprises at least one of 1,3-propanediol, 1,2-propanediol, methylpropanediol, and dipropylene glycol; the extractant of the thistle glycerin extract comprises glycerin; and the extractant of the cardamom alcohol extract comprises at least one of 1,3-propanediol, 1,2-propanediol, methylpropanediol, and dipropylene glycol.

[0006] In any embodiment of this application, the extractant concentration of the trumpet creeper alcohol extract is 20% to 70%; and / or, the extractant concentration of the thistle glycerol extract is 20% to 70%; and / or, the extractant concentration of the cardamom alcohol extract is 70% to 100%.

[0007] In any embodiment of this application, based on the total mass of the plant extract composition, the concentration of verbascoside in the plant extract composition is 0.2 mg / mL to 0.7 mg / mL, and / or; the concentration of isoverascoside in the plant extract composition is 0.02 mg / mL to 0.05 mg / mL, and / or; the concentration of strychnine in the plant extract composition is 0.03 mg / mL to 0.06 mg / mL, and / or; the total flavonoid content in the plant extract composition is 1.3 mg / mL to 1.7 mg / mL, and / or; and the total polyphenol content in the plant extract composition is 0.3 mg / mL to 1.2 mg / mL.

[0008] Secondly, this application provides a method for preparing a plant extract composition, comprising: mixing trumpet creeper powder with an alcohol extractant and extracting the mixture, followed by post-treatment to obtain a trumpet creeper alcohol extract; mixing thistle powder with a glycerol extractant and extracting the mixture, followed by post-treatment to obtain a thistle glycerol extract; mixing cardamom powder with an alcohol extractant and extracting the mixture, followed by post-treatment to obtain cardamom alcohol extract; wherein the alcohol extractant includes at least one selected from 1,3-propanediol, 1,2-propanediol, methylpropanediol, and dipropylene glycol, and the glycerol extractant includes glycerol; mixing the trumpet creeper alcohol extract, thistle glycerol extract, and cardamom alcohol extract in a mass ratio of 100:(28~305):(28~305) to obtain the plant extract composition.

[0009] In any embodiment of this application, in the step of mixing and extracting the trumpet creeper flower powder with an alcohol extractant to obtain a trumpet creeper flower alcohol extract, the mass concentration of the alcohol extractant is 20% to 70%; and / or, the extraction temperature is maintained at 75°C to 85°C; and / or, the extraction time is 1.5h to 3h; and / or, the mass ratio of the trumpet creeper flower powder to the alcohol extractant is 1:(6 to 15).

[0010] In any embodiment of this application, 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 20% to 70%; and / or, the extraction temperature is maintained at 75°C to 85°C; and / or, the extraction time is 1.5 h to 3 h; and / or, the mass ratio of *Cirsium japonicum* powder to glycerol extractant is 1:(6 to 15).

[0011] In any embodiment of this application, in the step of mixing and extracting cardamom powder with an alcohol extractant to obtain cardamom alcohol extract, the mass concentration of the alcohol extractant is 70%~100%; and / or, the extraction temperature is maintained at 75℃~85℃; and / or, the extraction time is 1.5h~3h; and / or, the mass ratio of cardamom powder to alcohol extractant is 1:(6~15).

[0012] Thirdly, this application provides an application of a plant extract composition, which includes the plant extract composition described in the first aspect or the plant extract composition prepared according to the preparation method described in the second aspect; the application includes at least one of (1) to (3): (1) application in the preparation of a product with the function of preventing ultraviolet light damage; (2) application in the preparation of a product with the function of repairing ultraviolet light damage; (3) application in the preparation of a product with the function of immediate soothing and repairing.

[0013] In any embodiment of this application, preventing or repairing ultraviolet light damage includes at least one of enhancing the activity of skin barrier-related proteins, increasing the content of skin barrier-related proteins, inhibiting the activity of inflammatory factors, and reducing the content of inflammatory factors, and / or; immediate soothing and repair includes at least one of inhibiting TRPV1 receptor activity, inhibiting calcium ion influx into HaCaT cells, reducing skin irritation, and reducing the skin erythema index.

[0014] 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; and / or, based on the total mass of the product, the plant extract composition in the product contains 0.05% to 3% by mass.

[0015] This application embodiment prepares a plant extract composition by mixing trumpet creeper alcohol extract, thistle glycerol extract, and cardamom alcohol extract in a mass ratio of 100:(28~305):(28~305). The combination of these three extracts in this composition exerts a synergistic effect, effectively protecting the skin from UV damage while providing immediate soothing and repair, achieving both core benefits and overcoming the limitations of traditional single-herb extraction methods that cannot fundamentally address skin allergies and fragility. Furthermore, the use of alcohol and glycerol extraction methods, compared to the irritating organic solvents used in traditional processes, is more suitable for sensitive skin, broadening the product's applicability and providing a more efficient, safe, and comprehensive solution for skin care. Attached Figure Description

[0016] 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.

[0017] Figure 1 This is a schematic diagram showing the effect of plant extracts prepared in Examples 1-4 and Comparative Examples 1-12 of this application on the viability of HaCaT cells (where mean ± SD, n=3; in the figure, "#" represents significant difference compared with the blank group, ####P<0.0001; "*" represents significant difference compared with the UVB group, *P<0.05, **P<0.01, ****P<0.0001; "ns" represents no significant difference compared with the UVB group).

[0018] Figure 2 This is a schematic diagram illustrating the effect of plant extracts prepared in Examples 1-4 and Comparative Examples 1-12 of this application on the ROS fluorescence intensity of HaCaT cells after UVB-induced damage (where mean ± SD, n=3; in the figure, "#" indicates a significant difference compared to the control group, ####P<0.0001; " "This indicates a significant difference compared to the UVB group." P<0.05, P<0.01, P<0.001, P<0.0001).

[0019] Figure 3 This diagram illustrates the effects of plant extracts prepared in Examples 1-4 and Comparative Examples 1-12 of this application on the mRNA expression levels of cell barrier repair-related genes secreted by HaCaT cells after UVB-induced damage (Figure A shows the effect of plant extracts prepared in Examples 1-4 and Comparative Examples 1-12 of this application on the ZO-1 mRNA expression level secreted by HaCaT cells after UVB-induced damage; Figure B shows the effect of plant extracts prepared in Examples 1-4 and Comparative Examples 1-12 of this application on the CLDN1 mRNA expression level secreted by HaCaT cells after UVB-induced damage; Figure C shows the effect of plant extracts prepared in Examples 1-4 and Comparative Examples 1-12 of this application on the FLG mRNA expression level secreted by HaCaT cells after UVB-induced damage; in the figures, "#" indicates a significant difference compared to the control group, ####P<0.0001; " "This indicates a significant difference compared to the UVB group." P<0.05, P<0.01, P<0.001, P<0.0001, “ns” means no significant difference compared with UVB group; “^” means significant difference compared with formulation A group, ^^P<0.01, ^^^P<0.001, ^^^^P<0.0001).

[0020] Figure 4 This is a schematic diagram showing the effects of plant extracts prepared in Examples 1-4 and Comparative Examples 1-12 of this application on inflammatory factors secreted by HaCaT cells after UVB-induced damage (wherein, Figure A shows the effect of plant extracts prepared in Examples 1-4 and Comparative Examples 1-12 of this application on TNF-α secreted by HaCaT cells after UVB-induced damage; Figure B shows the effect of plant extracts prepared in Examples 1-4 and Comparative Examples 1-12 of this application on NO secreted by HaCaT cells after UVB-induced damage; Figure C shows the effect of plant extracts prepared in Examples 1-4 and Comparative Examples 1-12 of this application on PEG2 secreted by HaCaT cells after UVB-induced damage; Figure D shows the effect of plant extracts prepared in Examples 1-4 and Comparative Examples 1-12 of this application on IL-6 secreted by HaCaT cells after UVB-induced damage; in the figures, "#" indicates a significant difference compared to the blank group, ####P<0.0001; " "This indicates a significant difference compared to the UVB group." P<0.05, P<0.01, P<0.001, P<0.0001, where “ns” indicates no significant difference compared to the UVB group; “^” indicates a significant difference compared to formulation A group, ^^^^P<0.0001).

[0021] Figure 5 These are fluorescence staining images of HaCaT cells irradiated with UVB after single-cell gel electrophoresis experiments using plant extracts prepared in Examples 1-4 and Comparative Examples 1-12 of this application.

[0022] Figure 6 This is a statistical graph showing the tail length of plant extracts prepared in Examples 1-4 and Comparative Examples 1-12 of this application after single-cell gel electrophoresis of HaCaT cells irradiated with UVB (where "#" indicates a significant difference compared to the blank group, ####P<0.0001; " "This indicates a significant difference compared to the model group." P<0.05, P<0.01, P<0.001, P<0.0001, “^” indicates that it is significant compared with formulation A (^^^^P<0.0001).

[0023] Figure 7 These are schematic diagrams illustrating the effects of plant extracts prepared in Examples 1-4 and Comparative Examples 1-12 of this application on preventing UVB-induced secretion of inflammatory factors in HaCaT cells (Figure A shows the effect of plant extracts prepared in Examples 1-4 and Comparative Examples 1-12 of this application on preventing UVB-induced secretion of TNF-α in HaCaT cells; Figure B shows the effect of plant extracts prepared in Examples 1-4 and Comparative Examples 1-12 of this application on preventing UVB-induced secretion of PGE2 in HaCaT cells, where mean ± SD, n=3; in the figures, "#" indicates a significant difference compared to the control group, ####P<0.0001; "This indicates a significant difference compared to the UVB group." P<0.05, P<0.01, P<0.001, P<0.0001, “^” indicates that it is significant compared with formulation A (^^^^P<0.0001).

[0024] Figure 8 This paper describes the effects of plant extracts prepared in Examples 1-4 and Comparative Examples 1-12 of this application on the relative expression levels of TRPV1 mRNA and protein secreted by HaCaT cells after capsaicin stimulation (Figure A is a schematic diagram of the effect of capsaicin stimulation on TRPV1 mRNA expression in HaCaT cells; Figure B is a schematic diagram of the effect of capsaicin stimulation on TRPV1 protein expression in HaCaT cells; where mean ± SD, n=3; in the figures, "#" indicates a significant difference compared to the control group, ####P<0.0001; " "This indicates a significant difference compared to the UVB group." P<0.05, P<0.01, P<0.001, P<0.0001; “^” indicates a significant difference compared to formulation A (^^^^P<0.0001).

[0025] Figure 9 This is a schematic diagram showing the effect of plant extracts prepared in Examples 1-4 and Comparative Examples 1-12 of this application on the fluorescence intensity of calcium ions in HaCaT cells.

[0026] Figure 10 This is a graph showing the immediate relief score of the plant extracts prepared in Examples 1-4 and Comparative Examples 1-12 of this application in the human lactic acid stinging test.

[0027] Figure 11 The graph shows the effect of plant extracts prepared in Examples 1-4 and Comparative Examples 1-12 of this application on the skin redness value in the human tape tearing test. Detailed Implementation

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] Trumpet creeper (dried flowers of the American trumpet creeper, a plant in the Bignoniaceae family) is recorded in the pharmacopoeia as being used for rashes, itchy skin, and acne. It is also recorded in traditional Chinese medicine books throughout history for its use in treating skin diseases. Thistle (dried aerial parts of thistle, a plant in the Asteraceae family) has hemostatic, anti-inflammatory, and detoxifying effects and is suitable for treating external bleeding and carbuncles. Red cardamom (dried mature fruit of Alpinia galanga, a plant in the Zingiberaceae family) has been proven to have antibacterial and anti-inflammatory pharmacological activities.

[0034] The first aspect of this application provides a plant extract composition comprising: 100 parts of trumpet creeper alcohol extract; 28 to 305 parts of thistle glycerol extract; and 28 to 305 parts of cardamom alcohol extract. The extractant for the trumpet creeper alcohol extract includes at least one of 1,3-propanediol, 1,2-propanediol, methylpropanediol, and dipropylene glycol. The extractant for the thistle glycerol extract includes glycerin. The extractant for the cardamom alcohol extract includes at least one of 1,3-propanediol, 1,2-propanediol, methylpropanediol, and dipropylene glycol.

[0035] Preferably, the plant extract composition comprises: 100 parts of trumpet creeper alcohol extract; 30 to 300 parts of thistle glycerol extract; and 30 to 300 parts of cardamom alcohol extract.

[0036] For example, when the mass fraction of the trumpet creeper alcohol extract in the plant extract composition is 100 parts, the mass fraction of the above-mentioned thistle glycerol extract can be 28 parts, 28.5 parts, 29 parts, 29.5 parts, 30 parts, 30.5 parts, 31 parts, 31.5 parts, 32 parts, 32.5 parts, 33 parts, 33.1 parts, 33.2 parts, 33.3 parts, 33.4 parts, 33.5 parts, 33.6 parts, 33.7 parts, 33.8 parts, 33.9 parts, 34 parts, 34.5 parts, 35 parts, 35.5 parts, 35.6 parts, 35.7 parts, 35.8 parts, 35.9 parts, 36 parts, 36.5 parts, 37 parts, 37.5 parts, 38 parts, 38 parts, 39.5 parts, 39.5 parts, 30 parts, 30.5 parts, 31 parts, 31.5 parts, 32 parts, 32.5 parts, 33 parts, 33.1 parts, 33.2 parts, 33.3 parts, 33.4 parts, 33.5 parts, 33.6 parts, 33.7 parts, 33.8 parts, 33.9 parts, 36 parts, 36.5 parts, 37 parts, 37.5 parts, 38 parts, 39.5 parts, 39.6 parts, 35.7 parts, 35.8 parts, 35.9 parts, 36 parts, 36.5 parts, 37 parts, 37.5 parts, 38 parts, 39.5 parts, 39.6 parts, 39.5 parts, 39.6 parts, 39.7 parts, 35.8 parts, 38.5 portions, 39 portions, 39.5 portions, 40 portions, 45 portions, 50 portions, 55 portions, 60 portions, 65 portions, 70 portions, 75 portions, 80 portions, 85 portions, 90 portions, 95 portions, 96 portions, 97 portions, 98 portions, 99 portions, 100 portions, 101 portions, 102 portions, 103 portions, 104 portions, 105 portions, 110 portions, 120 portions, 130 portions, 140 portions, 150 portions, 180 portions, 200 portions, 220 portions, 240 portions, 260 portions, 280 portions, 290 portions, 295 portions, 296 portions, 297 portions, 298 portions, 299 portions, 300 portions, 301 portions, 302 portions, 303 portions, 304 portions, or 305 portions.

[0037] For example, when the mass fraction of the trumpet creeper alcohol extract in the plant extract composition is 100 parts, the mass fraction of the cardamom alcohol extract can be 28 parts, 28.5 parts, 29 parts, 29.5 parts, 30 parts, 30.5 parts, 31 parts, 31.5 parts, 32 parts, 32.5 parts, 33 parts, 33.1 parts, 33.2 parts, 33.3 parts, 33.4 parts, 33.5 parts, 33.6 parts, 33.7 parts, 33.8 parts, 33.9 parts, 34 parts, 34.5 parts, 35 parts, 35.5 parts, 35.6 parts, 35.7 parts, 35.8 parts, 35.9 parts, 36 parts, 36.5 parts, 37 parts, 37.5 parts, 38 parts, 38 parts, 39.5 parts, 39.5 parts, 30 parts, 30.5 parts, 31 parts, 31.5 parts, 32 parts, 32.5 parts, 33 parts, 33.1 parts, 33.2 parts, 33.3 parts, 33.4 parts, 33.5 parts, 33.6 parts, 33.7 parts, 33.8 parts, 33.9 parts, 36 parts, 36.5 parts, 37 parts, 37.5 parts, 38 parts, 39.5 parts, 39.6 parts, 35.7 parts, 35.8 parts, 35.9 parts, 36 parts, 36.5 parts, 37 parts, 37.5 parts, 38 parts, 39.5 parts, 39.6 parts, 39.5 parts, 39.6 parts, 39.7 parts, 39.8 parts, 39.9 38.5 portions, 39 portions, 39.5 portions, 40 portions, 45 portions, 50 portions, 55 portions, 60 portions, 65 portions, 70 portions, 75 portions, 80 portions, 85 portions, 90 portions, 95 portions, 96 portions, 97 portions, 98 portions, 99 portions, 100 portions, 101 portions, 102 portions, 103 portions, 104 portions, 105 portions, 110 portions, 120 portions, 130 portions, 140 portions, 150 portions, 180 portions, 200 portions, 220 portions, 240 portions, 260 portions, 280 portions, 290 portions, 295 portions, 296 portions, 297 portions, 298 portions, 299 portions, 300 portions, 301 portions, 302 portions, 303 portions, 304 portions, or 305 portions.

[0038] For example, the extractant for the alcoholic extract of trumpet vine includes 1,3-propanediol.

[0039] For example, the extractant for thistle glycerol extracts includes glycerol.

[0040] For example, the extractant for cardamom alcohol extracts includes 1,3-propanediol.

[0041] This composition combines three plant extracts of specific types and in specific proportions, extracted using a gentle and safe extraction agent, to synergistically provide effective protection against UV damage and immediate soothing and repair. Specifically, the preparation of the trumpet creeper extract utilizes gentle polyols such as 1,3-propanediol, 1,2-propanediol, methylpropanediol, or dipropylene glycol, which fully preserves its anti-inflammatory and anti-allergic active ingredients, effectively inhibiting UV-induced erythema, itching, and inflammatory reactions, and alleviating skin sensitivity after photodamage. The preparation of the thistle glycerin extract uses glycerin as the extraction solvent, gently enriching its hemostatic, anti-inflammatory, and repairing components, enhancing skin barrier function, reducing microvascular damage and oxidative stress caused by UV radiation, and promoting rapid repair of damaged skin. The preparation of the cardamom extract uses polyols such as 1,3-propanediol and 1,2-propanediol, maximizing the preservation of its antibacterial and anti-inflammatory activities, synergistically with trumpet creeper and thistle to exert antioxidant effects, reducing UV-induced free radical damage and lowering the risk of photoaging. When these three components are combined within a specific ratio range, they form a synergistic effect: the trumpet creeper extract primarily provides anti-inflammatory and soothing effects, the thistle extract strengthens the skin barrier and repairs the skin barrier, and the cardamom extract enhances antioxidant protection, thereby blocking the chain reaction of skin damage caused by ultraviolet radiation at multiple targets. Simultaneously, the selected extracts, such as glycerin and propylene glycol, are not only highly safe, avoiding the irritation of sensitive skin caused by traditional organic solvents, but also improve the transdermal absorption efficiency of the active ingredients, further enhancing the immediate soothing and long-lasting protective effects of the composition.

[0042] In some embodiments, the extractant concentration of the trumpet creeper alcohol extract is 20% to 70%; and / or, the extractant concentration of the thistle glycerol extract is 20% to 70%; and / or, the extractant concentration of the cardamom alcohol extract is 70% to 100%.

[0043] For example, the extractant mass concentration of the trumpet creeper alcohol extract can be 20%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%.

[0044] For example, the extractant mass concentration of thistle glycerol extract can be 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, or 70%.

[0045] For example, the extractant mass concentration of cardamom alcohol extract can be 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, or 100%.

[0046] The mass concentrations of the three extracts were precisely set to suit the characteristics of their active ingredients, ensuring both efficient dissolution and stable activity of each component. Through synergistic concentration optimization, the combined formula maximizes its efficacy in protecting against UV damage and providing immediate soothing and repair to the skin. Specifically, for the trumpet creeper alcohol extract, the mass concentration was set at 20%–70%. This concentration range effectively balances the dissolution efficiency and gentleness of the active ingredients in trumpet creeper: too low a concentration would fail to fully dissolve its anti-inflammatory and soothing lipid-soluble active ingredients, such as flavonoids, thus failing to effectively intervene in the skin inflammation pathways caused by UV radiation; too high a concentration might lead to the dissolution of some irritating components, potentially exacerbating the skin's burden. A concentration of 20%–70% ensures that the extracted active ingredients effectively reduce the release of inflammatory factors, achieving immediate relief. It soothes and avoids damaging the skin barrier, making it suitable for sensitive skin in its vulnerable state after UV damage. The extract concentration of thistle glycerin is 20%~70%. If the glycerin concentration is too low, the ability to dissolve water-soluble repairing ingredients such as polysaccharides and saponins in thistle is limited, making it difficult to activate the proliferation and migration of skin keratinocytes and affecting the healing of epidermal damage caused by UV rays. If the concentration is too high, it may inhibit the dissolution of effective ingredients due to excessive osmotic pressure. At the same time, excessive glycerin may increase the viscosity of the extract, hindering the skin's absorption of repairing ingredients. A concentration of 20% to 70% ensures the full dissolution of water-soluble repairing ingredients. Simultaneously, the moisturizing film formed by glycerin reduces skin moisture evaporation, providing a humid environment for damage repair and enhancing repair efficacy. The extract concentration of cardamom alcohol extract is set at 70% to 100%. This higher concentration maximizes the dissolution of these fat-soluble components, effectively inhibiting secondary infections when the skin's microbiome is imbalanced after UV exposure. Furthermore, the high concentration of extract enhances the stability of active ingredients, ensuring they maintain strong anti-inflammatory capabilities even when combined with other extracts, strengthening the skin's resistance to UV damage, and forming a protective closed loop with trumpet creeper and thistle extracts.

[0047] In some embodiments, based on the total mass of the plant extract composition, the concentration of verbascoside in the plant extract composition is 0.2 mg / mL to 0.7 mg / mL, and / or; the concentration of isoverascoside in the plant extract composition is 0.02 mg / mL to 0.05 mg / mL, and / or; the concentration of strychnine in the plant extract composition is 0.03 mg / mL to 0.06 mg / mL, and / or; the total flavonoid content in the plant extract composition is 1.3 mg / mL to 1.7 mg / mL, and / or; and the total polyphenol content in the plant extract composition is 0.3 mg / mL to 1.2 mg / mL.

[0048] For example, the concentration of verbascoside in the plant extract composition is 0.2 mg / mL, 0.25 mg / mL, 0.30 mg / mL, 0.31 mg / mL, 0.32 mg / mL, 0.34 mg / mL, 0.35 mg / mL, 0.36 mg / mL, 0.37 mg / mL, 0.38 mg / mL, 0.39 mg / mL, 0.40 mg / mL, 0.45 mg / mL, 0.5 mg / mL, 0.6 mg / mL, or 0.7 mg / mL.

[0049] For example, the concentration of isobarbiturin in the plant extract composition is 0.02 mg / mL, 0.03 mg / mL, 0.032 mg / mL, 0.034 mg / mL, 0.035 mg / mL, 0.036 mg / mL, 0.037 mg / mL, 0.038 mg / mL, 0.039 mg / mL, 0.04 mg / mL, 0.045 mg / mL, or 0.05 mg / mL.

[0050] For example, the concentration of strychnine in the plant extract composition may be 0.03 mg / mL, 0.035 mg / mL, 0.040 mg / mL, 0.041 mg / mL, 0.0412 mg / mL, 0.0414 mg / mL, 0.0416 mg / mL, 0.0417 mg / mL, 0.0418 mg / mL, 0.0419 mg / mL, 0.042 mg / mL, 0.045 mg / mL, 0.05 mg / mL, or 0.06 mg / mL.

[0051] For example, the total flavonoid content in the plant extract composition is 1.3 mg / mL, 1.35 mg / mL, 1.4 mg / mL, 1.45 mg / mL, 1.5 mg / mL, 1.55 mg / mL, 1.6 mg / mL, 1.65 mg / mL or 1.7 mg / mL.

[0052] For example, the total polyphenol content in the plant extract composition is 0.3 mg / mL, 0.35 mg / mL, 0.40 mg / mL, 0.45 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.81 mg / mL, 0.82 mg / mL, 0.83 mg / mL, 0.84 mg / mL, 0.841 mg / mL, 0.842 mg / mL, 0.843 mg / mL, 0.844 mg / mL, 0.845 mg / mL, 0.85 mg / mL, 0.88 mg / mL, 0.90 mg / mL, 0.95 mg / mL, 1.0 mg / mL, 1.05 mg / mL, 1.1 mg / mL, or 1.2 mg / mL.

[0053] The second aspect of this application provides a method for preparing a plant extract composition, comprising: mixing trumpet creeper powder with an alcohol extractant and extracting the mixture, followed by post-treatment to obtain a trumpet creeper alcohol extract; mixing thistle powder with a glycerol extractant and extracting the mixture, followed by post-treatment to obtain a thistle glycerol extract; mixing cardamom powder with an alcohol extractant and extracting the mixture, followed by post-treatment to obtain cardamom alcohol extract; wherein the alcohol extractant includes at least one selected from 1,3-propanediol, 1,2-propanediol, methylpropanediol, and dipropylene glycol, and the glycerol extractant includes glycerol; mixing the trumpet creeper alcohol extract, thistle glycerol extract, and cardamom alcohol extract in a mass ratio of 100:(28~305):(28~305) to obtain the plant extract composition.

[0054] Preferably, the post-treatment includes centrifugation and filtration.

[0055] For example, the plant extract composition is obtained by mixing the trumpet creeper alcohol extract, the thistle glycerol extract and the cardamom alcohol extract in a mass ratio of 1:1:1.

[0056] For example, the plant extract composition is obtained by mixing the extracts of trumpet creeper, the extracts of thistle glycerides, and the extracts of cardamom in a mass ratio of 3:1:1.

[0057] For example, the plant extract composition is obtained by mixing the alcohol extract of trumpet creeper, the glycerol extract of thistle, and the alcohol extract of cardamom in a mass ratio of 1:3:1.

[0058] For example, the plant extract composition is obtained by mixing the alcohol extract of trumpet creeper, the glycerol extract of thistle, and the alcohol extract of cardamom in a mass ratio of 1:1:3.

[0059] In some embodiments, in the step of mixing and extracting the trumpet vine powder with an alcohol extractant to obtain a trumpet vine alcohol extract, the mass concentration of the alcohol extractant is 20% to 70%; and / or, the extraction temperature is maintained at 75°C to 85°C; and / or, the extraction time is 1.5 h to 3 h; and / or, the mass ratio of the trumpet vine powder to the alcohol extractant is 1:(6 to 15).

[0060] For example, in the step of mixing and extracting trumpet vine powder with an alcohol extractant to obtain trumpet vine alcohol extract, the mass concentration of the alcohol extractant can be 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%.

[0061] For example, in the step of mixing and extracting trumpet vine powder with an alcohol extractant to obtain trumpet vine alcohol extract, the extraction temperature is maintained at 75℃, 76℃, 77℃, 78℃, 79℃, 80℃, 81℃, 82℃, 83℃, 84℃ or 85℃.

[0062] For example, in the step of mixing and extracting the powder of trumpet creeper with an alcohol extractant to obtain the alcohol extract of trumpet creeper, the extraction time can be 1.5h, 1.8h, 2.0h, 2.2h, 2.4h, 2.6h, 2.8h or 3h.

[0063] For example, the mass ratio of trumpet vine powder to alcohol extractant can be 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14 or 1:15 (unit: g / g).

[0064] In some embodiments, 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 20% to 70%; and / or, the extraction temperature is maintained at 75°C to 85°C; and / or, the extraction time is 1.5 h to 3 h; and / or, the mass ratio of *Cirsium japonicum* powder to glycerol extractant is 1:(6 to 15).

[0065] For example, 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 can be 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%.

[0066] For example, in the step of mixing and extracting thistle powder with a glycerol extractant to obtain thistle glycerol extract, the extraction temperature is maintained at 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, or 85°C.

[0067] For example, in the step of mixing and extracting thistle powder with a glycerol extractant to obtain thistle glycerol extract, the extraction time can be 1.5h, 1.8h, 2.0h, 2.2h, 2.4h, 2.6h, 2.8h or 3h.

[0068] For example, the mass ratio of thistle powder to glycerol extract can be 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14 or 1:15 (in g / g).

[0069] In some embodiments, in the step of mixing and extracting cardamom powder with an alcohol extractant to obtain cardamom alcohol extract, the mass concentration of the alcohol extractant is 70% to 100%; and / or, the extraction temperature is maintained at 75°C to 85°C; and / or, the extraction time is 1.5 h to 3 h; and / or, the mass ratio of cardamom powder to alcohol extractant is 1:(6 to 15).

[0070] For example, in the step of mixing and extracting cardamom powder with an alcohol extractant to obtain cardamom alcohol extract, the mass concentration of the alcohol extractant can be 70%, 72%, 74%, 76%, 78%, 79%, 80%, 81%, 82%, 84%, 86%, 88%, 90%, 92%, 95%, 98%, 99%, or 100%.

[0071] For example, the extraction temperature is maintained at 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, or 85°C.

[0072] For example, in the step of mixing cardamom powder with an alcohol extractant and extracting it to obtain cardamom alcohol extract, the extraction time can be 1.5h, 1.8h, 2.0h, 2.2h, 2.4h, 2.6h, 2.8h or 3h.

[0073] For example, the mass ratio of cardamom powder to alcohol extract can be 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14 or 1:15 (in g / g).

[0074] Preferably, the 1,3-propanediol extract of trumpet creeper, the glycerin extract of thistle, and the 1,3-propanediol extract of cardamom are mixed in a mass ratio of 1:1:1 to obtain a plant extract composition.

[0075] The third aspect of the embodiments of this application provides an application of a plant extract composition, which includes the plant extract composition described in the first aspect or a plant extract composition prepared according to the preparation method described in the second aspect; the application includes at least one of (1) to (3): (1) application in the preparation of a product having the effect of preventing ultraviolet light damage; (2) application in the preparation of a product having the effect of repairing ultraviolet light damage; (3) application in the preparation of a product having the effect of immediate soothing and repairing.

[0076] In some embodiments, preventing or repairing ultraviolet (UV) photodamage includes at least one of enhancing the activity of skin barrier-related proteins, increasing the content of skin barrier-related proteins, inhibiting the activity of inflammatory factors, and reducing the content of inflammatory factors, and / or; immediate soothing and repair includes at least one of inhibiting TRPV1 receptor activity, inhibiting calcium ion influx into HaCaT cells, reducing skin irritation, and reducing the skin erythema index.

[0077] For example, skin barrier-related proteins include at least one of connexins, filaggrin (FLG), smearin, epidermal proteins, and desmosomes.

[0078] For example, the connecting proteins include at least one of ZO1 protein (tight junction protein 1), CLDN1 protein (closure protein 1), and CLDN4 protein (closure protein 4).

[0079] For example, inflammatory factors include at least one of pro-inflammatory cytokines and prostaglandin cytokines; and / or, pro-inflammatory cytokines include at least one of TNF-α, IL-1β, IL-6 and IL-8; and / or, prostaglandin cytokines include PGE2.

[0080] For example, inhibiting TRPV1 receptor activity includes reducing TRPV1 mRNA and protein activity.

[0081] In some embodiments, 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; and / or, based on the total mass of the product, the plant extract composition contains 0.05% to 3% of the product by mass.

[0082] Example

[0083] 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] The sources of some of the reagents and equipment used in Examples 1-4 and Comparative Examples 1-12 are as follows:

[0085] 1. HaCaT cells, manufacturer FuHeng, catalog number FH0186;

[0086] 2. PBS, manufacturer Gibco, catalog number 2002050, 500 mL;

[0087] 3. Fetal bovine serum (FBS) manufacturer MRC, catalog number CCS30009.02, 500 mL;

[0088] 4. Bispecific antibiotics (penicillin & streptomycin) Gibco15140122;

[0089] 5. DMEM, Gibco 11995-065 500 mL;

[0090] 6. Trypsin-EDTA (0.25%), containing phenol red Thermo Fisher 25200114;

[0091] 7. Petri dish: Thermo Fisher 150464;

[0092] 8. 6-well plate, Thermo Fisher 140675;

[0093] 9. CCK-8 Admas life C8022-10000T;

[0094] 10. ROS probe DCFH DA (D6883);

[0095] 11. Merck, D6883-50mg;

[0096] 12. DMSO, Merck, 472301-500mL.

[0097] Example 1

[0098] Example 1 of this application provides a plant extract composition, the preparation method of which includes:

[0099] S1, Campsis grandiflora powder was mixed with a 30% aqueous solution of 1,3-propanediol and extracted. The extraction temperature was maintained at 80℃ and the extraction time was 2h. The mass ratio of Campsis grandiflora powder to extractant (30% aqueous solution of 1,3-propanediol) was 1:10 (in g / g), and Campsis grandiflora alcohol extract (LXH30BEC) was obtained.

[0100] S2, thistle powder was mixed with a 60% glycerol aqueous solution and extracted. The extraction temperature was maintained at 85℃ and the extraction time was 2h. The mass ratio of thistle powder to extractant (60% glycerol aqueous solution) was 1:10 (in g / g), and thistle glycerol extract (DJ60GY) was obtained.

[0101] S3, mix cardamom powder with an 80% aqueous solution of 1,3-propanediol and extract the mixture. The extraction temperature is maintained at 80℃ and the extraction time is 2 hours. The mass ratio of cardamom powder to extractant (80% aqueous solution of 1,3-propanediol) is 1:10 (in g / g), to obtain cardamom alcohol extract (HDK80BEC).

[0102] S4. The above-mentioned trumpet creeper alcohol extract, thistle glycerol extract and cardamom alcohol extract are mixed in a mass ratio of 1:1:1 to obtain a plant extract composition. The specific formula is shown in Table 1.

[0103] Example 2

[0104] Example 2 of this application provides a plant extract composition, which differs from Example 1 only in step S4, specifically:

[0105] The plant extract composition was obtained by mixing the alcohol extract of trumpet creeper, the glycerol extract of thistle, and the alcohol extract of cardamom in a mass ratio of 6:2:2. The specific formula is shown in Table 1.

[0106] Example 3

[0107] Example 3 of this application provides a plant extract composition, which differs from Example 1 only in step S4, specifically:

[0108] The plant extract composition was obtained by mixing the alcohol extract of trumpet creeper, the glycerol extract of thistle, and the alcohol extract of cardamom in a mass ratio of 2:6:2. The specific formula is shown in Table 1.

[0109] Example 4

[0110] Example 4 of this application provides a plant extract composition, which differs from Example 1 only in step S4, specifically:

[0111] The plant extract composition was obtained by mixing the alcohol extract of trumpet creeper, the glycerol extract of thistle, and the alcohol extract of cardamom in a mass ratio of 2:2:6. The specific formula is shown in Table 1.

[0112] Comparative Example 1

[0113] Comparative Example 1 of this application provides a plant extract composition, which differs from Example 1 only in the extractant in step S2. Specifically, 60% glycerol is used instead of 60% 1,3-butanediol (DJ60DEC). The specific formulation is shown in Table 1.

[0114] Comparative Example 2

[0115] Comparative Example 2 of this application provides a plant extract composition, which differs from Example 1 only in the extractant in step S1. Specifically, 1,3-propanediol with a mass concentration of 30% is replaced with 1,3-butanediol (LXH60DEC) with a mass concentration of 60% in the extraction process of trumpet creeper. The specific formula is shown in Table 1.

[0116] Comparative Example 3

[0117] Comparative Example 3 of this application provides a plant extract composition, which differs from Example 1 only in the extractants in steps S1 and S2. Specifically, 1,3-propanediol with a mass concentration of 30% is replaced with 1,3-butanediol (LXH30DEC) with a mass concentration of 60% in step S1 of the trumpet creeper extraction process, and glycerol with a mass concentration of 60% is replaced with 1,3-butanediol (DJ60DEC) with a mass concentration of 60% in step S2 of the thistle extraction process. The specific formulation is shown in Table 1.

[0118] Comparative Example 4

[0119] Comparative Example 4 of this application provides a plant extract, the preparation method of which includes:

[0120] The powder of trumpet vine was mixed with 30% 1,3-propanediol and extracted. The extraction temperature was maintained at 80℃ and the extraction time was 2h. The mass ratio of trumpet vine powder to 1,3-propanediol was 1:10 (in g / g). The resulting alcohol extract of trumpet vine (LXH30BEC) is shown in Table 1.

[0121] Comparative Example 5

[0122] Comparative Example 5 of this application provides a plant extract, the preparation method of which includes:

[0123] The powder of trumpet vine was mixed with 60% 1,3-butanediol and extracted. The extraction temperature was maintained at 80℃ and the extraction time was 2h. The mass ratio of trumpet vine powder to 1,3-butanediol was 1:10 (in g / g). The resulting alcohol extract of trumpet vine (LXH60DEC) is shown in Table 1.

[0124] Comparative Example 6

[0125] Comparative Example 6 of this application provides a plant extract, the preparation method of which includes:

[0126] Thistle powder was mixed with 60% glycerol and extracted. The extraction temperature was maintained at 85℃ and the extraction time was 2 hours. The mass ratio of thistle powder to glycerol was 1:10 (in g / g) to obtain thistle glycerol extract (DJ60 GY). The specific formula is shown in Table 1.

[0127] Comparative Example 7

[0128] Comparative Example 7 of this application provides a plant extract, the preparation method of which includes:

[0129] Thistle powder was mixed with 60% 1,3-butanediol and extracted. The extraction temperature was maintained at 85℃ and the extraction time was 2 hours. The mass ratio of thistle powder to 1,3-butanediol was 1:10 (in g / g), resulting in thistle alcohol extract (DJ60DEC). The specific formula is shown in Table 1.

[0130] Comparative Example 8

[0131] Comparative Example 8 of this application provides a plant extract, the preparation method of which includes:

[0132] Red cardamom powder was mixed with 1,3-propanediol with a mass concentration of 80% and extracted. The extraction temperature was maintained at 80℃ and the extraction time was 2 hours. The mass ratio of red cardamom powder to alcohol extractant was 1:10 (in g / g) to obtain red cardamom alcohol extract (HDK80BEC). The specific formula is shown in Table 1.

[0133] Comparative Example 9

[0134] Comparative Example 9 of this application provides a plant extract composition, the preparation method of which includes:

[0135] S1, Campsis grandiflora powder was mixed with 30% 1,3-propanediol and extracted. The extraction temperature was maintained at 80℃ and the extraction time was 2h. The mass ratio of Campsis grandiflora powder to 1,3-propanediol was 1:10 (in g / g), and Campsis grandiflora alcohol extract (LXH30BEC) was obtained.

[0136] S2, thistle powder was mixed with 60% glycerol and extracted. The extraction temperature was maintained at 85℃ and the extraction time was 2h. The mass ratio of thistle powder to glycerol extractant was 1:10 (in g / g) to obtain thistle glycerol extract (DJ60GY).

[0137] S3, the above-mentioned trumpet creeper alcohol extract and thistle glycerol extract are mixed in a mass ratio of 1:1 to obtain a plant extract composition. The specific formula is shown in Table 1.

[0138] Comparative Example 10

[0139] Comparative Example 10 of this application provides a plant extract composition, the preparation method of which includes:

[0140] S1, Campsis grandiflora powder was mixed with 30% 1,3-propanediol and extracted. The extraction temperature was maintained at 80℃ and the extraction time was 2h. The mass ratio of Campsis grandiflora powder to 1,3-propanediol was 1:10 (in g / g), and Campsis grandiflora alcohol extract (LXH30BEC) was obtained.

[0141] S2, red cardamom powder was mixed with 1,3-propanediol with a mass concentration of 80% and extracted. The extraction temperature was maintained at 80℃ and the extraction time was 2h. The mass ratio of red cardamom powder to alcohol extractant was 1:10 (in g / g) to obtain red cardamom alcohol extract (HDK80BEC).

[0142] S3, the above-mentioned trumpet creeper alcohol extract and cardamom alcohol extract are mixed in a mass ratio of 1:1 to obtain a plant extract composition. The specific formula is shown in Table 1.

[0143] Comparative Example 11

[0144] Comparative Example 11 of this application provides a plant extract composition, the preparation method of which includes:

[0145] S1, thistle powder was mixed with 60% glycerol and extracted. The extraction temperature was maintained at 85℃ and the extraction time was 2h. The mass ratio of thistle powder to glycerol extractant was 1:10 (in g / g) to obtain thistle glycerol extract (DJ60GY).

[0146] S2, mix red cardamom powder with 1,3-propanediol with a mass concentration of 80% and extract the mixture. The extraction temperature is maintained at 80℃ and the extraction time is 2h. The mass ratio of red cardamom powder to alcohol extractant is 1:10 (in g / g) to obtain red cardamom alcohol extract (HDK80BEC). The specific formula is shown in Table 1.

[0147] Comparative Example 12

[0148] Comparative Example 12 of this application provides a plant extract composition, the preparation method of which includes:

[0149] S1, Purslane powder was mixed with a 30% aqueous solution of 1,3-propanediol and extracted. The extraction temperature was maintained at 80℃ for 2 hours. The mass ratio of purslane powder to 1,3-propanediol was 1:10 (in g / g), yielding purslane alcohol extract (MCX30BEC).

[0150] S2, thistle powder was mixed with a 60% glycerol aqueous solution and extracted. The extraction temperature was maintained at 85℃ and the extraction time was 2h. The mass ratio of thistle powder to glycerol extract was 1:10 (in g / g) to obtain thistle glycerol extract (DJ60GY).

[0151] S3, mix cardamom powder with an 80% (w / w) 1,3-propanediol aqueous solution and extract the mixture. The extraction temperature is maintained at 80℃ and the extraction time is 2 hours. The mass ratio of cardamom powder to alcohol extractant is 1:10 (in g / g), to obtain cardamom alcohol extract (HDK80BEC).

[0152] S4. The above-mentioned purslane alcohol extract, thistle glycerol extract and cardamom alcohol extract are mixed in a mass ratio of 1:1:1 to obtain a plant extract composition. The specific formula is shown in Table 1.

[0153] Table 1

[0154]

[0155] Performance testing

[0156] The performance of the plant extract compositions prepared in Examples 1-4 and Comparative Examples 1-12 was tested:

[0157] I. Physical and chemical performance testing

[0158] 1. Determination of active ingredient content by HPLC

[0159] 1.1 Chromatographic conditions

[0160] Column: Agilent ZORBAX SB-C18 (250 mm × 4.6 mm, 5 µm);

[0161] Mobile phase: acetonitrile-0.1% phosphoric acid solution (volume ratio 22:78), isocratic elution;

[0162] Flow rate: 1.0 mL / min;

[0163] Column temperature: 30℃;

[0164] Detection wavelength: 330 nm;

[0165] Injection volume: 10 μL.

[0166] 1.2 Preparation of reference solution

[0167] Accurately weigh 1.0 mg each of citronellol, mongholic acid, verbascoside and isorhuscoside reference standards, place them in a 10 mL volumetric flask, dissolve them in methanol and dilute to the mark, shake well to prepare a single reference standard stock solution containing 0.1 mg per mL.

[0168] 1.3 Plotting the Standard Curve

[0169] Accurately measure 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of each reference standard stock solution, place them in 10 mL stoppered test tubes, add water to bring the volume to 1.0 mL, mix well, and obtain a series of standard solutions of different concentrations. Inject and determine the concentrations under the chromatographic conditions described above, and establish a standard curve by performing linear regression of peak area (Y) against concentration (X, μg / mL).

[0170] 1.4 Preparation of test solution

[0171] Take an appropriate amount of the sample to be tested, dilute it directly or with the extraction solvent, centrifuge at 10,000 rpm for 10 min, filter the supernatant through a 0.22 μm microporous membrane, and take the filtrate as the test solution.

[0172] 1.5 Determination Method

[0173] Accurately pipette 10 μL each of the reference solution and the test solution, inject them into the liquid chromatograph, record the chromatograms, and calculate the content of each active ingredient using the external standard method.

[0174] The test results are shown in Table 2.

[0175] 2. Determination of total flavonoid content by ultraviolet spectrophotometry

[0176] 2.1 Preparation of Reagents and Solutions

[0177] (1) Rutin reference solution: Accurately weigh 4.05 mg of rutin reference standard, place it in a 10 mL volumetric flask, dissolve it in methanol and dilute to the mark, shake well to prepare a reference standard stock solution with a concentration of 0.405 mg / mL.

[0178] (2) 5% sodium nitrite solution (prepare fresh before use).

[0179] (3) 10% aluminum nitrate solution.

[0180] (4) 4% sodium hydroxide solution.

[0181] (5) 60% ethanol solution.

[0182] 2.2 Preparation of Standard Curve

[0183] (1) Accurately pipette 0, 0.4, 0.8, 1.2, 1.6 and 2.0 mL of rutin reference stock solution into six 10 mL volumetric flasks respectively;

[0184] (2) Add 60% ethanol solution to each volumetric flask to make the total volume of the solution in each flask 2.0 mL;

[0185] (3) Add 0.5 mL of 5% sodium nitrite solution in sequence, shake well, and let stand for 6 min;

[0186] (4) Add 0.5 mL of 10% aluminum nitrate solution, shake well, and let stand for 6 min;

[0187] (5) Add 4.0 mL of 4% sodium hydroxide solution, add 60% ethanol to the mark, shake well, and let stand for 15 min;

[0188] (6) Using 60% ethanol solution as a blank control, the absorbance of each solution was measured at a wavelength of 510 nm.

[0189] (7) Plot a standard curve with rutin concentration (μg / mL) on the x-axis and absorbance on the y-axis.

[0190] 2.3 Determination of the test solution

[0191] (1) Accurately pipette 2.0 mL of the test solution into a 10 mL volumetric flask;

[0192] (2) Follow the steps (3)-(6) under "2.2";

[0193] (3) Calculate the total flavonoid content (calculated as rutin) in the test sample from the standard curve based on the measured absorbance value.

[0194] The test results are shown in Table 3.

[0195] 3. Determination of total polyphenol content by ultraviolet spectrophotometry

[0196] 3.1 Preparation of Reagents and Solutions

[0197] (1) Gallic acid standard stock solution: Accurately weigh 20.92 mg of gallic acid reference standard, place it in a 100 mL volumetric flask, add 60% ethanol to dissolve and dilute to the mark, shake well to prepare a standard stock solution with a concentration of 0.2092 mg / mL.

[0198] (2) Working solution:

[0199] 10% Folin-Ciocalteu reagent (prepare fresh before use);

[0200] 7.5% sodium carbonate solution;

[0201] 60% ethanol solution.

[0202] 3.2 Preparation of Standard Curve

[0203] (1) Accurately pipette 0, 1, 2, 3, 4, 5, and 6 mL of gallic acid standard stock solution into seven 10 mL volumetric flasks;

[0204] (2) Dilute to the mark with 60% ethanol solution, shake well, and obtain a series of working solutions of different concentrations;

[0205] (3) Transfer 1.0 mL of each working solution into a 10 mL colorimetric tube;

[0206] (4) Add 5.0 mL of 10% Folin-Ciocalteu reagent to each tube, shake well, and react at room temperature for 5 min;

[0207] (5) Add 4.0 mL of 7.5% Na2CO3 solution and shake well;

[0208] (6) Place at room temperature away from light for 60 min;

[0209] (7) Using the 0 mL group as a blank control, the absorbance was measured at a wavelength of 765 nm;

[0210] (8) Plot a standard curve with gallic acid concentration (μg / mL) on the x-axis and absorbance on the y-axis.

[0211] 3.3 Determination of the test solution

[0212] (1) Accurately transfer 1.0 mL of the test solution into a 10 mL colorimetric tube;

[0213] (2) Follow the steps (4)-(7) under "3.2";

[0214] (3) Calculate the total polyphenol content (calculated as gallic acid) in the test sample from the standard curve based on the measured absorbance value.

[0215] The test results are shown in Table 4.

[0216] II. In vitro efficacy testing

[0217] 1. Determination of DPPH free radical scavenging capacity

[0218] 1.1 Reagent Preparation

[0219] (1) DPPH working solution:

[0220] Accurately weigh 3.2 mg of DPPH, add 4 mL of anhydrous ethanol, sonicate for 5 min, vortex until completely dissolved, store at 4°C protected from light, and dilute 10 times with anhydrous ethanol before use to prepare 0.2 mM DPPH working solution.

[0221] (2) Sample solution:

[0222] Dilute the sample to be tested 25 times with deionized water, and then dilute it 2 times with anhydrous ethanol;

[0223] Vitamin C reference standard was prepared into a 100 μg / mL standard solution using anhydrous ethanol.

[0224] 1.2 Determination Method

[0225] (1) Add 100 μL of sample solution to a 96-well plate;

[0226] (2) Add 100 μL of DPPH working solution and react in the dark for 30 min;

[0227] (3) Set up the following control group:

[0228] Sample control: 100 μL of sample solution mixed with 100 μL of anhydrous ethanol;

[0229] Blank control: 100 μL DPPH working solution and 100 μL anhydrous ethanol.

[0230] (4) After the reaction is complete, the absorbance of each well is measured at a wavelength of 540 nm.

[0231] 1.3 Calculation Formula

[0232] DPPH radical scavenging rate (%) = [(Ac-(As-Ad)) / Ac] × 100%

[0233] In the formula:

[0234] Ac: Absorbance value of blank control (100 μL DPPH + 100 μL anhydrous ethanol);

[0235] As: Absorbance values ​​of the sample group (100 μL sample and 100 μL DPPH);

[0236] Ad: Absorbance value of sample control (100 μL sample and 100 μL anhydrous ethanol).

[0237] The test results are shown in Table 5.

[0238] 2. Proliferative activity of HaCaT cells damaged by UVB (UVB modeling first, then sample addition)

[0239] With 2×10 5 HaCaT cells were quantitatively seeded into 96-well cell culture plates at a density of cells / well. After culturing in a cell culture incubator for 24 h, HaCaT cells were irradiated with UVB at 0.1 J / cm², followed by the addition of the test sample diluted 100-fold with cell culture medium (i.e., sample concentration of 1%). PBS buffer or culture medium was added to the wells around the perimeter of the 96-well cell culture plate to avoid edge effects and experimental errors. After further culturing in a 37 ℃, 5% CO2 incubator for 24 or 48 h, the culture medium in each well was discarded, and 110 µL of premixed 100 µL complete culture medium + 10 µL LCK-8 solution was added to each well. The plates were incubated at 37 ℃ for 1–2 h. The optical density at 450 nm was measured using a microplate reader. Cell viability (%) = (OD experimental group - OD blank control group) / (OD normal control group - OD blank control group) × 100%. The effect of 1% concentration of the sample on the changes in HaCaT cell proliferation activity induced by UVB irradiation was detected by CCK-8 assay. The experiment was divided into 3 groups: blank group, UVB irradiation group, and UVB + sample group (sample applied after UVB irradiation).

[0240] Experimental results are as follows Figure 1 As shown.

[0241] 3. UVB damages ROS levels in HaCaT cells (UVB modeling is performed first, followed by sample addition).

[0242] HaCaT cells were seeded into 6-well cell culture plates and cultured in a constant-temperature cell culture incubator at 37 ℃ and 5.0% CO2. After 24 h, the cells were irradiated with UVB (0.1 J / cm²) and then treated with 1% of the sample to be tested for 48 h. The 2,7-dichlorofluorescein diacetate (DCFH-DA) probe was diluted 1:1000 with serum-free culture medium. The original cell culture medium was discarded, and 1 mL of the diluted DCFH-DA probe was added to each well. The cells were incubated at 37 ℃ for 20 min. The cells were washed three times with PBS, and the fluorescence intensity of DCFH-DA was measured using a fluorescence microplate reader at an excitation wavelength of 500 nm and an emission wavelength of 525 nm. The relative ROS content in the cells of each group was calculated using the following formula.

[0243] Experimental results are as follows Figure 2 As shown.

[0244] 4. UVB-induced barrier function repair experiment in HaCaT cells

[0245] HaCaT cells were fed at a dose of 2.0 × 10⁻⁶. 5 Cells were seeded at a density of 1 cell / mL in 6-well plates (2 mL per well); cultured at 37°C in a 5% CO2 incubator for 24 h; irradiated with UVB (0.1 J / cm²); the blank control group was protected from light by wrapping with aluminum foil; after irradiation, the experimental group was replaced with 2 mL of sample solution diluted 100 times with DEME maintenance medium (i.e., sample concentration of 1%); 2 mL of DEME maintenance medium was added to the model control group; and cultured for another 24 h.

[0246] Add 1 mL of Trizol to each well of a 6-well plate to lyse cells, and transfer the sample to a 1.5 mL enzyme-free centrifuge tube. Add 200 μL of chloroform to the centrifuge tube, vortex until emulsified, and incubate at 4°C for 10 min. After the upper and lower layers separate, centrifuge at 12000 r / min at 4°C for 15 min. Gently aspirate 400 μL of the supernatant to a new enzyme-free centrifuge tube, add an equal volume of isopropanol and mix by inverting, incubate at 4°C for 10 min, and centrifuge at 12000 r / min at 4°C for 10 min. Discard the supernatant, add 1 mL of 75% anhydrous ethanol to wash the RNA precipitate, centrifuge at 12000 r / min at 4°C for 5 min, discard the ethanol, and after the precipitate is dry and transparent, add an appropriate amount of DEPC-ddH2O. Measure the RNA concentration using a micro UV spectrophotometer, and assess RNA quality at 260 / 280.

[0247] RNA was reverse transcribed into cDNA using the Evo M-MLV reverse transcription premix kit. The mixture was gently mixed and briefly centrifuged; incubated at 37°C for 15 min; then at 85°C for 5 s; and stored on ice for subsequent experiments or frozen. The resulting cDNA was diluted 10-fold, and a PCR reaction system was prepared using the diluted cDNA as a template: reaction conditions: 95°C pre-denaturation for 30 s, 95°C denaturation for 15 s, 60°C annealing and extension for 30 s, 40 cycles. β-actin was used as an internal control. Calculate the relative expression levels of the target gene mRNA. Perform statistical analysis and plotting on the data using GraphPad Prism 8.0, and conduct significance analysis of the experimental results using one-way ANOVA.

[0248] Experimental results are as follows Figure 3 As shown.

[0249] 5. Experiment on repairing UVB-induced inflammatory damage in HaCaT cells

[0250] HaCaT cells were fed at a dose of 2.0 × 10⁻⁶. 5 Cells were seeded at a density of 2 mL / well in 6-well plates and cultured at 37°C in a 5% CO2 incubator for 24 h. Cells were then irradiated with UVB (0.5 J / cm²). The blank control group was protected from light by wrapping the cells in aluminum foil. After irradiation, the experimental group was replaced with 2 mL of sample solution diluted 100 times with DEME maintenance medium (i.e., sample concentration of 1%). The model control group was given 2 mL of DEME maintenance medium and cultured for another 24 h.

[0251] Cell culture supernatant was collected, and the levels of TNF-α, PGE2, and IL-6 were detected using ELISA; NO levels were detected using the Griess method. Statistical analysis and plotting of the data were performed using GraphPad Prism 8.0, and one-way ANOVA was used to analyze the significance of the experimental results. The experimental results are as follows: Figure 4 As shown.

[0252] 6. DNA damage prevention performance test experiment

[0253] 6.1 Experimental Materials and Reagents

[0254] Samples: Each sample to be tested;

[0255] Cell line: HaCaT human immortalized keratinocytes;

[0256] Reagents: DMEM medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics), 0.25% trypsin-EDTA digestion solution, phosphate buffer (PBS, pH 7.4), cell lysis buffer, unwinding buffer, electrophoresis buffer, neutralization buffer, DNA staining reagent (such as ethidium bromide or SYBR Green I).

[0257] Experimental equipment: 24-well cell culture plates, 0.22 μm microporous filter membrane and filtration device, cell culture incubator (37℃, 5% CO2), UVB irradiation instrument (wavelength 312 nm), confocal laser scanning microscope, centrifuge, electrophoresis apparatus and electrophoresis tank.

[0258] 6.2 Experimental Procedure

[0259] Sample pretreatment: Each sample was diluted 100 times (i.e., sample concentration was 1%) using DMEM medium to prepare sample working solutions; the sample working solutions were aseptically filtered using a 0.22 μm microporous membrane, and the treated samples were stored at 4℃ for later use.

[0260] Cell seeding and culture: HaCaT cells in logarithmic growth phase were seeded at a rate of 1 × 10⁻⁶ cells / year. 5 Cells were seeded at a concentration of 1 cell / mL into 24-well cell culture plates, with each well containing 1 mL of the seeded cell. After seeding, the 24-well cell culture plates were placed in a cell culture incubator at 37°C and 5% CO2 for 24 h of cell adhesion before proceeding with subsequent experiments.

[0261] Cell pretreatment: Add 2 mL of the above-prepared working solution to adherent HaCaT cells for cell pretreatment for 2 h.

[0262] UVB modeling: After pretreatment, the sample working solution in the wells was aspirated and the cells were irradiated with UVB at a dose of 5 J / cm² using a UVB irradiator (wavelength 312 nm) to establish a cell DNA damage model.

[0263] Cell culture continued: After UVB irradiation, fresh DMEM medium was added to each well, and the cells were placed in a cell culture incubator at 37°C and 5% CO2 for 20 hours.

[0264] Cell collection and suspension preparation: After culture, aspirate the culture medium from the wells, add an appropriate amount of 0.25% trypsin-EDTA digestion solution to each well, and place the wells in a cell culture incubator to digest the cells until they detach; add an appropriate amount of DMEM culture medium to stop digestion, collect the cell suspension into a centrifuge tube, centrifuge at 1000 r / min for 5 min, and discard the supernatant; add 1 mL of PBS to the centrifuge tube, gently pipette to mix, and prepare a single-cell suspension.

[0265] 6.3 DNA damage detection

[0266] After cell lysis, unwinding, electrophoresis, neutralization, and staining, the cells were observed and photographed under a confocal microscope.

[0267] Experimental results are as follows Figure 5-6 As shown.

[0268] 7. Evaluation experiment on the ability of HaCaT cells to prevent UVB-induced inflammatory damage.

[0269] 7.1 Experimental Materials and Instruments

[0270] (1) Cell line: HaCaT human immortalized keratinocytes

[0271] (2) Main reagents:

[0272] DEME maintenance medium (containing 1% penicillin-streptomycin), PBS buffer (pH 7.4), TNF-α ELISA kit, and PGE2 ELISA kit.

[0273] (3) Main instruments: UVB lamp, 24-well cell culture plate, microplate reader, CO2 incubator (37℃, 5% CO2).

[0274] 7.2 Experimental Methods

[0275] (1) Sample pretreatment: Each sample was diluted 100 times (i.e., sample concentration 1%) using DMEM maintenance medium to prepare sample working solution; the sample working solution was sterile filtered using a 0.22 μm microporous membrane, and the treated sample was stored at 4℃ for later use.

[0276] (2) Cell seeding and culture: HaCaT cells in the logarithmic growth phase were seeded at 2.0 × 10⁻⁶ cells / year. 5 Cells were seeded at a concentration of 500 μL / mL into 24-well cell culture plates. After seeding, the 24-well cell culture plates were placed in a cell culture incubator at 37°C and 5% CO2 for 24 h before subsequent experiments were performed.

[0277] (3) Cell washing: After culturing for 24 h, carefully aspirate the culture medium from each well and gently wash the cells 3 times with PBS (pH 7.4) to remove residual culture medium and metabolic waste.

[0278] (4) Group processing and sample pretreatment:

[0279] Blank control group: Add 500 μL of DMEM maintenance medium to the wells.

[0280] Model group: Add 500 μL of DMEM maintenance medium to the wells.

[0281] Sample group: Add 500 μL of the sample working solution prepared above into the well.

[0282] All groups were pretreated in a cell culture incubator at 37℃ and 5% CO2 for 2 hours.

[0283] (5) UVB modeling treatment: After pretreatment, the liquid in the wells was aspirated. Except for the blank control group which was completely wrapped with tin foil (to avoid UV irradiation), both the model group and the sample group were irradiated with UVB at a dose of 2.5 J / cm² using a UVB irradiator (wavelength 312 nm) to establish a cell inflammatory damage model.

[0284] (6) Continued cell culture: After UVB irradiation, the foil of the blank control group was removed, and 500 μL of fresh DMEM maintenance medium was added to each well of all groups. Then, the 24-well cell culture plate was placed in a cell culture incubator at 37°C and 5% CO2 for 24 h.

[0285] (7) Collection of supernatant: After the culture is completed, carefully aspirate the culture supernatant from each well and transfer it to a centrifuge tube. Centrifuge at 12000 r / min for 10 min, collect the supernatant, and store it in a -80℃ refrigerator for later testing.

[0286] (8) Inflammatory factor detection: NO content was detected using the Griess method and the PGE2 content in each supernatant was detected according to the instructions of the PGE2 ELISA kit.

[0287] 7.4 Data Analysis

[0288] The experimental data were statistically analyzed and plotted using GraphPad Prism 8.0 software. One-way ANOVA was used to test the significance of the experimental results for each group.

[0289] Experimental results are as follows Figure 7 As shown.

[0290] 8. Capsaicin-stimulated TRPV1 in HaCaT cells experiment

[0291] 8.1 Experimental Materials and Instruments

[0292] (1) Cell line: HaCaT human immortalized keratinocytes

[0293] (2) Main reagents: Capsaicin (purity ≥98%), DEME maintenance medium (containing 1% antibiotics), Trizol lysis buffer, chloroform, isopropanol, Evo M-MLV reverse transcription kit, SYBR Green qPCR premixed reagent

[0294] (3) Main instruments: 24-well / 6-well cell culture plates, real-time fluorescence quantitative PCR instrument, enzyme-linked immunosorbent assay (ELISA) reader (detection wavelength: 540nm / 450nm), micro-volume ultraviolet spectrophotometer.

[0295] 8.2 Experimental Methods

[0296] (1) Sample solution preparation: Each sample was diluted 100 times (i.e., the mass concentration of the sample was 1%) using DMEM maintenance medium, and then aseptically filtered through a 0.22 μm microporous membrane and stored at 4℃ for later use.

[0297] (2) Capsaicin solution preparation: dilute capsaicin to a final concentration of 20 μM with DMEM maintenance medium and prepare fresh each time.

[0298] (3) Cell seeding and culture (24-well plate):

[0299] HaCaT cells in logarithmic growth phase were fed a dose of 1×10⁻⁶. 5 The cells were seeded at a concentration of 500 μL / mL into 24-well plates and cultured in a 37°C, 5% CO2 cell culture incubator for 24 h until the cells adhered and grew.

[0300] (4) Cell treatment (24-well plate):

[0301] After culturing for 24 h, carefully aspirate the culture medium from each well and gently wash the cells three times with PBS (pH 7.4) to remove any residual culture medium.

[0302] (5) Experimental Grouping

[0303] The cells obtained in step (4) are grouped into different groups.

[0304] Blank control group: Add 500 μL of DMEM maintenance medium to each well.

[0305] Model group: Add 500 μL of capsaicin solution with a final concentration of 20 μM to each well.

[0306] Sample group: Add 500 μL of capsaicin solution with a final concentration of 20 μM to each well.

[0307] Each group was incubated in a 37℃, 5% CO2 cell culture incubator for 2 h.

[0308] After incubation, the sample group was added to the sample solution prepared above and incubated for another 24 h at 37°C and 5% CO2.

[0309] (6) Cell seeding and culture (6-well plate, for gene expression detection):

[0310] HaCaT cells in logarithmic growth phase were fed a dose of 1×10⁻⁶. 5 The cells were seeded at a concentration of 2 mL / well into 6-well plates and cultured at 37°C in a 5% CO2 incubator for 24 h until they adhered to the plate and grew.

[0311] (7) Cell treatment (6-well plate):

[0312] After culturing for 24 h, carefully aspirate the culture medium from each well and gently wash the cells three times with PBS (pH 7.4).

[0313] (8) Experimental Grouping

[0314] The cells obtained in step (7) are grouped into different groups.

[0315] Blank control group: Add 2 mL of DMEM maintenance medium to each well.

[0316] Model group: Add 2 mL of capsaicin solution with a final concentration of 20 μM to each well.

[0317] Sample group: Add 2 mL of capsaicin solution with a final concentration of 20 μM to each well.

[0318] Each group was incubated in a 37℃, 5% CO2 cell culture incubator for 2 h.

[0319] After incubation, the sample group was added to the sample solution prepared above and incubated for another 24 h at 37°C and 5% CO2.

[0320] (9) Total RNA extraction (6-well plate):

[0321] After incubation for 24 h, aspirate the supernatant from each well, add 1 mL of Trizol reagent to each well, gently shake the culture plate to ensure the reagent fully contacts the cells, and incubate at room temperature for 5 min to lyse the cells.

[0322] Transfer the lysis buffer to 1.5 mL enzyme-free centrifuge tubes, add 200 μL of chloroform to each tube, vortex for 15-30 s until the solution becomes emulsified, and incubate at 4°C for 10 min. Discard the supernatant, add 1 mL of 75% anhydrous ethanol to wash the RNA precipitate, centrifuge at 12000 rpm at 4°C for 5 min, discard the ethanol, and wait for the precipitate to dry and become clear. Add an appropriate amount of DEPC-ddH2O, and determine the RNA concentration using a micro UV spectrophotometer, assessing RNA quality at 260 / 280. After RNA concentration determination, reverse transcribe the RNA into cDNA using the Evo M-MLV reverse transcription premix kit, mix gently, and centrifuge briefly; incubate at 37°C for 15 min; incubate at 85°C for 5 s; place on ice for subsequent experiments or freeze for storage. The cDNA obtained from reverse transcription was diluted 10-fold. PCR was configured using the diluted cDNA as a template under the following conditions: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 15 s, 60℃ annealing and extension for 30 s, for 40 cycles. β-actin was used as an internal control. Calculate the relative expression levels of the target gene mRNA. Perform statistical analysis and plotting on the data using GraphPad Prism 8.0, and perform significance analysis on the experimental results using one-way ANOVA.

[0323] Experimental results are as follows Figure 8 As shown.

[0324] 9. Experiment on calcium ion influx in HaCaT cells

[0325] 9.1 Experimental Materials and Instruments

[0326] (1) Cell line: HaCaT human immortalized keratinocytes

[0327] (2) Main reagents: Capsaicin (purity ≥98%), DMSO (cell culture grade), Fluo-4 AM calcium ion fluorescent probe (0.5 mM stock solution), DMEM complete culture medium (containing 10% FBS), 0.22 μm sterile filter membrane.

[0328] (3) Main instruments: 96-well plates with black transparent bottom (Corning® 3904), fluorescence microplate reader (equipped with 488 nm excitation / 529 nm emission filter), cell culture centrifuge, biosafety cabinet (Class II).

[0329] 9.2 Experimental Methods

[0330] (1) Preparation of capsaicin storage solution and working solution:

[0331] Weigh 50 mg of capsaicin, add 1.673 mL of DMSO, dissolve thoroughly to prepare a 100 mM stock solution, dispense into centrifuge tubes, and store at 4°C protected from light for later use.

[0332] When ready to use, dilute the capsaicin stock solution with PBS to a final concentration of 20 μM working solution, and prepare fresh each time.

[0333] (2) Sample solution preparation:

[0334] Take appropriate amounts of the samples to be tested according to formulations A, B, C, D, E, F, and G, and add 9 times the volume of DMEM culture medium at a ratio of 1:9. Mix well and then dilute 10 times (i.e., the sample concentration is 1%). Filter through a 0.22 μm microporous membrane to remove bacteria and store at 4℃ for later use.

[0335] (3) Cell seeding and culture:

[0336] HaCaT cells in logarithmic growth phase were injected with 1.0 × 10⁻⁶ cells. 5 The cells were seeded at a concentration of 100 μL / mL into black transparent 96-well plates. After seeding, the 96-well plates were incubated in a cell culture incubator at 37°C and 5% CO2 for 24 h until the cells adhered and grew.

[0337] (4) Sample incubation:

[0338] After culturing for 24 hours, carefully aspirate the culture medium from each well and gently wash the cells twice with PBS to remove any residual culture medium.

[0339] Add 100 μL of sample working solution of different concentrations (10-fold, 20-fold, 40-fold dilution) to each well, and incubate the 96-well plate in a cell culture incubator for 6 h.

[0340] (5) Fluo-4 AM load:

[0341] After incubation, aspirate the working solution from the wells and gently wash the cells three times with PBS.

[0342] Add 100 μL of Fluo-4 AM working solution diluted with PBS (final concentration 0.5 μM) to each well, and incubate the 96-well plate at 37°C in the dark for 30 min to allow Fluo-4 AM to fully enter the cells.

[0343] (6) Fluorescent probe conversion:

[0344] After incubation for 30 min, aspirate the Fluo-4 AM working solution from the wells and gently wash the cells three times with PBS to remove any fluorescent probes that have not entered the cells. After washing, place the 96-well plate in a cell culture incubator at 37°C and 5% CO2 for another 20 min to ensure that Fluo-4 AM is completely converted to Fluo-4 within the cells.

[0345] (7) Capsaicin treatment and fluorescence detection:

[0346] Add capsaicin working solution with a final concentration of 20 μM to each well, and immediately use a fluorescence microplate reader to detect the change in fluorescence intensity within 2 min. The excitation wavelength is set to 488 nm and the emission wavelength is set to 529 nm. Record the change curve of fluorescence intensity over time.

[0347] Experimental results are as follows Figure 9 As shown.

[0348] 10. Human Experiment Lactic Acid Stinging Relief Score

[0349] Before the test, all participants cleaned their faces with water and gently patted them dry with lint-free absorbent paper towels. They sat quietly in a standardized testing environment for at least 30 minutes, during which time they could not drink water or beverages, remained relaxed, and the tested area was exposed and avoided being touched.

[0350] Lactic acid stimulation phase: Filter paper (0.8 ± 0.1 cm in diameter) soaked in 50 μL of 10% lactic acid solution was applied to both sides of the subject's nasolabial folds. When the subject's subjective sensation (stinging and / or itching) in both sides of the nasolabial folds was at or above the moderate level, the filter paper was removed and the subject's stimulation sensation at this time was recorded (i.e., the stimulation endpoint, T0).

[0351] Following the principles of randomization and control, a self-controlled trial was conducted, with test products randomly distributed. Testers used the test product in the test area of ​​the sample group according to the sample distribution table. The control group used a corresponding test product base formulation without soothing ingredients or served as a blank control. Each test product was used once, and the application area should completely cover the lactic acid irritation area. The sample usage amount should refer to the sample instructions. If the control group was set as a corresponding test product base formulation without soothing ingredients, the usage method and dosage were the same as the test product; if set as a blank control, no product was used in the test area. At the lactic acid irritation endpoint (T0) and at different test time points after sample use, subjects conducted a semi-subjective assessment of their sensations in the test area. Multiple time points and observation durations could be set according to product needs, usually not exceeding 30 minutes. The semi-subjective assessment sensations were itching and stinging. The subjective assessment was scored using a 4-point scale: 0 points for no sensation, 1 point for mild sensation, 2 points for moderate sensation, and 3 points for severe sensation. Median scores, such as 0.5 or the median of the score, could be used to describe the degree of sensation.

[0352] Statistical analysis software was used to perform statistical analysis of the data; appropriate descriptive statistics were performed according to the data classification. The difference between each test time point and the stimulus endpoint was calculated for each group, and this difference was used to statistically analyze the differences between the sample group and the control group at each visit point. Semi-subjective ratings were ordinal data: comparisons before and after use were performed using a two-sample rank-sum test; comparisons between the sample group and the control group were performed using an independent samples rank-sum test. All statistical analyses were two-tailed tests, with a significance level of α = 0.05.

[0353] The formulation is shown in Table 6. Experimental results are as follows: Figure 10 As shown.

[0354] 11. Human body soothing redness value measurement experiment

[0355] The inner forearm was selected as the test site. The test area was marked on qualified participants. A skin barrier damage model was established by inducing skin erythema through tape peeling. The induction parameters were standardized: constant pressure (e.g., 200g weight or 200N pressure device), tape application time controlled at 10 seconds, followed by rapid peeling. This peeling was repeated 20-30 times, with the actual number determined based on the thickness and degree of redness of the volunteers' skin. The endpoint of repeated peeling was defined as a clearly visible erythema (referring to the skin reaction grading standard for closed patch tests in human skin patch testing, grade 2 erythema standard). The actual number of peels was recorded. The peeling area was randomly divided into a product test area and a blank control area. After establishing the skin barrier damage model, participants sat quietly in a suitable environment for at least 30 minutes for baseline skin value testing before product use. In the product test area, staff applied the product using latex finger cots at a dosage of (2.0±0.1) mg / cm³. 2 The blank control area is left unprocessed. The same test is performed again at the set test time points. Test data can be normalized.

[0356] The formulation is shown in Table 6. Experimental results are as follows: Figure 11 As shown.

[0357] II. Test Results

[0358] 1. Determination of active ingredient content by HPLC

[0359] Table 2 shows that Formula B indicates that *Campsis grandiflora* is rich in verbascoside. A comparison of Formula A and Formula F shows that the verbascoside content obtained by extracting *Campsis grandiflora* with a 30% propylene glycol aqueous solution is higher than that obtained by extracting with a 60% butylene glycol aqueous solution. Formula C shows that *Cirsium japonicum* contains scutellarin. A comparison of Formula A and Formula E shows that the scutellarin content obtained by extracting *Cirsium japonicum* with a 60% glycerol aqueous solution is higher than that obtained by extracting with a 60% butylene glycol aqueous solution.

[0360] A comparison of formulas A, B, C, and D reveals that different proportions of trumpet creeper, thistle, and cardamom can yield varying amounts of verbascoside, isoverascoside, and buddlejaside. Overall, the combination of trumpet creeper, thistle, and cardamom extracts in a 1:1:1 mass ratio exhibits moderate levels of the active ingredients verbascoside, isoverascoside, and buddlejaside, thus enabling them to exert their respective biological effects.

[0361] Table 2. Comparison of concentrations of verbascoside, isoverascoside, and buddlejaside in different formulations.

[0362]

[0363] 2. Determination of total flavonoid content by ultraviolet spectrophotometry

[0364] Table 3 shows that formulas B, C, and D have higher total flavonoid content, indicating that *Campsis grandiflora*, *Cirsium japonicum*, and *Amomum villosum* are rich in flavonoids. A comparison of formulas A and F shows that the total flavonoid content obtained by extracting *Campsis grandiflora* with a 30% propylene glycol aqueous solution is higher than that obtained with a 60% butylene glycol aqueous solution. A comparison of formulas A and E shows that the total flavonoid content obtained by extracting *Cirsium japonicum* with a 60% glycerol aqueous solution is higher than that obtained with a 60% butylene glycol aqueous solution. A comparison of formulas A, B, C, and D shows that different proportions of *Campsis grandiflora*, *Cirsium japonicum*, and *Amomum villosum* yield different total flavonoid contents. Overall, the combination of *Campsis grandiflora*, *Cirsium japonicum*, and *Amomum villosum* extracts in a 1:1:1 mass ratio has a moderate total flavonoid content. Because flavonoids have an unstable chemical structure, they are easily affected by environmental factors, undergoing oxidation, hydrolysis, and other chemical reactions, thus changing their color. Typically, the color deepens at high temperatures and fades under light. Significant color changes can greatly limit the application of active ingredients. Therefore, considering both bioactivity and application, formula A with a moderate total flavonoid content should be given priority.

[0365] Table 3 Results of total flavonoid content in different formulations

[0366]

[0367] 3. Determination of total polyphenol content by ultraviolet spectrophotometry

[0368] Table 4 shows that Formula B has a higher total polyphenol content, indicating that Trumpet Vine is rich in polyphenols. A comparison of Formulas A and F shows that the total polyphenol content obtained by extracting Trumpet Vine with a 30% propylene glycol aqueous solution is higher than that obtained with a 60% butylene glycol aqueous solution. A comparison of Formulas A and E shows that the total polyphenol content obtained by extracting Thistle with a 60% glycerol aqueous solution is higher than that obtained with a 60% butylene glycol aqueous solution. A comparison of Formulas A, B, C, and D shows that different proportions of Trumpet Vine, Thistle, and Cardamom all yield different total polyphenol contents. Overall, the combination of Trumpet Vine, Thistle, and Cardamom extracts in a 1:1:1 mass ratio has a moderate total polyphenol content. Because polyphenols have unstable chemical structures, they are easily affected by environmental factors, undergoing oxidation, hydrolysis, and other chemical reactions, thus changing their color. Typically, high temperature and light exposure lead to a darker color. Significant color changes can greatly limit the application of active ingredients. Therefore, considering both bioactivity and application, formulation A with a moderate total polyphenol content should be given priority.

[0369] Table 4 Comparison of total polyphenol content

[0370]

[0371] 4. Determination of DPPH free radical scavenging ability

[0372] Table 5 shows that the compositions composed of trumpet creeper, thistle, and cardamom all exhibit good antioxidant and DPPH free radical scavenging abilities. A comparison of formulations A, E, and F reveals that the composition formed by extracting trumpet creeper with a 30% propylene glycol aqueous solution and thistle with a 60% glycerol aqueous solution has a better antioxidant effect.

[0373] Table 5 DPPH scavenging ability results

[0374]

[0375] 5. Proliferative activity of HaCaT cells damaged by UVB (UVB modeling first, then sample addition)

[0376] Figure 1 The results showed that the combination of trumpet creeper, thistle, and cardamom could promote the proliferation of HaCaT cells after UVB irradiation.

[0377] 6. UVB damages ROS levels in HaCaT cells (UVB modeling is performed first, followed by sample addition).

[0378] Figure 2 Experimental results showed that the compositions of extracts from trumpet vine, thistle, and cardamom could reduce ROS production in HaCaT cells after UVB stimulation. Furthermore, it was clearly observed that formulations A-D significantly reduced ROS production in HaCaT cells after UVB stimulation compared to formulations E-P. Comparing formulations H-L and M-O, the combination of the two extracts was significantly more effective than the single plant extracts. Comparing formulations M-O and A-G, the composition of the three extracts (trumpet vine, thistle, and cardamom) was significantly more effective than the combination of the two extracts. Replacing the trumpet vine extract with purslane extract and combining it with thistle and cardamom also inhibited ROS production in HaCaT cells after UVB stimulation, but its inhibitory effect was weaker than that of formulations A-D.

[0379] 7. UVB-induced barrier function repair experiment in HaCaT cells

[0380] Figure 3Experimental results showed that the compositions of extracts from trumpet creeper, thistle, and cardamom could all promote the production of barrier-related proteins ZO-1, CLDN1, and FLG in HaCaT cells after UVB stimulation at the mRNA level. Furthermore, formulations A-D showed a significantly stronger ability to promote the expression of barrier-related proteins at the mRNA level in HaCaT cells after UVB stimulation, compared to formulations E-P. Comparing formulations H-L and M-O, the combination of each pair of extracts was significantly more effective than the single plant extracts. Comparing formulations M-O and A-G, the combination of the three extracts (trumpet creeper, thistle, and cardamom) was significantly more effective than the combination of any two extracts. Replacing the trumpet creeper extract with purslane extract and combining it with thistle and cardamom also promoted the expression of barrier-related proteins in HaCaT cells after UVB stimulation, but its inhibitory effect was weaker than that of formulations A-D.

[0381] 8. Experiment to inhibit UVB-induced inflammatory damage in HaCaT cells

[0382] Figure 4 Experimental results showed that the compositions of extracts from trumpet vine, thistle, and cardamom could inhibit the production of inflammatory factors TNF-α, NO, PGE2, and IL-6 in HaCaT cells after UVB stimulation. Furthermore, formulations A-D showed a significantly stronger ability to inhibit the production of inflammatory factors in HaCaT cells after UVB stimulation compared to formulations E-P. Comparing formulations H-L and M-O, the combinations of the two extracts were significantly more effective than the single plant extracts. Comparing formulations M-O and A-G, the compositions of the three extracts (trumpet vine, thistle, and cardamom) were significantly more effective than the combinations of the two extracts. Replacing the trumpet vine extract with purslane extract and combining it with thistle and cardamom also inhibited the production of inflammatory factors in HaCaT cells after UVB stimulation, but its inhibitory effect was weaker than that of formulations A-D.

[0383] 9. DNA damage prevention performance test experiment

[0384] Figure 5-6Experimental results showed that after UVB irradiation of HaCaT cells, the average cell tail length in the model group significantly increased compared to the control group, indicating that the UVB damage model was successfully established. Adding 1% concentrations of formulations A, B, C, and D significantly reduced the average tail length of HaCaT cells, showing a significant difference compared to formulations E, F, G, and P, indicating that formulations A-D have a prominent protective effect on UVB-irradiated HaCaT cells and can reduce DNA damage after UVB irradiation. Adding 1% concentrations of formulations E, F, G, and P increased the average tail length of HaCaT cells, indicating that these four formulations have no protective effect on UVB-irradiated HaCaT cells.

[0385] 10. Evaluation experiment on the prevention of UVB-induced inflammatory damage to HaCaT cells

[0386] Figure 7 Experimental results showed that after UVB irradiation of blank HaCaT cells, the inflammatory-related indicators in the model group were significantly increased compared with the blank group, indicating that UVB caused certain damage to HaCaT cells. After incubation with formulations A, B, C, D, and E-P, followed by UVB irradiation, the inflammatory-related indicators produced by HaCaT cells were all lower than those of the blank cells. In particular, after incubation with the positive control dexamethasone and formulations A-D, the levels of NO and PGE2 secreted by HaCaT cells after UVB irradiation were significantly reduced by half. This indicates that dexamethasone and formulations A-D have excellent ability to prevent photodamage to HaCaT cells, and are significantly superior to formulations E-P, meaning that formulations A-D are significantly different from formulations E-P. Comparing formulations H-L and M-O, the combined effects of the two formulations were significantly better than those of single plant extracts. Comparing formulations M-O and A-G, the combined effects of the three extracts (Campsis grandiflora, Cirsium japonicum, and Amomum villosum) were significantly better than those of the two-in-one combinations. A combination of trumpet creeper extract, purslane extract, and this herb, cardamom, can also prevent photodamage to HaCaT cells, but its inhibitory effect is weaker than that of formulations A through D.

[0387] 11. Capsaicin-stimulated TRPV1 experiment in HaCaT cells

[0388] Figure 8Experimental results showed that after capsaicin stimulation of HaCaT cells, TRPV1 expression in the model group was significantly increased at both the mRNA and protein levels compared to the control group, indicating successful model establishment. Formulas A, B, C, and D all significantly reduced TRPV1 expression at both the mRNA and protein levels after capsaicin stimulation of HaCaT cells, with their reduction ability being significantly better than formulas E-P. Comparing formulas H-L and M-O, the combination of the two extracts was significantly more effective than the single plant extracts. Comparing formulas M-O and A-G, the combination of the three extracts (Campsis grandiflora, Cirsium japonicum, and Amomum villosum) was significantly more effective than the combination of the two extracts. Replacing the Campsis grandiflora extract with Portulaca oleracea extract and combining it with Cirsium japonicum and Amomum villosum also inhibited the expression of TRPV1 mRNA and the production of TRPV1 in HaCaT cells after capsaicin stimulation, but its inhibitory effect was weaker than that of formulas A-D.

[0389] 12. Experiment on calcium ion influx in HaCaT cells

[0390] Figure 9 Experimental results showed that capsaicin stimulation of HaCaT cells significantly increased calcium ion influx, indicating successful model establishment. Extracts composed of *Campsis grandiflora*, *Cirsium japonicum*, and cardamom were all able to reduce calcium ion influx levels in HaCaT cells after capsaicin stimulation, with formulations A-D showing significantly better results than formulations E-P. Comparing formulations H-L and M-O, the combination of the two extracts was significantly more effective than the single plant extracts. Comparing formulations M-O and A-G, the combination of the three extracts (*Campsis grandiflora*, *Cirsium japonicum*, and cardamom) was significantly more effective than the combination of the two extracts. Replacing the *Campsis grandiflora* extract with *Portulaca oleracea* extract and combining it with *Cirsium japonicum* and cardamom also reduced calcium ion influx levels in HaCaT cells after capsaicin stimulation, but its inhibitory effect was weaker than that of formulations A-G.

[0391] 13. Experiment on timely relief of lactic acid stinging in the human body

[0392] Figure 10The experimental results showed that after lactic acid stimulation, the subjects' scores were around 3.5, indicating that the lactic acid stimulation was quite severe, classified as a high-intensity stimulation. Adding products containing 5% of formulas A-D, E, M, and P significantly reduced the stimulation after about 2.5 minutes. In the untreated area, the stimulation also decreased due to the activation of the body's own immune regulatory system, dropping from 3.5 to around 2.0. The untreated matrix also decreased from 3.5 to 1.98, which is considered to be due to the body's own regulatory mechanisms, similar to the untreated area. Applying products containing 5% of formulas A-D reduced the irritation score from 3.5 to 1.1-1.3 (mild irritation). Applying products containing 5% of formulas E, M, and P reduced the irritation score from 3.5 to 1.72, 1.84, and 1.70 respectively. This indicates that the combined effect of two formulas is weaker than the combined effect of the three extracts (Campsis grandiflora, Cirsium japonicum, and Amomum villosum). Formulas A-D are significantly better at reducing irritation than formulas E, M, and P, meaning that the effect of formulas A-D in reducing irritation is significantly different from that of formulas E, M, and P. Replacing the Campsis grandiflora extract with Portulaca oleracea extract and combining it with Cirsium japonicum and Amomum villosum also reduced lactic acid irritation after 2.5 minutes, but its effect was weaker than that of formulas A-D. After 5 minutes, the score of the untreated control decreased from 2.0 to 1.7, and the score of the control matrix also decreased from 1.98 to 1.7, which is considered to be a result of the body's own regulation, comparable to the control. Applying products containing 5% of formulations A through D reduced the time to 0.6-0.8 minutes from 1.1-1.3 minutes (2.5 minutes). Applying products containing 5% of formulations E, M, and P reduced the time to 1.17, 1.25, and 1.18 minutes, respectively. This indicates that the combined effects of these two formulations are weaker than the combined effects of the three extracts: trumpet creeper, thistle, and cardamom. Replacing the trumpet creeper extract with purslane extract and combining it with thistle and cardamom also reduced lactic acid irritation, but its effect was weaker than that of formulations A through D.

[0393] Table 6 Product Formulas (Including Formulas)

[0394]

[0395] 14. Experiment on the immediate relief of redness and irritation caused by peeling off human body tape.

[0396] Figure 11Experimental results showed that the red value increased after the tape was torn. Adding products containing 5% of formulations A-D, E, M, and P significantly reduced the red value after about 30 minutes, with formulations A-D showing a significantly better effect than formulations E, M, and P. Applying products containing 5% of formulations A-D reduced the highest red value to 40-52, while applying products containing 5% of formulations E, M, and P reduced the highest red value to 67, 73, and 69 respectively. This indicates that the effect of combinations of two formulations is weaker than the effect of a combination of the three extracts of trumpet creeper, thistle, and cardamom. Replacing the trumpet creeper extract with purslane extract and combining it with thistle and cardamom also reduced the red value caused by tape tearing after 30 minutes, but its effect was weaker than that of formulations A-D. After 60 minutes, the pH of products containing 5% of formulations A-D decreased to 20-30, while the pH of products containing 5% of formulations E, M, and P decreased to 45, 49, and 50, respectively. This indicates that the combined effects of the two-in-one combinations are weaker than the combined effects of the three extracts: trumpet creeper, thistle, and cardamom. Replacing the trumpet creeper extract with purslane extract and combining it with thistle and cardamom also reduced lactic acid irritation, but its effect was weaker than that of formulations A-D.

Claims

1. A plant extract composition, characterized by, The plant extract composition comprises: a campsis flower alcohol extract, 100 parts; a radix cirsii japonici glycerol extract, 28-305 parts; a hainanorchis alcohol extract, 28-305 parts; The extraction agent of the campsis flower alcohol extract is 1,3-propanediol with a mass concentration of 30%; the extraction agent of the radix cirsii japonici glycerol extract is glycerol with a mass concentration of 60%; and the extraction agent of the hainanorchis alcohol extract is 1,3-propanediol with a mass concentration of 80%.

2. The plant extract composition according to claim 1, characterized by, The concentration of verbascoside in the plant extract composition is 0.2-0.7 mg / mL, and / or; The concentration of isoverbascoside in the plant extract composition is 0.02-0.05 mg / mL, and / or; The concentration of bauhinia purpurea glycoside in the plant extract composition is 0.03-0.06 mg / mL, and / or; The total flavonoid content in the plant extract composition is 1.3-1.7 mg / mL, and / or; The total polyphenol content in the plant extract composition is 0.3-1.2 mg / mL.

3. A method of preparing the plant extract composition of claim 1, characterized by, The plant extract composition comprises: a campsis flower alcohol extract, 100 parts; a radix cirsii japonici glycerol extract, 28-305 parts; a hainanorchis alcohol extract, 28-305 parts; The extraction agent of the campsis flower alcohol extract is 1,3-propanediol with a mass concentration of 30%; the extraction agent of the radix cirsii japonici glycerol extract is glycerol with a mass concentration of 60%; and the extraction agent of the hainanorchis alcohol extract is 1,3-propanediol with a mass concentration of 80%.

4. The production method according to claim 3, characterized by, The plant extract composition comprises: The temperature of the extraction is maintained at 75-85 DEG C, and / or; The extraction time is 1.5-3 h, and / or; The mass ratio of the campsis flower powder to the alcohol extraction agent is 1: (6-15).

5. The preparation method according to claim 3, characterized in that, The temperature of the extraction is maintained at 75-85 DEG C, and / or; The extraction time is 1.5-3 h, and / or; The mass ratio of the campsis flower powder to the alcohol extraction agent is 1: (6-15).

6. The preparation method according to claim 3, characterized in that, The temperature of the extraction is maintained at 75-85 DEG C, and / or; The extraction time is 1.5-3 h, and / or; The mass ratio of the campsis flower powder to the alcohol extraction agent is 1: (6-15). ​ 7. Use of a plant extract composition, characterized in that, The plant extract composition comprises the plant extract composition of any one of claims 1-2 or the plant extract composition prepared according to the preparation method of any one of claims 3-6; and the application at least comprises one of (1)-(3): (1) the application in preparing a product having the efficacy of preventing ultraviolet light damage; (2) the application in preparing a product having the efficacy of repairing ultraviolet light damage; (3) the application in preparing a product having the efficacy of instant soothing and repairing.

8. Use according to claim 7, characterized in that, The prevention of ultraviolet light damage or the repair of ultraviolet light damage comprises at least one of enhancing the activity of skin barrier-related proteins, increasing the content of skin barrier-related proteins, inhibiting the activity of inflammatory factors, and reducing the content of inflammatory factors, and / or; The instant soothing and repairing comprises at least one of inhibiting the production of TRPV1, inhibiting the calcium ion influx of HaCaT cells, reducing the skin tingling sensation, and reducing the skin erythema index.

9. Use according to claim 7, characterized in that, The product comprises at least one of a pharmaceutical product and a cosmetic product; and / or, The dosage form of the product comprises at least one of a cream, a liquid, a gel, a spray, an aerosol, a film, and a lyophilized agent; and / or, The mass content of the plant extract composition in the product is 0.05%-5% based on the total mass of the product.

Images