Application of fructus galangae extract in preparation of product for preventing or repairing skin light injury

Abstract

The invention discloses application of a fructus galangae extract in preparation of a product for preventing or repairing skin light injury. The effect of preventing or repairing the skin light injury at least comprises at least one of improvement of the activity of skin barrier related protein and up-regulation of the content of the skin barrier related protein. In addition, the product also has an instant soothing effect on light-damaged skin. The effect of preventing or repairing the skin light injury and the effect of instantly relieving the light injury skin are remarkably improved by purposefully selecting an extracting agent of the fructus galangae extract.

Description

Technical Field

[0001] This application belongs to the field of cosmetic technology, and in particular relates to the use of a cardamom extract in the preparation of products for preventing or repairing photodamage to the skin. Background Technology

[0002] Red cardamom is the dried, mature fruit of Alpinia galanga, a plant in the ginger family. Its main chemical components are volatile oils, flavonoids, diphenylheptane compounds, and glycosides. According to the pharmacopoeia, it can be used for abdominal pain due to cold, indigestion and bloating, vomiting and diarrhea.

[0003] Traditional extraction processes have significant technical limitations. The extracts are highly irritating to the skin and pose solvent safety issues. Currently, there are no reports of using cardamom extract to protect against photodamage to the skin. Summary of the Invention

[0004] This application provides new uses for cardamom extract in the field of dermatology.

[0005] Specifically, this application provides the use of cardamom extract in the preparation of products for preventing or repairing photodamage to the skin.

[0006] In any embodiment of this application, preventing or repairing photodamage to the skin includes at least one of increasing the activity of skin barrier-related proteins and upregulating the content of skin barrier-related proteins, including at least one of connective proteins, filaggrin, nautiloid proteins, epidermal proteins, and desmosomes.

[0007] In any embodiment of this application, skin barrier-related proteins include junction proteins, which include at least one of tight junction protein 1, closure protein 1, and closure protein 4.

[0008] In any embodiment of this application, the product also has an immediate soothing effect on photodamaged skin.

[0009] In any embodiment of this application, the cardamom extract includes an alcoholic extract of cardamom.

[0010] In any embodiment of this application, the extractant for the alcoholic extract of cardamom includes a 1,3-propanediol solution.

[0011] In any embodiment of this application, the mass concentration of the 1,3-propanediol solution is 75% to 100%.

[0012] In any embodiment of this application, the product includes at least one of pharmaceuticals and cosmetics.

[0013] In any embodiment of this application, the cosmetic also includes excipients, which include at least one of moisturizers, preservatives, and thickeners.

[0014] This application provides a use of cardamom extract, including its use in the preparation of products with the efficacy of preventing and repairing ultraviolet photodamage; the product also has an immediate soothing effect on photodamaged skin.

[0015] This application is the first to realize the synergistic application of cardamom extract in both protecting against and immediately soothing skin photodamage. It can not only effectively prevent skin photodamage and promote skin barrier repair after photodamage occurs, but also immediately soothe skin discomfort, achieving integrated protection and soothing repair. This application overcomes the limitations of existing technologies where cardamom extract has a single function and cannot comprehensively address the combined problems of skin sensitivity and photodamage, providing an innovative solution for the daily care of sensitive skin. 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 diagram illustrates the effects of various cardamom extracts from Examples 1 and 1-6 of this application on HaCaT cell viability (wherein, "#" indicates a significant difference compared to the blank control group, ####P<0.0001; "*" indicates a significant difference compared to the UVB group, *P<0.05).

[0018] **P<0.01, ***P<0.001, ***P<0.0001, “ns” indicates no significant difference compared to the UVB group; mean ± SD, n = 3).

[0019] Figure 2 This is a schematic diagram showing the effect of each cardamom extract from Example 1 and Comparative Examples 1-6 of this application on the ROS fluorescence intensity of HaCaT cells after UVB-induced damage (in the figure, "#" indicates a significant difference compared with the blank control group, ####P<0.0001; "*" indicates a significant difference compared with the UVB group, ****P<0.0001; "^" indicates a significant difference compared with the formulation "UVB+HDK80BEC" group, ^^^^P<0.0001; mean±SD, n=3).

[0020] Figure 3This is a schematic diagram showing the effect of each cardamom extract from Example 1 and Comparative Examples 1-6 of this application on the expression level of ZO-1 mRNA secreted by HaCaT cells after UVB-induced damage (in the figure, "#" indicates a significant difference compared with the blank control group, ###P<0.001; "*" indicates a significant difference compared with the UVB group, ****P<0.0001; "^" indicates a significant difference compared with the formulation "UVB+HDK80BEC" group, ^^^^P<0.0001; mean±SD, n=3).

[0021] Figure 4 This is a schematic diagram illustrating the effect of various cardamom extracts from Example 1 and Comparative Examples 1-6 of this application on the expression level of CLDN1 mRNA secreted by HaCaT cells after UVB-induced damage (in the figure, "#" indicates a significant difference compared to the blank control group, ###P<0.001; "*" indicates a significant difference compared to the blank control group).

[0022] The difference was statistically significant compared to the UVB group (**P<0.01, **P<0.0001), with "^" indicating a significant difference compared to the "UVB+HDK80BEC" formulation group (^^^^P<0.0001); mean ±

[0023] SD, n=3).

[0024] Figure 5 This is a schematic diagram illustrating the effect of each cardamom extract from Example 1 and Comparative Examples 1-6 of this application on the expression level of FLG mRNA secreted by HaCaT cells after UVB-induced damage (in the figure, "#" indicates a significant difference compared with the blank control group, ####P<0.0001; "*" indicates a significant difference compared with the UVB group, *P<0.05, ****P<0.0001; "ns" indicates no significant difference compared with the UVB group; "^" indicates a significant difference compared with the formulation "UVB+HDK80BEC", ^^^^P<0.0001; mean±SD, n=3).

[0025] Figure 6 This is a statistical graph showing the tail length of HaCaT cells after UVB irradiation by the various cardamom extracts of Examples 1 and Comparative Examples 1-6 of this application, as determined by single-cell gel electrophoresis. (In the graph, "#" indicates a significant difference compared to the blank control group (P < 0.0001), "ns" indicates no significant difference compared to the UVB group, "^" indicates a significant difference compared to the "UVB+HDK80BEC" formulation (P < 0.0001), and "*" indicates a significant difference compared to the UVB group.)

[0026] P<0.0001).

[0027] Figure 7This is a schematic diagram illustrating the effect of each cardamom extract from Example 1 and Comparative Examples 1-6 of this application on preventing UVB-induced TNF-α secretion by HaCaT cells (in the figure, "#" indicates a significant difference compared to the blank control group, ####P<0.0001; "*" indicates a significant difference compared to the UVB group, *P<0.05, ****P<0.0001; "^" indicates a significant difference compared to the formulation "UVB+HDK80BEC" group, ^^^^P<0.0001; mean±SD, n=3).

[0028] Figure 8 This is a schematic diagram illustrating the effect of various cardamom extracts from Examples 1 and Comparative Examples 1-6 of this application on preventing UVB-induced PEG2 secretion in HaCaT cells (in the figure, "#" indicates a significant difference compared to the blank control group, ####P<0.0001; "*" indicates a significant difference compared to the UVB group).

[0029] *P<0.05, ****P<0.0001, “^” indicates significant difference compared to the “UVB+HDK80BEC” group, ^^^^P<0.0001; mean±SD, n=3).

[0030] Figure 9 This is a schematic diagram showing the effect of each cardamom extract from Example 1 and Comparative Examples 1-6 of this application on NO secretion by HaCaT cells after UVB-induced damage (in the figure, "#" indicates a significant difference compared with the blank control group, ####P<0.0001; "*" indicates a significant difference compared with the UVB group, ***P<0.001, ***P<0.0001; "^" indicates a significant difference compared with the formulation "UVB+HDK80BEC" group, ^^^^P<0.0001; mean±SD, n=3).

[0031] Figure 10 This is a schematic diagram illustrating the effect of each cardamom extract from Example 1 and Comparative Examples 1-6 of this application on IL-6 secreted by HaCaT cells after UVB-induced damage (in the figure, "#" indicates a significant difference compared to the blank control group, ####P<0.0001; "*" indicates a significant difference compared to the UVB group, **P<0.01, ***P<0.001, ***P<0.0001; "^" indicates a significant difference compared to the formulation "UVB+HDK80BEC", ^^^^P<0.0001; mean±SD, n=3).

[0032] Figure 11This is a schematic diagram illustrating the effect of each cardamom extract from Example 1 and Comparative Examples 1-6 of this application on the expression of TRPV1 mRNA secreted by HaCaT cells after capsaicin stimulation (in the figure, "#" indicates a significant difference compared with the blank control group, ####P<0.0001; "*" indicates a significant difference compared with the UVB group, *P<0.05, ****P<0.0001; "^" indicates a significant difference compared with the formulation "UVB+HDK80BEC" group, ^^^^P<0.0001; mean±SD, n=3).

[0033] Figure 12 This is a schematic diagram showing the effect of each cardamom extract from Example 1 and Comparative Examples 1-6 of this application on the expression of TRPV1 protein secreted by HaCaT cells after capsaicin stimulation (in the figure, "#" indicates a significant difference compared with the blank control group, ####P<0.0001; "*" indicates a significant difference compared with the UVB group, ****P<0.0001; "^" indicates a significant difference compared with the formulation "UVB+HDK80BEC" group, ^^^^P<0.0001; mean±SD, n=3).

[0034] Figure 13 This is a schematic diagram showing the effect of various cardamom extracts from Examples 1 and Comparative Examples 1-6 of this application on the fluorescence intensity of intracellular calcium ions in HaCaT cells. Detailed Implementation

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] Red cardamom is the dried, mature fruit of *Alpinia galanga*, a plant in the ginger family. Its main chemical components are volatile oils, flavonoids, diphenylheptane compounds, and glycosides. Pharmacopoeia records its use for abdominal pain due to cold, indigestion, bloating, vomiting, and diarrhea. Currently, there are no reports of red cardamom extract being used to protect the skin from UV damage or to provide immediate soothing and repair.

[0041] In view of the deficiencies of the prior art, this application provides a new use for cardamom extract, which can prevent and repair photodamage to the skin and provide immediate soothing and repair for photodamaged skin.

[0042] Embodiments of this application provide the use of cardamom extract in the preparation of products for preventing or repairing photodamage to the skin.

[0043] In some implementations, preventing and repairing photodamage to the skin includes at least one of enhancing the activity of skin barrier-related proteins and increasing the content of skin barrier-related proteins.

[0044] In some embodiments, skin barrier-related proteins include at least one of connective proteins, filaggrin (FLG), smear proteins, epidermal proteins, and desmosomes.

[0045] In some embodiments, skin barrier-related proteins include junction proteins, which include at least one of tight junction protein 1 (ZO1 protein), closure protein 1 (CLDN1 protein), and closure protein 4 (CLDN4 protein).

[0046] In some implementations, products containing cardamom extract also have an immediate soothing effect on photodamaged skin.

[0047] In some embodiments, the cardamom extract includes an alcoholic extract of cardamom.

[0048] In some embodiments, the extractant for the cardamom alcohol extract includes a 1,3-propanediol solution.

[0049] In some embodiments, the mass concentration of the 1,3-propanediol solution is 75% to 100%.

[0050] The mass concentration of the 1,3-propanediol solution can be 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0051] In some implementations, the product includes at least one of pharmaceuticals and cosmetics.

[0052] In some embodiments, the cosmetic also includes excipients, which include at least one of humectants, preservatives, and thickeners.

[0053] Example

[0054] 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.

[0055] The sources of some of the reagents and equipment used in Example 1 and Comparative Examples 1-6 are as follows:

[0056] 1. HaCaT cells, manufacturer FuHeng, catalog number FH0186;

[0057] 2. PBS, manufacturer Gibco, catalog number 2002050, 500mL;

[0058] 3. Fetal bovine serum (FBS) manufacturer MRC, catalog number CCS30009.02, 500mL;

[0059] 4. Bispecific antibiotics (penicillin & streptomycin) Gibco15140122;

[0060] 5. DMEM, Gibco 11995-065 500mL;

[0061] 6. Trypsin-EDTA (0.25%), containing phenol red Thermo Fisher 25200114;

[0062] 7. Petri dish: Thermo Fisher 150464;

[0063] 8. 6-well plate, Thermo Fisher 140675;

[0064] 9. CCK-8 Admas life C8022-10000T;

[0065] 10. ROS probe DCFH DA (D6883);

[0066] 11. Merck, D6883-50mg;

[0067] 12. DMSO, Merck, 472301-500mL.

[0068] Example 1

[0069] Example 1 of this application provides a cardamom extract, the preparation method of which includes:

[0070] S1 Weigh out dried cardamom raw material, pulverize it, and pass it through a 50-mesh sieve to obtain cardamom powder.

[0071] S2 accurately weigh 1 kg of the cardamom powder, add 10 kg of 80% wt 1,3-propanediol aqueous solution, and mix well.

[0072] S3 extracts the mixture by heating it to 85°C and stirring it continuously at that temperature for 2 hours.

[0073] S4 Then, the system was cooled to 50°C and preliminarily filtered using a 200-mesh filter bag, and all the filtrate was collected.

[0074] S5. Add 1 wt% diatomaceous earth filter aid to the filtrate (based on the mass of the filtrate), stir thoroughly, and filter again until the filtrate is clear and transparent to obtain the cardamom extract.

[0075] Comparative Example 1

[0076] Comparative Example 1 of this application provides a cardamom extract, which differs from Example 1 only in the extractant in step S2. Specifically, 10 kg of 80% wt 1,3-propanediol aqueous solution is replaced with 10 kg of caprylic acid triglyceride (100 wt%).

[0077] Comparative Example 2

[0078] Comparative Example 2 of this application provides a cardamom extract, which differs from Example 1 only in the extractant in step S2. Specifically, 10 kg of 80% wt 1,3-propanediol aqueous solution is replaced with 10 kg of 80% wt glycerol aqueous solution.

[0079] Comparative Example 3

[0080] Comparative Example 3 of this application provides a cardamom extract, which differs from Example 1 only in the extractant in step S2. Specifically, 10 kg of 80% wt 1,3-propanediol aqueous solution is replaced with 10 kg of 100% wt glycerol aqueous solution.

[0081] Comparative Example 4

[0082] Comparative Example 4 of this application provides a cardamom extract, which differs from Example 1 only in the extractant in step S2. Specifically, 10 kg of 80% wt 1,3-propanediol aqueous solution is replaced with 10 kg of 100% wt 1,3-propanediol aqueous solution.

[0083] Comparative Example 5

[0084] Comparative Example 5 of this application provides a cardamom extract, which differs from Example 1 only in the extractant in step S2. Specifically, 10 kg of 80% wt 1,3-propanediol aqueous solution is replaced with 10 kg of 80% wt 1,3-butanediol aqueous solution.

[0085] Comparative Example 6

[0086] Comparative Example 6 of this application provides a cardamom extract, which differs from Example 1 only in the extractant in step S2. Specifically, 10 kg of 80% wt 1,3-propanediol aqueous solution is replaced with 10 kg of 100% wt 1,3-butanediol aqueous solution.

[0087] Table 1 summarizes the types of extractants and their concentration parameters used in Example 1 and Comparative Examples 1-6.

[0088] Table 1

[0089]

[0090]

[0091] Performance testing

[0092] The performance of the cardamom extracts prepared in Example 1 and Comparative Examples 1-6 was tested:

[0093] 1. Determination of DPPH free radical scavenging ability

[0094] 1.1 Reagent Preparation

[0095] (1) DPPH solution: Accurately weigh 3.2 mg of DPPH reagent and place it in a 5 mL volumetric flask. Add 4 mL of anhydrous ethanol and sonicate for 5 min (300 W, 40 kHz). Then vortex for 1 min to dissolve completely. Transfer the resulting solution to a brown reagent bottle and store it in a refrigerator at 4°C protected from light. Before use, dilute the above solution 10 times with anhydrous ethanol to prepare a 0.2 mM DPPH working solution. Continue to store it at 4°C protected from light. The shelf life is 24 h.

[0096] (2) Sample solution: Take the cardamom extract prepared in each example and comparative example, dilute it 25 times with deionized water, and then dilute it 2 times with anhydrous ethanol to obtain the test sample solution with a final concentration of 1%wt.

[0097] (3) Positive control solution: Weigh an appropriate amount of vitamin C standard, dissolve it in anhydrous ethanol and dilute to volume to prepare a vitamin C standard solution with a concentration of 100 μg / mL as a positive control.

[0098] 1.2 Determination Method

[0099] The determination was performed using the 96-well plate method, and the specific procedures are as follows:

[0100] Sample group: Add 100 μL of the test sample solution to the 96-well plate, then add 100 μL of DPPH working solution, mix well, and let stand in the dark for 30 min to react.

[0101] Sample control group: Add 100 μL of the test sample solution to a 96-well plate, then add 100 μL of anhydrous ethanol, mix well, and let stand in the dark for 30 min.

[0102] Blank control group (Control group): Add 100 μL of anhydrous ethanol to a 96-well plate, then add 100 μL of DPPH working solution, mix well, and let stand in the dark for 30 min.

[0103] After the reaction was completed, the absorbance of each group was measured at a wavelength of 540 nm using an ELISA reader. Each group was measured in triplicate, and the average value was taken as the final result.

[0104] 1.3 Calculation Formula

[0105] The scavenging rate of the sample against DPPH free radicals was calculated using the following formula:

[0106] Clearance rate (%) = [(Ac - (As - Ad)) / Ac] × 100%

[0107] In the formula:

[0108] Ac represents the average absorbance of the blank control group (100 μL anhydrous ethanol + 100 μL DPPH working solution);

[0109] As represents the average absorbance of the sample group (100 μL of test sample solution + 100 μL of DPPH working solution);

[0110] Ad represents the average absorbance of the sample control group (100 μL of test sample solution + 100 μL of anhydrous ethanol).

[0111] 1.4 Experimental Results

[0112] The DPPH free radical scavenging rate results of the cardamom extracts prepared in each example and comparative example and the positive control are shown in Table 2.

[0113] 2. Assay of the proliferative activity of HaCaT cells damaged by UVB

[0114] 2.1 Experimental Materials and Grouping

[0115] 2.1.1 Cell line: HaCaT cells (human immortalized keratinocytes) were cultured in DMEM complete medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin. The cells were passaged in a 37℃, 5% CO2 incubator, and cells in the logarithmic growth phase were used for experiments.

[0116] 2.1.2 Group Setup: The experiment was randomly divided into 3 groups, namely:

[0117] Blank control group (Control group): cultured in complete culture medium only, without UVB irradiation or drug treatment;

[0118] UVB irradiation group (model group): A cell damage model was established by UVB irradiation and cultured in complete culture medium only;

[0119] Red cardamom extract intervention group: After UVB irradiation, the group was treated with a 1 wt% red cardamom extract solution.

[0120] 2.2 Experimental Methods

[0121] 2.2.1 Cell Seeding and Culture: HaCaT cells in logarithmic growth phase were harvested and their density adjusted to 1×10⁻⁶ cells using complete culture medium. 5Cells / mL were seeded at 100 μL per well in a 96-well cell culture plate and cultured at 37°C in a 5% CO2 incubator. Further processing was carried out when the cell confluence reached about 70%.

[0122] 2.2.2 Pre-treatment: The corresponding concentration of cardamom extract solution (diluted with complete culture medium) was added to each well of the Cirsium japonicum extract intervention group, while the same amount of complete culture medium was added to the normal group and the UVB irradiation group. All groups were pre-cultured in a 37℃, 5% CO2 incubator for 24h.

[0123] 2.2.3 UVB Irradiation Treatment: Discard all culture medium from the 96-well plate, gently wash cells twice with PBS buffer, and add 100 μL of PBS to each well to cover the cell surface. Irradiate using a UVB phototherapy device (wavelength 311 nm) at a dose of 60 mJ / cm². 2 During irradiation, keep the culture plate horizontal to prevent cells from drying out.

[0124] 2.3 Culture and Detection: After irradiation, discard the PBS in the wells, add 100 μL of complete culture medium to each well, and continue incubation at 37℃, 5% CO2 for 24 h. Cell proliferation activity was detected using the CCK-8 assay: add 10 μL of CCK-8 reagent to each well, incubate in the dark for 2 h, and then measure the absorbance (OD value) of each well at 450 nm using a microplate reader. Each group had 6 replicates, and the experiment was repeated 3 times.

[0125] Cell proliferation rate calculation: With the normal group cell proliferation rate as 100%, the relative cell proliferation rate of each group is calculated using the following formula:

[0126] Relative proliferation rate (%) = (OD value of experimental group ÷ OD value of normal group) × 100%

[0127] 2.4 Experimental Results

[0128] The effects of different concentrations of cardamom extract on the proliferation and repair of HaCaT cells damaged by UVB are as follows: Figure 1 As shown.

[0129] 3. Measurement of intracellular ROS content in HaCaT cells damaged by UVB

[0130] 3.1 Experimental Materials and Grouping

[0131] Consistent with 2.1 above.

[0132] 3.2 Experimental Methods

[0133] Consistent with section 2.2 above.

[0134] 3.3 Subsequent Culture and ROS Detection: After irradiation, the PBS in the dishes was discarded, and 2 mL of complete culture medium was added to each dish. The dishes were then incubated at 37°C with 5% CO2 for 24 h. Intracellular ROS levels were detected by flow cytometry.

[0135] 3.4 Experimental Results

[0136] The effects of different treatment groups on intracellular ROS levels in HaCaT cells damaged by UVB are as follows: Figure 2 As shown.

[0137] 4. UVB-induced barrier function repair experiment in HaCaT cells

[0138] 4.1 Experimental Materials and Grouping

[0139] 4.1.1 Cell line: Same as HaCaT cells described in 2.1.1, with consistent culture conditions, and logarithmic growth phase cells were used for experiments.

[0140] 4.1.2 Reagents and Instruments: DMEM maintenance medium (serum-free), Trizol reagent, chloroform, isopropanol, 75% anhydrous ethanol (prepared with DEPC water), DEPC-ddH2O, Evo M-MLV reverse transcription premix kit, PCR primers (targeting barrier function-related genes and β-actin internal reference gene), micro UV spectrophotometer, PCR instrument, GraphPad Prism 8.0 software, etc.

[0141] 4.1.3 Group Setup: The experiment was randomly divided into 3 groups, namely:

[0142] Blank control group (Control group): Cells were cultured in DMEM maintenance medium only, wrapped in aluminum foil throughout the process to avoid ultraviolet radiation, and were not treated with drugs;

[0143] Model group: A cell barrier function damage model was established by UVB irradiation, and DMEM was added to the culture medium after irradiation for maintenance.

[0144] Sample group: After UVB irradiation, a solution of cardamom extract (concentration of 1wt%) diluted 100 times with DMEM maintenance medium was added for culture.

[0145] 4.2 Experimental Methods

[0146] 4.2.1 Cell Seeding and UVB Irradiation: HaCaT cells were seeded at 2.0 × 10⁻⁶ cells / day. 5Cells were seeded at a density of 10 cells / mL in 6-well plates, with 2 mL of cell suspension added to each well. The plates were then incubated at 37°C in a 5% CO2 incubator for 24 hours. After incubation, except for the control group which was completely wrapped in aluminum foil, the other two groups were irradiated with UVB lamps (wavelength 311 nm) at a dose of 0.1 J / cm². 2 .

[0147] 4.2.2 Subsequent culture: After irradiation, cells in each group were gently washed three times with PBS buffer. 2 mL of DMEM maintenance medium was added to each well in the blank and model groups, and 2 mL of cardamom extract solution (1 wt%) diluted 100 times with DMEM maintenance medium was added to each well in the sample groups. The cells were then incubated at 37°C in a 5% CO2 incubator for 24 h.

[0148] 4.3 Total RNA extraction:

[0149] After culture, carefully aspirate and discard the supernatant from each well. Add 1 mL of Trizol reagent to each well to fully lyse the cells. Transfer the lysate 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 rpm for 15 min at 4°C. Gently aspirate 400 μL of the supernatant to a new enzyme-free centrifuge tube, add an equal volume of isopropanol, invert and mix well, incubate at 4°C for 10 min, and centrifuge at 12000 rpm for 10 min at 4°C. Discard the supernatant, add 1 mL of 75% anhydrous ethanol to wash the RNA precipitate, centrifuge at 12000 rpm for 5 min at 4°C, 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.

[0150] 4.4 Real-time quantitative PCR detection:

[0151] After RNA concentration determination, 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 placed on ice for subsequent experiments or frozen for storage. The reverse-transcribed cDNA was diluted 10-fold, and the diluted cDNA was used as a template to prepare the PCR reaction system: 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. Using β-actin as an internal control, the relative expression level of the target gene mRNA was calculated using 2-ΔΔCt.

[0152] 4.5 Data Analysis: GraphPad Prism 8.0 software was used to perform statistical analysis on the experimental data. Quantitative data were expressed as mean ± standard deviation (x ± s), and one-way ANOVA was used for inter-group comparisons.

[0153] 4.6 Experimental Results

[0154] The effects of cardamom extract on the mRNA expression levels of genes related to barrier function in UVB-induced HaCaT cells are as follows: Figure 3 , Figure 4 , Figure 5 As shown.

[0155] 5. Experiments on the prevention of DNA damage

[0156] 5.1 Experimental Materials and Grouping

[0157] 5.1.1 Cell line: Same as HaCaT cells described in 2.1.1, with consistent culture conditions, and logarithmic growth phase cells were used for experiments.

[0158] 5.1.2 Reagents and Instruments: DMEM complete culture medium, 0.22μm microporous filter membrane, trypsin (0.25%), PBS buffer, cell lysis buffer, unwinding buffer, electrophoresis buffer, neutralization buffer, DNA staining reagent (such as EB or SYBR Green), 24-well cell culture plate, UVB irradiation instrument (wavelength 312nm), confocal microscope, etc.

[0159] 5.1.3 Group Setup: The experiment was randomly divided into 3 groups, namely:

[0160] Blank control group (Control group): Cells were cultured in DMEM complete medium only, without UVB irradiation or sample processing;

[0161] UVB damage group: A DNA damage model was established by UVB irradiation of cells. Cells were cultured in DMEM complete medium before and after irradiation without adding any samples.

[0162] Sample pretreatment group: Cells were pretreated with cardamom extract sample solution and then irradiated with UVB, followed by continued culture.

[0163] 5.2 Experimental Methods

[0164] 5.2.1 Sample solution preparation: The cardamom extracts prepared in each example and comparative example were diluted 100 times with DMEM complete culture medium (concentration of 1wt%), filtered through a 0.22μm microporous membrane for sterilization, and stored in a refrigerator at 4℃ in the dark for later use.

[0165] 5.2.2 Cell Seeding and Culture: HaCaT cells were seeded at a rate of 1×10⁻⁶ cells / cells. 5 The cells were seeded at a density of 1 cell / mL in 24-well cell culture plates, with 1 mL of cell suspension added to each well. The plates were then incubated at 37°C in a 5% CO2 cell culture incubator for 24 hours. After the cells had fully adhered to the plate, further processing was performed.

[0166] 5.2.3 Sample pretreatment: 1 mL of the prepared sample diluent was added to each well of the sample pretreatment group, and 1 mL of DMEM complete culture medium was added to each well of the blank control group and the UVB damage group. All groups were pretreated in a 37℃, 5% CO2 incubator for 2 h.

[0167] 5.2.4 UVB Irradiation Treatment: After pretreatment, the culture medium for each group was discarded. Cells in the UVB-damaged group and the sample pretreatment group were gently washed once with PBS buffer. 0.5 mL of PBS was added to each well to cover the cell surface. Irradiation was then performed using a UVB irradiator (wavelength 312 nm) at a dose of 5 J / cm². 2 The normal control group was wrapped in aluminum foil throughout the process to avoid ultraviolet radiation.

[0168] 5.2.5 Subsequent Culture and Cell Collection: After irradiation, discard the PBS in each well, add 1 mL of DMEM complete medium to each well, and continue culturing in a 37℃, 5% CO2 incubator for 20 h. After culture, aspirate the medium, add 0.5 mL of trypsin to each well, and incubate for 3-5 min to digest. After the cells have completely detached from the cell wall, add 0.5 mL of DMEM complete medium to stop digestion, gently pipette to prepare a single-cell suspension, transfer to a 1.5 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 1 mL of PBS to resuspend the cells, and set aside for later use.

[0169] 5.3 DNA Damage Detection

[0170] After cell lysis, unwinding, electrophoresis, neutralization, and staining, the above cell suspension was observed and photographed under a confocal microscope.

[0171] 5.4 Experimental Results

[0172] The preventive effect of cardamom extract pretreatment on UVB-induced DNA damage in HaCaT cells is as follows: Figure 6 As shown.

[0173] 6. Experiment on the ability of HaCaT cells to prevent UVB-induced inflammatory damage

[0174] 6.1 Experimental Materials and Grouping

[0175] 6.1.1 Cell line: Same as HaCaT cells described in 2.1.1, with consistent culture conditions, and logarithmic growth phase cells were used for experiments.

[0176] 6.1.2 Reagents and Instruments: DMEM maintenance medium (serum-free), TNF-α ELISA kit, PGE2 ELISA kit, PBS buffer, 24-well cell culture plate, UVB irradiation instrument, microplate reader, GraphPad Prism 8.0 software, etc.

[0177] 6.1.3 Group Setup: The experiment was randomly divided into 3 groups, namely:

[0178] Blank control group (Control group): Cells were cultured in DMEM maintenance medium only, wrapped in aluminum foil throughout the process to avoid ultraviolet radiation, and no sample processing was performed;

[0179] Model group: Cells were irradiated with UVB to establish an inflammatory injury model. They were cultured in DMEM maintenance medium before and after irradiation, without adding any samples.

[0180] Sample group: Cells were pretreated with cardamom extract sample solution and then irradiated with UVB, and then cultured for a period of time.

[0181] 6.2 Experimental Methods

[0182] 6.2.1 Sample solution preparation: The cardamom extracts prepared in each example and comparative example were diluted 100 times with DMEM maintenance medium (concentration of 1wt%), filtered through a 0.22μm microporous membrane for sterilization, and stored in a refrigerator at 4℃ in the dark for later use.

[0183] 6.2.2 Cell Seeding and Culture: HaCaT cells were seeded at a density of 2.0 × 10⁶ cells / cell. 5 Cells were seeded at a density of cells / mL in 24-well cell culture plates, with 500 μL of cell suspension added to each well. The plates were then incubated at 37°C in a 5% CO2 cell culture incubator for 24 h. After the cells had fully adhered to the plate, further processing was performed.

[0184] 6.2.3 Sample pretreatment and UVB irradiation:

[0185] After the culture is completed, discard the culture medium from each well and gently wash the cells three times with PBS buffer.

[0186] Add 500 μL of DMEM maintenance medium to each well of the blank control group and the model group, and add 500 μL of the above-prepared sample dilution to each well of the sample group. All groups were pretreated in a 37℃, 5% CO2 incubator for 2 h.

[0187] After pretreatment, except for the blank control group which was wrapped in tin foil, the model group and sample group were irradiated with a UVB irradiator (wavelength 312nm) at a dose of 2.5J / cm². 2 .

[0188] 6.2.4 Subsequent Culture and Supernatant Collection: After irradiation, all culture plates were placed in a 37℃, 5% CO2 incubator for 24 hours. After culture, the supernatant from each well was carefully aspirated and transferred to a 1.5mL centrifuge tube. The tube was centrifuged at 3000r / min for 10 minutes at 4℃. The supernatant was then transferred to a new centrifuge tube and stored at -80℃ for later use in the detection of inflammatory factors.

[0189] 6.2.5 Inflammatory factor detection

[0190] The levels of TNF-α and PGE2 were detected using ELISA.

[0191] 6.3 Data Analysis: GraphPad Prism 8.0 software was used to perform statistical analysis on the experimental data. Quantitative data were expressed as mean ± standard deviation (x ± s), and one-way ANOVA was used for inter-group comparisons.

[0192] 6.4 Experimental Results

[0193] The effects of cardamom extract on UVB-induced secretion of inflammatory factors TNF-α and PGE2 in HaCaT cells are as follows: Figure 7 , Figure 8 As shown.

[0194] 7. Experiment on inhibiting UVB-induced inflammatory damage (repair) in HaCaT cells

[0195] 7.1 Experimental Materials and Grouping

[0196] 7.1.1 Cell line: Same as HaCaT cells described in 2.1.1, with consistent culture conditions, and logarithmic growth phase cells were used for experiments.

[0197] 7.1.2 Reagents and Instruments: DMEM maintenance medium (serum-free), TNF-α ELISA kit, PGE2 ELISA kit, PBS buffer, 24-well cell culture plate, UVB irradiation instrument, microplate reader, GraphPadPrism 8.0 software, etc.

[0198] 7.1.3 Group Setup: The experiment was randomly divided into 3 groups, namely:

[0199] Blank control group (Control group): Cells were cultured in DMEM maintenance medium only, wrapped in aluminum foil throughout the process to avoid ultraviolet radiation, and no sample processing was performed;

[0200] Model group: Cells were irradiated with UVB to establish an inflammatory injury model. After irradiation, they were cultured in DMEM maintenance medium only, without adding any samples.

[0201] Sample repair group: After UVB irradiation, cells were treated with thistle extract sample solution to assess their ability to repair inflammatory damage.

[0202] 7.2 Experimental Methods

[0203] 7.2.1 Sample solution preparation: The cardamom extracts prepared in each example and comparative example were diluted 100 times with DMEM maintenance medium (concentration of 1wt%), filtered through a 0.22μm microporous membrane for sterilization, and stored in a refrigerator at 4℃ in the dark for later use.

[0204] 7.2.2 Cell Seeding and Culture: HaCaT cells were seeded at a density of 2.0 × 10⁶ cells / cell. 5 Cells were seeded at a density of cells / mL in 24-well cell culture plates, with 500 μL of cell suspension added to each well. The plates were then incubated at 37°C in a 5% CO2 cell culture incubator for 24 h. After the cells had fully adhered to the plate, further processing was performed.

[0205] 7.2.3 UVB Irradiation Treatment: After the culture period, except for the blank control group which was wrapped in tin foil, the model group and the sample repair group were irradiated with a UVB irradiator at a dose of 0.5 J / cm². 2 .

[0206] 7.2.4 Sample repair treatment: After irradiation, the residual liquid in each well was aspirated and the cells were gently washed three times with PBS buffer. 500 μL of the prepared sample dilution solution was added to each well of the sample repair group, and 500 μL of DMEM maintenance medium was added to each well of the blank control group and the model group. All groups were placed in a 37℃, 5% CO2 incubator for 24 h to achieve the repair effect of the samples on UVB-induced inflammatory damage.

[0207] 7.3 Inflammatory factor detection

[0208] 7.3.1 NO content detection (Griess method): Mix 50 μL of supernatant with 50 μL of Griess reagent, incubate at room temperature in the dark for 10 min, and measure the absorbance value at a wavelength of 540 nm using an ELISA reader. Calculate the NO concentration according to the sodium nitrite standard curve.

[0209] 7.3.2 Cytokine detection (ELISA method): Follow the instructions of the IL-6 ELISA kit, and follow the same steps as the inflammatory factor detection in 6.2.5. Calculate the cytokine concentration, set up 3 replicates for each group, and repeat the experiment 3 times.

[0210] 7.4 Data Analysis

[0211] Statistical analysis of the experimental data was performed using GraphPad Prism 8.0 software. Quantitative data were expressed as mean ± standard deviation (x ± s), and one-way ANOVA was used for inter-group comparisons.

[0212] 7.5 Experimental Results

[0213] The repair effect of cardamom extract on UVB-induced inflammatory damage to HaCaT cells (indicating NO and IL-6 secretion levels) is as follows: Figure 9 , Figure 10 As shown.

[0214] 8. Experiment on TRPV1 stimulation in HaCaT cells by capsaicin

[0215] 8.1 Experimental Materials and Grouping

[0216] 81.1 Cell line: Same as HaCaT cells described in 2.1.1, with consistent culture conditions, and logarithmic growth phase cells were used for experiments.

[0217] 8.1.2 Reagents and Instruments: DMEM maintenance medium (serum-free), capsaicin (purity ≥95%), Griess reagent, IL-6 ELISA kit, IL-8 ELISA kit, PGE2 ELISA kit, Trizol reagent, chloroform, isopropanol, 75% anhydrous ethanol (prepared with DEPC water), DEPC-ddH2O, Evo M-MLV reverse transcription premix kit, PCR primers (targeting TRPV1 gene and β-actin internal reference gene), 24-well cell culture plates, 6-well cell culture plates, micro-ultraviolet spectrophotometer, PCR instrument, microplate reader, GraphPad Prism 8.0 software, etc.

[0218] 8.1.3 Group Setup: The experiment was randomly divided into 3 groups, namely:

[0219] Blank control group (Control group): Cells were cultured in DMEM maintenance medium only, without the addition of capsaicin or samples;

[0220] Model group: A TRPV1 activation model was established by stimulating cells with capsaicin and cultured in DMEM maintenance medium only, without adding any samples;

[0221] Sample group: After cells were stimulated with capsaicin, they were treated with a sample solution of cardamom extract to evaluate its effect on TRPV1-related responses.

[0222] 8.2 Experimental Methods

[0223] 8.2.1 Preparation of sample solution and capsaicin solution:

[0224] Sample solution: Take the cardamom extract prepared in each example and comparative example, dilute it to a concentration of 1 wt% with DMEM maintenance medium, filter it through a 0.22 μm microporous membrane for sterilization, and store it at 4°C in the dark for later use.

[0225] Capsaicin solution: Accurately weigh capsaicin, dissolve it in anhydrous ethanol, and then dilute it with DMEM maintenance medium to prepare a capsaicin working solution with a final concentration of 20 μM. Prepare and use immediately.

[0226] 8.2.2 Cell Seeding and Pretreatment:

[0227] 24-well plate seeding: HaCaT cells were seeded at a rate of 1 × 10⁻⁶ cells / well. 5 The cells were seeded at a density of 1 cell / mL in 24-well cell culture plates, with 500 μL of cell suspension added to each well. The plates were then incubated at 37°C in a 5% CO2 cell culture incubator for 24 hours until the cells were fully attached to the plate.

[0228] 6-well plate seeding: HaCaT cells were seeded at a rate of 1 × 10⁻⁶ cells / well. 5 The cells were seeded at a density of 1 cell / mL in 6-well cell culture plates, with 2 mL of cell suspension added to each well, and cultured for 24 h under the same conditions.

[0229] After the culture was completed, both culture plates were gently washed three times with PBS buffer to remove residual culture medium.

[0230] 8.2.3 Capsaicin Irritation and Sample Processing:

[0231] Blank control group: 500 μL of DMEM maintenance medium was added to each well of a 24-well plate, and 2 mL of DMEM maintenance medium was added to each well of a 6-well plate.

[0232] Model group and sample group: 500 μL of capsaicin solution with a final concentration of 20 μM was added to each well of a 24-well plate, and 2 mL of capsaicin solution with a final concentration of 20 μM was added to each well of a 6-well plate. The plates were incubated at 37°C and 5% CO2 for 2 h.

[0233] After incubation, the sample group was replaced with DMEM maintenance medium containing cardamom extract, i.e., the sample solution prepared above (500 μL per well of a 24-well plate and 2 mL per well of a 6-well plate), and the model group was replaced with an equal amount of DMEM maintenance medium. Both groups were then incubated for another 24 h under the same conditions.

[0234] 8.3 Collection and Detection of Supernatant (24-well plate):

[0235] After incubation, carefully aspirate the supernatant from each well of the 24-well plate and transfer it to a 1.5 mL centrifuge tube. Centrifuge at 3000 rpm for 10 min at 4 °C. Transfer the supernatant to a new centrifuge tube and freeze at -80 °C for later use.

[0236] 8.4 Total RNA Extraction

[0237] Discard the supernatant from each well of the 6-well plate, add 1 mL of Trizol reagent to each well to lyse the cells, and then perform the same total RNA extraction steps as in 4.3 to determine the RNA concentration and purity.

[0238] 8.5 Real-time quantitative PCR detection:

[0239] After RNA concentration determination, 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 placed on ice for subsequent experiments or frozen for storage. The resulting cDNA was diluted 10-fold, and PCR was configured using the diluted cDNA as a template. The reaction conditions were: 95°C pre-denaturation for 30 s, 95°C denaturation for 15 s, 60°C annealing and extension for 30 s, for 40 cycles. β-actin was used as an internal control, and the relative expression level of the target gene mRNA was calculated using 2-ΔΔCt.

[0240] 8.6 Data Analysis:

[0241] Statistical analysis was performed using GraphPad Prism 8.0 software. Quantitative data were expressed as mean ± standard deviation (x ± s), and one-way ANOVA was used for inter-group comparisons.

[0242] 8.7 Experimental Results

[0243] The effects of cardamom extract on TRPV1 gene expression in capsaicin-stimulated HaCaT cells are as follows: Figure 11-12 As shown.

[0244] 9. Experiment on calcium ion influx in HaCaT cells

[0245] 9.1 Experimental Materials and Grouping

[0246] 9.1.1 Cell line: Same as HaCaT cells described in 2.1.1, with consistent culture conditions, and logarithmically growing cells were used for experiments.

[0247] 9.1.2 Reagents and Instruments: DMEM culture medium, DMSO (analytical grade), capsaicin (Cap, purity ≥95%), Fluo-4AM fluorescent probe, PBS buffer, black transparent 96-well plate, 0.22μm microporous filter membrane, fluorescent microplate reader, etc.

[0248] 9.1.3 Group Setup: The experiment was randomly divided into 4 groups, namely:

[0249] Blank control group (Control group): Cells were cultured in DMEM medium only, without the addition of capsaicin or other samples;

[0250] Model group: Cells were treated with 20 μM capsaicin to induce calcium ion influx, without adding any sample;

[0251] Sample group: Cells were pretreated with diluted sample solutions from each example and comparative example, and then treated with 20 μM capsaicin to evaluate the effect of the samples on calcium ion influx.

[0252] 9.2 Preparation of Experimental Solutions

[0253] 9.2.1 Capsaicin solution:

[0254] Storage solution: Accurately weigh 50 mg capsaicin, add 1.673 mL DMSO to dissolve, prepare a 100 mM capsaicin storage solution, dispense into sterile centrifuge tubes, and store at 4°C in the dark for later use.

[0255] Working solution: When ready to use, dilute the capsaicin storage solution with DMEM medium to prepare a capsaicin working solution with a final concentration of 20 μM. Prepare and use immediately.

[0256] 9.2.2 Sample solution:

[0257] Stock solution: Take an appropriate amount of each example and comparative sample, add 9 times the volume of DMEM culture medium at a ratio of 1:9, dilute 10 times and mix thoroughly, filter through a 0.22μm microporous membrane for sterilization, and store at 4℃ in the dark for later use.

[0258] 9.2.3 Fluo-4 AM working solution: Take the Fluo-4 AM fluorescent probe, dilute it with PBS buffer to a final concentration of 0.5 μM, store it in the dark, and prepare it fresh each time.

[0259] 9.3 Experimental Methods

[0260] 9.3.1 Cell Seeding and Culture: HaCaT cells were seeded at a concentration of 1.0 × 10⁶ cells / cell. 5 The cells were seeded at a density of 100 μL / mL in black transparent 96-well plates, and 100 μL of cell suspension was added to each well. The plates were then incubated at 37°C in a 5% CO2 cell culture incubator for 24 h until the cells were fully attached to the plate.

[0261] 9.3.2 Sample Pretreatment: After culture, the cells were gently washed twice with PBS buffer to remove residual culture medium. Sample groups 1-3 were added with the corresponding concentration of sample working solution (100 μL per well), and the blank control group and model group were added with an equal volume of DMEM culture medium. The samples were incubated at 37℃ and 5% CO2 for 6 h.

[0262] 9.3.3 Fluorescent probe loading: After incubation, aspirate the culture medium from each well and gently wash the cells three times with PBS buffer; add 100 μL of Fluo-4 AM working solution (final concentration 0.5 μM) to each well and incubate at 37°C in the dark for 30 min to allow the probe to fully enter the cells.

[0263] 9.3.4 Probe transformation and washing: After incubation, wash the cells three times with PBS buffer to remove free probes that have not entered the cells; then continue to incubate in the dark for 20 min at 37℃ and 5% CO2 to ensure that Fluo-4 AM in the cells is completely hydrolyzed into Fluo-4 (active fluorescent form).

[0264] 9.3.5 Capsaicin stimulation and fluorescence detection: Add capsaicin working solution (model group and sample group) with a final concentration of 20 μM to each well, and add an equal volume of PBS buffer to the blank control group; immediately place the 96-well plate in a fluorescence microplate reader and detect the dynamic changes in fluorescence intensity within 2 min. The detection parameters are: excitation wavelength 488 nm, emission wavelength 529 nm, and the fluorescence value is recorded every 10 s. Each group has 6 replicate wells, and the experiment is repeated 3 times.

[0265] 9.5 Experimental Results

[0266] The effects of different concentrations of samples from various examples and comparative examples on capsaicin-induced calcium ion influx in HaCaT cells are as follows: Figure 12 As shown.

[0267] Experimental results

[0268] 1. Results of DPPH free radical scavenging capacity measurement

[0269] Table 2 shows that all cardamom extracts have the ability to inhibit DPPH free radicals.

[0270] Table 2 Results of DPPH scavenging ability of extracts

[0271]

[0272]

[0273] 2. Results of UVB-damaged HaCaT cell proliferation activity assay

[0274] Figure 1The experimental results showed that the cardamom extract had no cytotoxicity on HaCaT cells. Among them, compared with the HDKOIL, HDK80GY, HDK100GY, HDK100BEC, HDK80DEC and HDK100DEC groups, the cardamom extract HDK80BEC could significantly promote the proliferation of HaCaT cells.

[0275] 3. Results of ROS content measurement in HaCaT cells damaged by UVB

[0276] All red cardamom extracts can reduce the production of ROS in HaCaT cells stimulated by UVB. Among them, the red cardamom extract HDK80BEC has the best effect, which is significantly better than HDKOIL, HDK80GY, HDK100GY, HDK100BEC, HDK80DEC and HDK100DEC groups.

[0277] 4. Results of UVB-induced barrier function repair in HaCaT cells

[0278] Figure 3 , Figure 4 , Figure 5 Experimental results showed that cardamom extract could increase the mRNA expression levels of skin barrier-related proteins ZO-1, CLDN1, and FLG proteins after UVB stimulation of HaCaT cells. Among them, cardamom extract HDK80BEC showed the best effect, which was significantly better than HDKOIL, HDK80GY, HDK100GY, HDK100BEC, HDK80DEC, and HDK100DEC groups.

[0279] 5. Experimental results on prevention of DNA damage

[0280] Figure 6 Experimental results showed that after UVB irradiation of HaCaT cells, the average cell tail length in the model group was significantly increased compared with the control group, and the UVB damage model was successfully established. The addition of cardamom extract HDK80BEC reduced the average tail length of HaCaT cells, indicating that cardamom extract has a significant protective effect on UVB-irradiated HaCaT cells and can reduce DNA damage after UVB irradiation. Cardamom extract HDK80BEC showed the best effect in preventing DNA damage, significantly superior to the HDKOIL, HDK80GY, HDK100GY, HDK100BEC, HDK80DEC, and HDK100DEC groups.

[0281] As can be seen from the experimental results 1-3 and 5 above, in terms of anti-oxidation and UV protection, cardamom extract has the ability to inhibit DPPH free radicals, indicating that it has excellent antioxidant activity. In the cell model, cardamom extract can effectively reduce the production of ROS in HaCaT cells stimulated by UVB, thereby alleviating oxidative stress damage and further reducing DNA damage caused by UVB irradiation, thus combating photodamage through a dual approach of oxidative defense and gene integrity protection.

[0282] 6. Experimental results on the ability of HaCaT cells to prevent UVB-induced inflammatory damage.

[0283] Figure 7 , Figure 8 Experimental results showed that the cardamom extract HDK80BEC could prevent inflammatory damage to HaCaT cells caused by UVB irradiation and significantly reduce the secretion of inflammatory factors TNF-α and PGE2. The cardamom extract HDK80BEC had the best effect in reducing the secretion of inflammatory factors TNF-α and PGE2, which was significantly better than the HDKOIL, HDK80GY, HDK100GY, HDK100BEC, HDK80DEC and HDK100DEC groups.

[0284] 7. Experimental results on inhibiting UVB-induced inflammatory damage (repair) in HaCaT cells

[0285] Figure 9 , Figure 10 Experimental results showed that after UVB irradiation of HaCaT cells, the inflammation-related indicators in the model group were significantly increased compared with the control group, and the UVB damage model was successfully established. The addition of cardamom extract HDK80BEC significantly reduced the production of inflammatory factors NO and IL-6. HDK80BEC showed the best effect in reducing the production of NO and IL-6, significantly superior to the HDKOIL, HDK80GY, HDK100GY, HDK100BEC, HDK80DEC, and HDK100DEC groups.

[0286] As can be seen from the experimental results 4, 6-7 above, cardamom extract exhibits outstanding barrier-enhancing function in terms of barrier repair and anti-inflammatory regulation. It can significantly upregulate the expression of tight junction key proteins ZO-1 and CLDN1, as well as differentiation marker protein FLG, at the mRNA level in HaCaT cells after UVB stimulation, thereby strengthening the physical barrier structure of the skin. At the same time, it can also effectively reduce the production and content of inflammatory factors TNF-α, IL-6, PGE2, and NO, and alleviate skin inflammatory responses induced by ultraviolet radiation and immune stress by inhibiting inflammatory pathways through multiple targets.

[0287] 8. Results of the experiment on TRPV1 in HaCaT cells stimulated by capsaicin

[0288] Figure 11-12 Experimental 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. The addition of cardamom extract HDK80BEC significantly reduced TRPV1 expression at both the mRNA and protein levels in HaCaT cells stimulated by capsaicin. HDK80BEC showed the best inhibitory effect on TRPV1 in HaCaT cells, significantly superior to the HDKOIL, HDK80GY, HDK100GY, HDK100BEC, HDK80DEC, and HDK100DEC groups.

[0289] Figure 13 Experimental results showed that capsaicin stimulation of HaCaT cells significantly increased calcium ion influx, indicating successful model establishment. The addition of cardamom extract HDK80BEC significantly inhibited calcium ion influx after capsaicin stimulation of HaCaT cells. HDK80BEC showed the best effect in inhibiting calcium ion influx after capsaicin stimulation of HaCaT cells, significantly superior to the HDKOIL, HDK80GY, HDK100GY, HDK100BEC, HDK80DEC, and HDK100DEC groups.

[0290] As can be seen from the above experimental results 8, cardamom extract also has a transient receptor potential regulation effect, which can reduce the expression of TRPV1 at the mRNA and protein levels in HaCaT cells after capsaicin stimulation, and effectively inhibit the calcium ion influx induced by capsaicin, thereby blocking the pain and burning signal transmission pathway. This provides a solid mechanistic basis for its application in relieving skin sensitivity, burning and stinging.

[0291] Therefore, the cardamom extract provided in this application can exhibit synergistic effects in terms of UV protection and immediate soothing. It can not only effectively prevent skin inflammation and cell damage caused by photodamage, but also promote the repair of skin barrier and cell function and reduce the secretion of inflammatory factors after photodamage occurs. It can also provide immediate soothing for skin discomfort such as redness and burning.

[0292] The above description is merely a specific implementation of this application. Those skilled in the art will clearly understand that, for the sake of convenience and brevity, the specific working process described above can be referred to the corresponding process in the foregoing method embodiments, and will not be repeated here. It should be understood that the protection scope of this application is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in this application, and these modifications or substitutions should all be covered within the protection scope of this application.

Claims

1. Use of cardamom extract in the preparation of products for preventing or repairing photodamage to the skin.

2. The use according to claim 1, characterized in that, The prevention or repair of photodamage to the skin includes at least one of increasing the activity of skin barrier-related proteins and upregulating the content of skin barrier-related proteins.

3. The use according to claim 2, characterized in that, The skin barrier-related proteins include at least one of the following: connective proteins, filaggrin, smear proteins, epidermal proteins, and desmosomes.

4. The use according to claim 3, characterized in that, The junctional proteins include at least one of tight junction protein 1, closure protein 1, and closure protein 4.

5. The use according to claim 1, characterized in that, The product also provides immediate relief for sun-damaged skin.

6. The use according to claim 1, characterized in that, The cardamom extract includes alcoholic extracts of cardamom.

7. The use according to claim 6, characterized in that, The extractant for the alcoholic extract of cardamom includes a 1,3-propanediol solution.

8. The use according to claim 1, characterized in that, The mass concentration of the 1,3-propanediol solution is 75% to 100%.

9. The use according to claim 1, characterized in that, The product includes at least one of pharmaceuticals and cosmetics.

10. The use according to claim 1, characterized in that, The cosmetic also includes excipients, which include at least one of moisturizers, preservatives, and thickeners.

Images