Application of campsis grandiflora extract

Abstract

The invention discloses an application of a campsis grandiflora extract, which at least comprises one of (1)-(3): (1) an application in preparation of a product for preventing ultraviolet light damage, and prevention of the ultraviolet light damage comprises prevention of skin light damage caused by UVB radiation; (2) application in preparation of a product for repairing ultraviolet light damage, wherein the step of repairing the ultraviolet light damage comprises performing after-sun repair on skin light damage caused by UVB radiation; and (3) application in preparation of a product capable of soothing the skin instantly. According to the application, collaborative application of the campsis grandiflora extract on dual effects of ultraviolet protection and instant soothing is realized for the first time, skin light injury caused by UVB can be effectively prevented, skin barrier repair after injury is promoted, an instant soothing effect is provided, and comprehensive requirements of skin on ultraviolet protection and soothing repair are met.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cosmetics, and particularly relates to a use of campsis grandiflora extract. BACKGROUND

[0002] Campsis grandiflora is the dried flower of Campsis grandiflora or Campsis radicans, and the chemical components mainly include flavonoids, phenylpropanoids, organic acids and volatile oils, etc. According to the Pharmacopoeia, it can be used for blood stasis and abdominal mass, traumatic injury, wind rashes and redness, and skin itching.

[0003] The traditional extraction process has significant technical limitations, the extraction agent has great irritation to the skin, and there are solvent safety problems. Currently, there is no related report on the use of campsis grandiflora extract to prevent and repair skin UV light damage and provide immediate soothing effect. SUMMARY

[0004] The embodiments of the present application provide a new use of campsis grandiflora extract, which can effectively prevent or repair UVB-induced skin light damage and provide immediate soothing effect.

[0005] In any embodiment of the present application, the use of campsis grandiflora extract at least includes one of (1) to (3): (1) the use in preparing a product for preventing UV light damage, the prevention of UV light damage including the prevention of skin light damage caused by UVB radiation; (2) the use in preparing a product for repairing UV light damage, the repair of UV light damage including post-sun repair of skin light damage caused by UVB radiation; and (3) the use in preparing a product capable of immediately soothing the skin.

[0006] In any embodiment of the present application, the extraction agent of campsis grandiflora extract includes propylene glycol, preferably, the extraction agent of campsis grandiflora extract includes 1,3-propylene glycol.

[0007] In any embodiment of the present application, the mass concentration of the extraction agent is 25% to 95%.

[0008] In any embodiment of the present application, the prevention of UV light damage includes up-regulating the expression level of skin barrier-related proteins; and / or, the repair of UV light damage includes up-regulating the expression level of skin barrier-related proteins.

[0009] In any embodiment of the present application, the prevention of UV light damage includes down-regulating the expression level of inflammatory factors; and / or, the repair of UV light damage includes down-regulating the expression level of inflammatory factors.

[0010] In any embodiment of the present application, the inflammatory factors include at least one of pro-inflammatory cytokines, prostaglandin cytokines and oxidative stress-related factors.

[0011] In any embodiment of the present application, the proinflammatory cytokine comprises at least one of tumor necrosis factor-alpha, interleukin-1 beta, interleukin-6 and interleukin-8.

[0012] In any embodiment of the present application, the prostaglandin cytokine comprises prostaglandin E2.

[0013] In any embodiment of the present application, the product comprises a cosmetic product.

[0014] In any embodiment of the present application, the dosage form of the cosmetic product comprises a cream, a paste, a milk, a patch, a mask.

[0015] In any embodiment of the present application, the product comprises a pharmaceutical product, and the dosage form of the pharmaceutical product comprises a tablet, a capsule, a granule, a pill, an oral solution, an emulsion, a dry suspension, a dry extract, an injection.

[0016] The present application realizes, for the first time, the synergistic application of campsis grandiflora extract in the field of ultraviolet light damage prevention, which integrates the triple effects of prevention, repair and instant relief. The extract not only can effectively prevent skin light damage caused by UVB, but also can promote skin barrier repair after damage and can immediately relieve the discomfort of the skin. This application breaks through the limitation of single effect of campsis grandiflora extract in the prior art, provides a new solution for solving the compound problem of light damage and skin sensitivity, and shows important application value in the daily care of sensitive skin. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiments of the present application will be briefly introduced. For those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0018] Figure 1 is a schematic diagram of the influence of each campsis grandiflora extract of embodiment 1 and comparative examples 1-9 on the activity of HaCaT cells (wherein "#" in the figure represents significant difference compared with the blank group,

[0019] ####P<0.0001; "*" represents significant difference compared with the UVB group, **P<0.01, ***P<0.001, ****P<0.0001, "ns" represents no significant difference compared with the blank group;

[0020] mean±SD, n=3).

[0021] Figure 2Figure 1 is a graph showing the effect of each of the Campsis grandiflora extracts of Example 1 and Comparative Examples 1-9 on the ROS fluorescence intensity of HaCaT cells after UVB-induced damage (in the figure, "#" represents a significant difference compared to the blank group, ###P<0.001; "*" represents a significant difference compared to the UVB group, ****P<0.0001, "^" represents a significant difference compared to the formulation "UVB+LXH30BEC" group, ^^^^P<0.0001; mean±SD, n=3).

[0022] Figure 3 Figure 2 is a graph showing the effect of each of the Campsis grandiflora extracts of Example 1 and Comparative Examples 1-9 on the ZO-1 mRNA expression level secreted from HaCaT cells after UVB-induced damage (in the figure, "#" represents a significant difference compared to the blank group, ###P<0.001; "*" represents a significant difference compared to the UVB group, **P<0.01, ***P<0.001, ****P<0.0001, "^" represents a significant difference compared to the formulation "UVB+LXH30BEC" group, ^^^^P<0.0001, "ns" represents no significant difference compared to the UVB group; mean±SD, n=3).

[0023] Figure 4 Figure 3 is a graph showing the effect of each of the Campsis grandiflora extracts of Example 1 and Comparative Examples 1-9 on the CLDN1 mRNA expression level secreted from HaCaT cells after UVB-induced damage (in the figure, "#" represents a significant difference compared to the blank group, ###P<0.001; "*" represents a significant difference compared to the UVB group, *P<0.05, **P<0.01, ****P<0.0001, "^" represents a significant difference compared to the formulation "UVB+LXH30BEC" group, ^^^^P<0.0001, "ns" represents no significant difference compared to the UVB group; mean±SD, n=3).

[0024] Figure 5 Figure 4 is a graph showing the effect of each of the Campsis grandiflora extracts of Example 1 and Comparative Examples 1-9 on the FLG mRNA expression level secreted from HaCaT cells after UVB-induced damage (in the figure, "#" represents a significant difference compared to the blank group, ###P<0.001; "*" represents a significant difference compared to the UVB group, ****P<0.0001, "^" represents a significant difference compared to the formulation "UVB+LXH30BEC" group, ^^^^P<0.0001; mean±SD, n=3).

[0025] Figure 6Figure 2 is a histogram showing the tail length of each Campsis grandiflora extract of Example 1 and Comparative Examples 1-9 after UVB irradiation on HaCaT cells after single cell gel electrophoresis experiment (in the figure, "#" represents significant compared with the blank group, ###P<0.001; "*" represents significant compared with the UVB group, **P<0.01, ****P<0.0001, "^" represents significant compared with the formula "UVB+LXH30BEC" group, ^^^P<0.0001, "ns" represents no significant compared with the UVB group; mean±SD, n=3).

[0026] Figure 7 Figure 3 is a diagram showing the effect of each Campsis grandiflora extract of Example 1 and Comparative Examples 1-9 on preventing UVB-induced HaCaT cells from secreting TNF-α (in the figure, "#" represents significant compared with the blank group, ###P<0.001; "*" represents significant compared with the UVB group, **P<0.01, ****P<0.0001, "^" represents significant compared with the formula "UVB+LXH30BEC" group, ^^^P<0.0001; mean±SD, n=3).

[0027] Figure 8 Figure 4 is a diagram showing the effect of each Campsis grandiflora extract of Example 1 and Comparative Examples 1-9 on preventing UVB-induced HaCaT cells from secreting PGE2 (in the figure, "#" represents significant compared with the blank group, #P<0.05; "*" represents significant compared with the UVB group, **P<0.01, ****P<0.0001, "^" represents significant compared with the formula "UVB+LXH30BEC" group, ^^^P<0.0001; mean±SD, n=3).

[0028] Figure 9 Figure 5 is a diagram showing the effect of each Campsis grandiflora extract of Example 1 and Comparative Examples 1-9 on the NO secretion of HaCaT cells after UVB-induced damage (in the figure, "#" represents significant compared with the blank group, ####P<0.0001; "*" represents significant compared with the UVB group, ****P<0.0001, "^" represents significant compared with the formula "UVB+LXH30BEC" group, ^^^P<0.0001; mean±SD, n=3).

[0029] Figure 10Figure 2 is a diagram showing the effect of each of the campsis grandiflora extracts of Example 1 and Comparative Examples 1-9 on IL-6 secretion by HaCaT cells after UVB-induced damage (in the figure, "#" represents a significant difference from the blank group, ###P<0.0001; "*" represents a significant difference from the UVB group, ****P<0.0001, "^" represents a significant difference from the formula "UVB+LXH30BEC" group, ^^^P<0.0001; mean±SD, n=3).

[0030] Figure 11 Figure 3 is a diagram showing the effect of each of the campsis grandiflora extracts of Example 1 and Comparative Examples 1-9 on TRPV1 mRNA expression secreted by HaCaT cells after capsaicin stimulation (in the figure, "#" represents a significant difference from the blank group, ###P<0.0001; "*" represents a significant difference from the UVB group, *P<0.05, ****P<0.0001, "^" represents a significant difference from the formula "UVB+LXH30BEC" group, ^^^P<0.0001; mean±SD, n=3).

[0031] Figure 12 Figure 4 is a diagram showing the effect of each of the campsis grandiflora extracts of Example 1 and Comparative Examples 1-9 on TRPV1 protein expression secreted by HaCaT cells after capsaicin stimulation (in the figure, "#" represents a significant difference from the blank group, ###P<0.0001; "*" represents a significant difference from the UVB group, *P<0.05, **P<0.01, ****P<0.0001, "^" represents a significant difference from the formula "UVB+LXH30BEC" group, ^^^P<0.0001; mean±SD, n=3).

[0032] Figure 13 Figure 5 is a diagram showing the effect of each of the campsis grandiflora extracts of Example 1 and Comparative Examples 1-9 on the fluorescence intensity of calcium ions in HaCaT cells. DETAILED DESCRIPTION

[0033] In order to make the application purposes, technical solutions and beneficial technical effects of the present application clearer, the present application will be further described in detail below in conjunction with the examples. It should be understood that the examples described in the present specification are only for the purpose of explaining the present application, and are not intended to limit the present application.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] Trumpet creeper flowers are the dried flowers of the trumpet creeper (Campsis grandiflora) or American trumpet creeper (Campsis radicans), belonging to the Bignoniaceae family. Their chemical components mainly include flavonoids, phenylpropanoids, organic acids, and volatile oils. Pharmacopoeia records their use for amenorrhea due to blood stasis, abdominal masses, traumatic injuries, rashes, and itchy skin. Currently, there are no reports on the application of cardamom extract in preventing or repairing UV damage to the skin or providing immediate soothing and repair.

[0039] In view of the deficiencies of the prior art, this application provides a new use for the extract of trumpet vine, which can effectively prevent or repair skin photodamage caused by UVB and provide an immediate soothing effect.

[0040] In some embodiments, the use of the trumpet creeper extract includes at least one of (1) to (3): (1) use in the preparation of products for preventing ultraviolet light damage, including preventing skin light damage caused by UVB radiation; (2) use in the preparation of products for repairing ultraviolet light damage, including post-sun exposure repair of skin light damage caused by UVB radiation; and (3) use in the preparation of products for instantly soothing the skin.

[0041] In some embodiments, the extractant for the trumpet vine extract includes propylene glycols, and preferably, the extractant for the trumpet vine extract includes 1,3-propanediol.

[0042] In some embodiments, the mass concentration of the extractant is 25% to 95%.

[0043] As an example, the mass concentration of the extractant can be 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 38%, 40%, 45%, 48%, 50%, 52%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 68%, 70%, 72%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%.

[0044] The extract of *Campsis grandiflora* prepared using the aforementioned types and concentrations of extractants exhibits several advantages. First, in terms of antioxidant and UV protection, it enhances the ability of *Campsis grandiflora* extract to inhibit DPPH free radicals, indicating its excellent direct free radical scavenging activity. In cell models, this extract effectively reduces ROS production 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 pathway of oxidative defense and gene integrity protection. Second, in terms of barrier repair and anti-inflammatory regulation, this extract demonstrates outstanding barrier-enhancing function, significantly upregulating 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 skin's physical barrier structure. Simultaneously, it effectively reduces the production of inflammatory factors TNF-α, IL-6, PGE2, and NO, alleviating skin inflammatory responses induced by UV radiation and immune stress through multi-target inhibition of inflammatory pathways. Finally, in terms of neurogenic soothing and sensory modulation, this extract exhibits excellent transient receptor potential regulation, reducing the expression of TRPV1 at both the mRNA and protein levels in HaCaT cells after capsaicin stimulation and effectively inhibiting capsaicin-induced calcium ion influx, thereby blocking pain and burning signal transduction pathways. This provides a solid mechanistic basis for its application in alleviating skin sensitivity, burning, and stinging. In summary, the trumpet creeper extract prepared using the above-mentioned types and concentrations of extractants not only overcomes the irritation and safety issues caused by traditional organic solvent extraction but also synergistically enhances the multi-dimensional bioefficacy of cardamom extract in anti-oxidation, photoprotection, barrier repair after photodamage, anti-inflammation, and transient soothing, making it particularly suitable for daily protection and repair of sensitive skin.

[0045] In some implementations, preventing UV photodamage includes upregulating the expression levels of skin barrier-related proteins; and / or, repairing UV photodamage includes upregulating the expression levels of skin barrier-related proteins.

[0046] As an example, skin barrier-related proteins include at least one of connexins, filaggrin (FLG), smearin, epidermal proteins, and desmosomes.

[0047] 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).

[0048] In some implementations, preventing UV light damage includes downregulating the expression levels of inflammatory factors; and / or, repairing UV light damage includes downregulating the expression levels of inflammatory factors.

[0049] In some embodiments, the inflammatory factors include at least one of pro-inflammatory cytokines, prostaglandin cytokines, and oxidative stress-related factors.

[0050] In some embodiments, the pro-inflammatory cytokines include at least one of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-8 (IL-8).

[0051] In some implementations, prostaglandin cytokines include prostaglandin E2 (PGE2).

[0052] In some implementations, immediate relief includes reducing intracellular calcium ion influx levels; and / or, inhibiting the expression level of TRPV1 (transient receptor potential vanillin subtype 1, also known as capsaicin receptor) mRNA; and / or, inhibiting the expression level of TRPV1 protein.

[0053] In some implementations, the product includes cosmetics.

[0054] In some implementations, the dosage forms of cosmetics include ointments, creams, lotions, patches, and masks.

[0055] In some embodiments, the product includes a pharmaceutical product, and the dosage form of the pharmaceutical product includes tablets, capsules, granules, pills, oral liquids, emulsions, dry suspensions, dry extracts, and injections.

[0056] Example

[0057] 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.

[0058] The sources of some of the reagents and equipment used in Example 1 and Comparative Examples 1-9 are as follows:

[0059] 1. Trumpet creeper flower extract, purchased from Kangweifu Pharmaceutical Co., Ltd., produced in Yunnan.

[0060] HaCaT cells, manufacturer FuHeng, catalog number FH0186;

[0061] 2. PBS, manufacturer Gibco, catalog number 2002050, 500mL;

[0062] 3. Fetal bovine serum (FBS) manufacturer MRC, catalog number CCS30009.02, 500mL;

[0063] 4. Bispecific antibiotics (penicillin & streptomycin) Gibco15140122;

[0064] 5. DMEM, Gibco 11995-065 500mL;

[0065] 6. Trypsin-EDTA (0.25%), containing phenol red Thermo Fisher 25200114;

[0066] 7. Petri dish: Thermo Fisher 150464;

[0067] 8. 6-well plate, Thermo Fisher 140675;

[0068] 9. CCK-8 Admas life C8022-10000T;

[0069] 10. ROS probe DCFH DA (D6883);

[0070] 11. Merck, D6883-50mg;

[0071] 12. DMSO, Merck, 472301-500mL.

[0072] Example 1

[0073] Example 1 of this application provides a trumpet vine flower extract, the preparation method of which includes:

[0074] S1 Weigh out the dried trumpet creeper flower raw material, pulverize it and pass it through a 50-mesh sieve to obtain trumpet creeper flower powder.

[0075] S2 accurately weigh 1 kg of the trumpet creeper powder, add 10 kg of 30% wt 1,3-propanediol aqueous solution as the extractant, and mix evenly.

[0076] S3 extracts the mixture by heating it to 85°C and stirring it continuously at that temperature for 2 hours.

[0077] S4 Then, the system was cooled to 50°C and preliminarily filtered using a 200-mesh filter bag, and all the filtrate was collected.

[0078] 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 trumpet creeper extract.

[0079] Comparative Example 1

[0080] Comparative Example 1 of this application provides a trumpet vine extract, which differs from Example 1 only in the extractant in step S2. Specifically, 10 kg of 30% wt 1,3-propanediol aqueous solution is replaced with 10 kg of pure water.

[0081] Comparative Example 2

[0082] Comparative Example 2 of this application provides a trumpet vine extract, which differs from Example 1 only in the extractant in step S2. Specifically, 10 kg of 30% wt 1,3-propanediol aqueous solution is replaced with 10 kg of 30% wt glycerol aqueous solution.

[0083] Comparative Example 3

[0084] Comparative Example 3 of this application provides a trumpet vine extract, which differs from Example 1 only in the extractant in step S2. Specifically, 10 kg of 30% wt 1,3-propanediol aqueous solution is replaced with 10 kg of 60% wt glycerol aqueous solution.

[0085] Comparative Example 4

[0086] Comparative Example 4 of this application provides a trumpet vine extract, which differs from Example 1 only in the extractant in step S2. Specifically, 10 kg of 30% wt 1,3-propanediol aqueous solution is replaced with 10 kg of 90% wt glycerol aqueous solution.

[0087] Comparative Example 5

[0088] Comparative Example 5 of this application provides a trumpet vine extract, which differs from Example 1 only in the extractant in step S2. Specifically, 10 kg of 30% wt 1,3-propanediol aqueous solution is replaced with 10 kg of 60% wt 1,3-propanediol aqueous solution.

[0089] Comparative Example 6

[0090] Comparative Example 6 of this application provides a trumpet vine extract, which differs from Example 1 only in the extractant in step S2. Specifically, 10 kg of 30% wt 1,3-propanediol aqueous solution is replaced with 10 kg of 90% wt 1,3-propanediol aqueous solution.

[0091] Comparative Example 7

[0092] Example 7 of this application provides a trumpet vine extract, which differs from Example 1 only in the extractant in step S2. Specifically, 10 kg of 30% wt 1,3-propanediol aqueous solution is replaced with 10 kg of 30% wt 1,3-butanediol aqueous solution.

[0093] Comparative Example 8

[0094] Comparative Example 8 of this application provides a trumpet vine extract, which differs from Example 1 only in the extractant in step S2. Specifically, 10 kg of 30% wt 1,3-propanediol aqueous solution is replaced with 10 kg of 60% wt 1,3-butanediol aqueous solution.

[0095] Comparative Example 9

[0096] Comparative Example 9 of this application provides a trumpet vine extract, which differs from Example 1 only in the extractant in step S2. Specifically, 10 kg of 30% wt 1,3-propanediol aqueous solution is replaced with 10 kg of 90% wt 1,3-butanediol aqueous solution.

[0097] Table 1 summarizes the types of extractants and their concentration parameters used in Example 1 and Comparative Examples 1-9.

[0098] Table 1

[0099]

[0100]

[0101] Performance testing

[0102] The performance of the trumpet creeper extracts prepared in Example 1 and Comparative Examples 1-9 was tested:

[0103] 1. Determination of DPPH free radical scavenging ability

[0104] 1.1 Reagent Preparation

[0105] (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.

[0106] (2) Sample solution: Take the extract of trumpet vine prepared in each example, dilute it 25 times with deionized water, and then dilute it 4 times with anhydrous ethanol to obtain a test sample solution with a final concentration of 1%wt.

[0107] (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.

[0108] 1.2 Determination Method

[0109] The determination was performed using the 96-well plate method, and the specific procedures are as follows:

[0110] 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.

[0111] 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.

[0112] Blank 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.

[0113] 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.

[0114] 1.3 Calculation Formula

[0115] The scavenging rate of the sample against DPPH free radicals was calculated using the following formula:

[0116] Clearance rate (%) = [(Ac - (As - Ad)) / Ac] × 100%

[0117] In the formula:

[0118] Ac represents the average absorbance of the blank control group (100 μL anhydrous ethanol + 100 μL DPPH working solution);

[0119] As represents the average absorbance of the sample group (100 μL of test sample solution + 100 μL of DPPH working solution);

[0120] Ad represents the average absorbance of the sample control group (100 μL of test sample solution + 100 μL of anhydrous ethanol).

[0121] 1.4 Experimental Results

[0122] The DPPH free radical scavenging rates of the extracts from *Campsis grandiflora* in each embodiment and the positive control are shown in Table 2.

[0123] 2. Assay of the proliferative activity of HaCaT cells damaged by UVB

[0124] 2.1 Experimental Materials and Grouping

[0125] 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.

[0126] 2.1.2 Group Setup: The experiment was randomly divided into 3 groups, namely:

[0127] Normal group (blank control group): cultured in complete culture medium only, without UVB irradiation or drug treatment;

[0128] UVB irradiation group (model group): A cell damage model was established by UVB irradiation and cultured in complete culture medium only;

[0129] Campsis grandiflora extract intervention group: After UVB irradiation, the patients were treated with a 1 wt% Campsis grandiflora extract solution.

[0130] 2.2 Experimental Methods

[0131] 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. 5 Cells / 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%.

[0132] 2.2.2 Pre-treatment: The corresponding concentration of Campsis grandiflora extract solution (diluted with complete culture medium) was added to each well of the Campsis grandiflora 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.

[0133] 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.

[0134] 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.

[0135] 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:

[0136] Relative proliferation rate (%) = (OD value of experimental group ÷ OD value of normal group) × 100%

[0137] 2.4 Experimental Results

[0138] The effects of different concentrations of Trumpet Vine extract on the proliferation and repair of HaCaT cells damaged by UVB are as follows: Figure 1 As shown.

[0139] 3. Measurement of intracellular ROS content in HaCaT cells damaged by UVB

[0140] 3.1 Experimental Materials and Grouping

[0141] Consistent with 2.1 above.

[0142] 3.2 Experimental Methods

[0143] Consistent with section 2.2 above.

[0144] 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.

[0145] 3.4 Experimental Results

[0146] The effects of different treatment groups on intracellular ROS levels in HaCaT cells damaged by UVB are as follows: Figure 2 As shown.

[0147] 4. UVB-induced barrier function repair experiment in HaCaT cells

[0148] 4.1 Experimental Materials and Grouping

[0149] 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.

[0150] 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.

[0151] 4.1.3 Group Setup: The experiment was randomly divided into 3 groups, namely:

[0152] 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.

[0153] 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.

[0154] Sample group: After UVB irradiation, the sample group was cultured with a solution of trumpet creeper extract diluted 100 times (concentration of 1wt%) in DMEM maintenance medium.

[0155] 4.2 Experimental Methods

[0156] 4.2.1 Cell Seeding and UVB Irradiation: HaCaT cells were seeded at 2.0 × 10⁻⁶ cells / day. 5 Cells 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 .

[0157] 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 a 1 wt% solution of Campsis grandiflora extract diluted 100 times with DMEM maintenance medium was added to each well in the sample groups. All wells were then incubated at 37°C in a 5% CO2 incubator for 24 h.

[0158] 4.3 Total RNA extraction:

[0159] After the culture is complete, carefully aspirate and discard the supernatant from each well, add 1 mL of Trizol reagent to each well to fully lyse the cells, and transfer the lysate to a 1.5 mL enzyme-free centrifuge tube;

[0160] Add 200 μL of chloroform to a centrifuge tube, vortex for 15 seconds until the solution is emulsified, let stand at 4°C for 10 minutes, and allow the solution to naturally separate into layers (the upper layer is a colorless aqueous phase and the lower layer is a red organic phase).

[0161] Centrifuge at 12000 r / min for 15 min at 4℃, and gently aspirate 400 μL of the upper aqueous phase into a new enzyme-free centrifuge tube, avoiding aspiration of the intermediate protein layer;

[0162] Add an equal volume of isopropanol, invert the centrifuge tube to mix gently, let stand at 4°C for 10 min, then centrifuge at 4°C and 12000 r / min for 10 min. A white RNA precipitate will be visible at the bottom of the tube.

[0163] Discard the supernatant, add 1 mL of 75% anhydrous ethanol (prepared with DEPC water) to wash the precipitate, centrifuge at 12000 r / min for 5 min at 4℃, carefully discard the ethanol, and let stand at room temperature for 5-10 min until the precipitate is dry and transparent.

[0164] Add 50 μL of DEPC-ddH2O to dissolve the RNA precipitate, and determine the RNA concentration (OD) using a micro-volume UV spectrophotometer. 260 (value), and in OD 260 / OD 280 The ratio (1.8-2.0 is acceptable) is used to assess RNA purity.

[0165] 4.4 Real-time quantitative PCR detection:

[0166] 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.

[0167] 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.

[0168] 4.6 Experimental Results

[0169] The effects of Trumpet creeper 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.

[0170] 5. Experiments on the prevention of DNA damage

[0171] 5.1 Experimental Materials and Grouping

[0172] 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.

[0173] 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.

[0174] 5.1.3 Group Setup: The experiment was randomly divided into 3 groups, namely:

[0175] Normal control group: Cells were cultured in DMEM complete medium only, without UVB irradiation or sample processing;

[0176] 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.

[0177] Sample pretreatment group: Cells were pretreated with a sample solution of trumpet creeper extract, then irradiated with UVB, and cultured after irradiation.

[0178] 5.2 Experimental Methods

[0179] 5.2.1 Sample solution preparation: Take the extracts of trumpet creeper prepared in each example and comparative example, dilute them 100 times with DMEM complete culture medium (concentration of 1wt%), filter them through a 0.22μm microporous membrane for sterilization, and store them in a refrigerator at 4℃ in the dark for later use.

[0180] 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.

[0181] 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 normal control group and the UVB damage group. All groups were pretreated in a 37℃, 5% CO2 incubator for 2 h.

[0182] 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.

[0183] 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.

[0184] 5.3 DNA Damage Detection

[0185] After cell lysis, unwinding, electrophoresis, neutralization, and staining, the above cell suspension was observed and photographed under a confocal microscope.

[0186] 5.4 Experimental Results

[0187] The preventive effect of pretreatment with trumpet creeper extract on UVB-induced DNA damage in HaCaT cells is as follows: Figure 6 As shown.

[0188] 6. Experiment on the ability of HaCaT cells to prevent UVB-induced inflammatory damage

[0189] 6.1 Experimental Materials and Grouping

[0190] 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.

[0191] 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.

[0192] 6.1.3 Group Setup: The experiment was randomly divided into 3 groups, namely:

[0193] Blank control group: Cells were cultured in DMEM maintenance medium only, wrapped in aluminum foil to avoid ultraviolet radiation throughout the process, and no sample processing was performed.

[0194] 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.

[0195] Sample group: Cells were pretreated with a sample solution of trumpet creeper extract, then irradiated with UVB, and then cultured for a period of time.

[0196] 6.2 Experimental Methods

[0197] 6.2.1 Sample solution preparation: Take the extracts of trumpet creeper prepared in each example and comparative example, dilute them 100 times with DMEM maintenance medium (concentration of 1wt%), filter them through a 0.22μm microporous membrane for sterilization, and store them in a refrigerator at 4℃ in the dark for later use.

[0198] 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.

[0199] 6.2.3 Sample pretreatment and UVB irradiation:

[0200] After the culture is completed, discard the culture medium from each well and gently wash the cells three times with PBS buffer.

[0201] 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.

[0202] 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 .

[0203] 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.

[0204] 6.2.5 Inflammatory factor detection

[0205] The levels of TNF-α and PGE2 were detected using ELISA.

[0206] 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.

[0207] 6.4 Experimental Results

[0208] The effects of Trumpet creeper extract on UVB-induced secretion of inflammatory factors TNF-α and PGE2 in HaCaT cells are as follows: Figure 7 , Figure 8 As shown.

[0209] 7. Experiment on inhibiting UVB-induced inflammatory damage (repair) in HaCaT cells

[0210] 7.1 Experimental Materials and Grouping

[0211] 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.

[0212] 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.

[0213] 7.1.3 Group Setup: The experiment was randomly divided into 3 groups, namely:

[0214] Blank control group: Cells were cultured in DMEM maintenance medium only, wrapped in aluminum foil to avoid ultraviolet radiation throughout the process, and no sample processing was performed.

[0215] 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.

[0216] Sample repair group: After UVB irradiation, cells were treated with a sample solution of trumpet creeper extract to evaluate their ability to repair inflammatory damage.

[0217] 7.2 Experimental Methods

[0218] 7.2.1 Sample solution preparation: Take the extracts of trumpet creeper prepared in each example and comparative example, dilute them 100 times with DMEM maintenance medium (concentration of 1wt%), filter them through a 0.22μm microporous membrane for sterilization, and store them in a refrigerator at 4℃ in the dark for later use.

[0219] 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.

[0220] 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 .

[0221] 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.

[0222] 7.3 Inflammatory factor detection

[0223] 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.

[0224] 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.

[0225] 7.4 Data Analysis

[0226] 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.

[0227] 7.5 Experimental Results

[0228] The repair effect of Campsis grandiflora extract on UVB-induced inflammatory damage in HaCaT cells (indicating NO and IL-6 secretion levels) is as follows: Figure 9 , Figure 10 As shown.

[0229] 8. Experiment on TRPV1 stimulation in HaCaT cells by capsaicin

[0230] 8.1 Experimental Materials and Grouping

[0231] 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.

[0232] 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.

[0233] 8.1.3 Group Setup: The experiment was randomly divided into 3 groups, namely:

[0234] Blank control group: Cells were cultured in DMEM maintenance medium only, without the addition of capsaicin or samples;

[0235] Model group: A TRPV1 activation model was established by stimulating cells with capsaicin and cultured in DMEM maintenance medium only, without adding any samples;

[0236] Sample group: After cells were stimulated with capsaicin, they were treated with a sample solution of trumpet creeper extract to evaluate its effect on TRPV1-related responses.

[0237] 8.2 Experimental Methods

[0238] 8.2.1 Preparation of sample solution and capsaicin solution:

[0239] Sample solution: Take the extracts of trumpet creeper prepared in each example and comparative example, dilute them to a concentration of 1 wt% with DMEM maintenance medium, filter them through a 0.22 μm microporous membrane for sterilization, and store them at 4°C in the dark for later use.

[0240] 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.

[0241] 8.2.2 Cell Seeding and Pretreatment:

[0242] 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.

[0243] 6-well plate seeding: HaCaT cells were seeded at a rate of 1 × 10⁻⁶ cells / well. 5The 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.

[0244] After the culture was completed, both culture plates were gently washed three times with PBS buffer to remove residual culture medium.

[0245] 8.2.3 Capsaicin Irritation and Sample Processing:

[0246] 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.

[0247] 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.

[0248] After incubation, the sample group was replaced with DMEM maintenance medium containing trumpet creeper extract (500 μL per well in 24-well plates and 2 mL per well in 6-well plates), and the model group was replaced with the same amount of DMEM maintenance medium. Both groups were then incubated for another 24 h under the same conditions.

[0249] 8.3 Collection and Detection of Supernatant (24-well plate):

[0250] 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.

[0251] 8.4 Total RNA Extraction

[0252] 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.

[0253] 8.5 Real-time quantitative PCR detection:

[0254] 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.

[0255] 8.6 Data Analysis:

[0256] 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.

[0257] 8.7 Experimental Results

[0258] The effect of trumpet creeper extract on TRPV1 gene expression in capsaicin-stimulated HaCaT cells is as follows: Figure 11-12 As shown.

[0259] 9. Experiment on calcium ion influx in HaCaT cells

[0260] 9.1 Experimental Materials and Grouping

[0261] 9.1.1 Cell line: Same as HaCaT cells described in 2.1.1, with consistent culture conditions, and logarithmic growth phase cells were used for experiments.

[0262] 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.

[0263] 9.1.3 Group Setup: The experiment was randomly divided into 4 groups, namely:

[0264] Blank control group: Cells were cultured in DMEM medium only, without the addition of capsaicin or other samples;

[0265] Model group: Cells were treated with 20 μM capsaicin to induce calcium ion influx, without adding any sample;

[0266] 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.

[0267] 9.2 Preparation of Experimental Solutions

[0268] 9.2.1 Capsaicin solution:

[0269] 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.

[0270] 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.

[0271] 9.2.2 Sample solution:

[0272] 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.

[0273] 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.

[0274] 9.3 Experimental Methods

[0275] 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.

[0276] 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.

[0277] 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.

[0278] 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).

[0279] 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.

[0280] 9.5 Experimental Results

[0281] 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 13 As shown.

[0282] Experimental results

[0283] 1. Results of DPPH free radical scavenging capacity measurement

[0284] Table 2 shows the experimental results.

[0285] Experimental results

[0286] 1. Results of DPPH free radical scavenging capacity measurement

[0287] Table 2 shows that all extracts of *Campsis grandiflora* have the ability to inhibit DPPH free radicals. Among them, the extract of *Campsis grandiflora* in a 90% wt aqueous solution of 1,3-propanediol has the strongest ability to inhibit DPPH free radicals.

[0288] Table 2 Results of DPPH scavenging ability of extracts

[0289] Sample Name Sample No. DPPH Scavenging Ability (%) Vitamin C 93.0±0.0 Campsis radicans 30% 1,3-propanediol extract LXH30BEC 84.3±2.0 Campsis radicans water extract LXHS 45.5±0.3 Campsis radicans 30% glycerol extract LXH30GY 62.0±1.3 Campsis radicans 60% glycerol extract LXH60GY 69.3±1.5 Campsis radicans 90% glycerol extract LXH90GY 82.9±0.7 Campsis radicans 60% 1,3-propanediol extract LXH60BEC 79.1±2.4 Campsis radicans 90% 1,3-propanediol extract LXH90BEC 82.1±0.7 Campsis radicans 30% 1,3-butanediol extract LXH30DEC 83.4±0.7 Campsis radicans 60% 1,3-butanediol extract LXH60DEC 83.6±2.8 Campsis radicans 90% 1,3-butanediol extract LXH90DEC 77.5±7.3

[0290] 2. Results of UVB-damaged HaCaT cell proliferation activity assay

[0291] Figure 1 Experimental results showed that the extract LXH30BEC from the trumpet creeper flower had no cytotoxic effect on HaCaT cells.

[0292] 3. Results of ROS content measurement in HaCaT cells damaged by UVB

[0293] All extracts of the trumpet creeper flower could reduce the production of ROS in HaCaT cells stimulated by UVB. Among them, the extract LXH30BEC had the best effect, which was significantly better than the LXHS, LXH30GY, LXH60GY, LXH90GY, LXH60BEC, LXH90BEC, LXH30DEC, LXH60DEC and LXH90EDC groups.

[0294] 4. Results of UVB-induced barrier function repair in HaCaT cells

[0295] Figure 3 , Figure 4 , Figure 5Experimental results showed that the extract of Campsis grandiflora flower could increase the expression of mRNA levels of skin barrier-related proteins ZO-1, CLDN1, and FLG proteins after UVB stimulation of HaCaT cells. Among them, the extract of Campsis grandiflora flower LXH30BEC had the best effect, which was significantly better than LXHS, LXH30GY, LXH60GY, LXH90GY, LXH60BEC, LXH90BEC, LXH30DEC, LXH60DEC, and LXH90EDC groups.

[0296] 5. Experimental results on prevention of DNA damage

[0297] 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 Campsis grandiflora extract LXH30BEC reduced the average tail length of HaCaT cells, indicating that Campsis grandiflora extract has a significant protective effect on UVB-irradiated HaCaT cells and can reduce DNA damage after UVB irradiation. Furthermore, LXH30BEC's effect on reducing DNA damage after UVB irradiation was significantly better than that of the LXHS, LXH30GY, LXH60GY, LXH90GY, LXH60BEC, LXH90BEC, LXH30DEC, LXH60DEC, and LXH90EDC groups.

[0298] 6. Experimental results on the ability of HaCaT cells to prevent UVB-induced inflammatory damage.

[0299] Figure 7 , Figure 8 Experimental results showed that the extract of trumpet creeper flower could prevent inflammatory damage to HaCaT cells caused by UVB irradiation and significantly reduce the secretion of inflammatory factors TNF-α and PGE2. Moreover, the effect of LXH30BEC in reducing the secretion of inflammatory factors TNF-α and PGE2 was significantly better than that of LXHS, LXH30GY, LXH60GY, LXH90GY, LXH60BEC, LXH90BEC, LXH30DEC, LXH60DEC, and LXH90EDC groups.

[0300] 7. Experimental results on inhibiting UVB-induced inflammatory damage (repair) in HaCaT cells

[0301] Figure 9 , Figure 10Experimental 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 Campsis grandiflora extract LXH30BEC significantly reduced the production of inflammatory factors NO and IL-6, and the effect of LXH30BEC in reducing the production of inflammatory factors NO and IL-6 was significantly better than that of the LXHS, LXH30GY, LXH60GY, LXH90GY, LXH60BEC, LXH90BEC, LXH30DEC, LXH60DEC, and LXH90EDC groups.

[0302] 8. Results of the experiment on TRPV1 in HaCaT cells stimulated by capsaicin

[0303] 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. Compared to the LXHS, LXH30GY, LXH60GY, LXH90GY, LXH60BEC, LXH90BEC, LXH30DEC, LXH60DEC, and LXH90EDC groups, the addition of Campsis grandiflora extract LXH30BEC significantly reduced TRPV1 expression at both the mRNA and protein levels in HaCaT cells after capsaicin stimulation.

[0304] Figure 13 Experimental results showed that capsaicin stimulation of HaCaT cells significantly increased calcium ion influx, indicating successful model establishment. Compared with the LXHS, LXH30GY, LXH60GY, LXH90GY, LXH60BEC, LXH90BEC, LXH30DEC, LXH60DEC, and LXH90EDC groups, the addition of Campsis grandiflora extract LXH30BEC significantly inhibited the calcium ion influx level after capsaicin stimulation of HaCaT cells.

[0305] In summary, the trumpet creeper extract provided in this application exhibits synergistic multiple effects in the prevention, repair, and immediate soothing of ultraviolet radiation. It can not only effectively prevent or repair skin inflammation and cell damage caused by UVB photodamage, but also promote the repair of skin barrier and cell function and reduce the secretion of inflammatory factors after photodamage occurs. At the same time, the extract can also quickly soothe skin redness, burning and other discomforts, achieving an immediate soothing effect.

[0306] The above description is merely a specific implementation of this application. Those skilled in the art will clearly understand that, for the sake of convenience and brevity, the specific working process described above can be referred to the corresponding process in the foregoing method embodiments, and will not be repeated here. It should be understood that the protection scope of this application is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in this application, and these modifications or substitutions should all be covered within the protection scope of this application.

Claims

1. The use of a trumpet vine flower extract, characterized in that, The stated uses include at least one of (1) to (3): (1) Use in the preparation of products for preventing ultraviolet light damage, wherein the prevention of ultraviolet light damage includes prevention of skin light damage caused by UVB radiation; (2) Use in the preparation of products for repairing ultraviolet light damage, wherein the repair of ultraviolet light damage includes post-sun exposure repair of skin light damage caused by UVB radiation. (3) Use in the preparation of products that can instantly soothe the skin.

2. The use according to claim 1, characterized in that, The extractant for the trumpet creeper flower extract includes propylene glycol. Preferably, the extractant of the trumpet creeper extract includes 1,3-propanediol.

3. The use according to claim 2, characterized in that, The extractant has a mass concentration of 25% to 95%.

4. The use according to claim 1, characterized in that, The prevention of UV photodamage includes upregulating the expression levels of skin barrier-related proteins; and / or, The repair of UV photodamage includes upregulating the expression levels of skin barrier-related proteins.

5. The use according to claim 1, characterized in that, The prevention of ultraviolet photodamage includes downregulating the expression levels of inflammatory factors; and / or, The repair of UV photodamage includes downregulating the expression levels of inflammatory factors.

6. The use according to claim 5, characterized in that, The inflammatory factors include at least one of pro-inflammatory cytokines, prostaglandin cytokines, and oxidative stress-related factors.

7. The use according to claim 6, characterized in that, The pro-inflammatory cytokines include at least one of tumor necrosis factor-α, interleukin-1β, interleukin-6, and interleukin-8.

8. The use according to claim 6, characterized in that, The prostaglandin cytokines include prostaglandin E2.

9. The use according to claim 1, characterized in that, The product includes cosmetics, and optionally, the dosage form of the cosmetics includes ointments, creams, lotions, patches, and masks.

10. The use according to claim 1, characterized in that, The product includes pharmaceuticals, and optionally, the dosage form of the pharmaceuticals includes tablets, capsules, granules, pills, oral liquids, emulsions, dry suspensions, dry extracts, and injections.

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