Application of liquorice yin-nourishing decoction in preparation of sunscreen product

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to application of liquorice yin-nourishing decoction in preparation of a sunscreen product. The invention provides an application of a traditional Chinese medicine composition in preparation of a sunscreen product. The traditional Chinese medicine composition comprises a traditional Chinese medicine composition for preparing a liquorice yin-nourishing decoction. Experiments prove that the feasibility of the liquorice yin-nourishing soup in enhancing the skin sunscreen capability, reducing UVB-induced skin injury and further reducing sunburn injury is provided, and the new application of the liquorice yin-nourishing soup is provided for constructing a more comprehensive sunscreen scheme, assisting daily sunscreen and reducing the adverse effect of sunburn on the skin. And an efficient and convenient new way is provided for preventing sunshine-related skin problems.

Description

Technical Field

[0002] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to the application of Licorice Nourishing Yin Decoction in the preparation of sunscreen products. Background Technology

[0003] As the body's first line of defense, the skin is vulnerable to ultraviolet (UV) damage. UV radiation consists of 95% UVA and 5% UVB, but UVB has significantly higher energy, up to 1000 times that of UVA. Therefore, UVB is a major cause of skin damage. Although acute photodamage caused by UVB only reaches the epidermis, it can lead to erythema, DNA damage, and even skin cancer, making it a primary cause of photodamage. Acute photodamage can cause skin laxity and wrinkles, primarily due to changes in the extracellular matrix (ECM) of the dermis: elastic fiber function is affected, and collagen (which accounts for 75% of the dermis) is degraded due to upregulation of matrix metalloproteinases (MMPs). Simultaneously, UVB excessively generates reactive oxygen species (ROS), disrupting oxidative balance, reducing the activity of endogenous antioxidant enzymes, and inducing inflammatory responses. This creates a vicious cycle of "free radicals-oxidative stress-inflammation" through NF-κB and AP-1, further activating the MAPK signaling pathway (including ERK, JNK, and p38), mediating keratinocyte apoptosis, and exacerbating the damage.

[0004] Currently, sun protection methods in this field mainly include physical sunscreens, chemical sunscreens, the use of antioxidants, medical aesthetic treatments, or the use of 0.05% all-trans retinoic acid cream. However, these methods generally have limitations: physical or chemical sunscreens can be burdensome for sensitive skin, antioxidants such as vitamin C / E have poor stability, and medical aesthetic treatments are expensive; the 0.05% all-trans retinoic acid cream approved by the US FDA is prone to causing retinoid dermatitis and has a slow effect. Therefore, how to provide a highly effective and stable sunscreen product has become a research hotspot.

[0005] Glycyrrhiza Nourishing Yin Decoction (GCNY) is developed based on Zhu Danxi's theory of "Yin is often insufficient, Yang is often excessive." It uses licorice as the principal ingredient (anti-inflammatory and antioxidant), and Polygonatum odoratum as the assistant ingredient (nourishing Yin and moisturizing dryness while alleviating the side effects of licorice), combined with ginseng, dandelion, etc., to nourish Yin, strengthen the spleen, and harmonize Yin and Yang. However, there are no reports on whether the above-mentioned herbal combination can be used for effective sun protection. Summary of the Invention

[0006] Based on the aforementioned background technology, and in order to address the lack of application of traditional Chinese medicine compositions in the preparation of highly effective and stable sunscreen products in existing technologies, this invention preliminarily confirms that Licorice Nourishing Yin Decoction has sunscreen effects, particularly showing a significant inhibitory effect on UVB-induced acute photodamage to the skin. This invention provides the application of Licorice Nourishing Yin Decoction in the preparation of sunscreen products, specifically including the following technical solutions: This invention provides an application of a traditional Chinese medicine composition in the preparation of sunscreen products, wherein the traditional Chinese medicine composition includes a traditional Chinese medicine composition used in the preparation of Licorice Nourishing Yin Decoction.

[0007] Preferably, the components of the traditional Chinese medicine composition used to prepare the licorice yin-nourishing decoction include 2-6 parts of licorice, 1-5 parts of ginseng, 1-3 parts of dried ginger, 1-3 parts of dandelion, 2-6 parts of monk fruit, 1-5 parts of Solomon's seal, and 2-6 parts of jujube.

[0008] Preferably, the traditional Chinese medicine composition includes Licorice Nourishing Yin Decoction.

[0009] Preferably, the sun protection includes protection against ultraviolet-induced acute photodamage to the skin; Preferably, the ultraviolet light includes medium-wave ultraviolet light.

[0010] Preferably, the symptoms of acute photodamage to the skin include any one or more of the following: 1) Skin develops erythema; 2) Wrinkles appear on the skin; 3) Increased thickness of the upper epidermis; 4) Increased thickness of the dermis; 5) Increased number of mast cells; 6) The elastic fiber network is flocculent and damaged, with thickening, curling and / or disordered arrangement, accompanied by breakage, twisting and / or hyperplasia; 7) Increased MMP-1 protein content.

[0011] Preferably, the product includes a drug.

[0012] Preferably, when the drug is applicable to mice, the ratio of the dosage of licorice yin-nourishing decoction to the mouse body weight is 2.5g / kg to 10g / kg.

[0013] Preferably, the product is used orally.

[0014] Preferably, the dosage form of the product includes a liquid formulation.

[0015] The beneficial effects of this invention are as follows: This invention provides an application of a traditional Chinese medicine composition in the preparation of sunscreen products. In the Licorice Nourishing Yin Decoction, licorice is the principal ingredient, exerting the effects of invigorating qi, tonifying the middle jiao, clearing heat, and detoxifying. Polygonatum odoratum is the assistant ingredient, not only synergizing with licorice to enhance its efficacy but also eliminating its side effects of water and sodium retention. Dandelion and monk fruit are adjuvant ingredients, and jujube is the guiding ingredient. The entire formula works synergistically to nourish yin, strengthen the spleen, eliminate dampness, harmonize yin and yang, and promote yang within yin. This invention experimentally verifies the feasibility of the Licorice Nourishing Yin Decoction in sun protection, particularly in preventing and treating acute photodamage to the skin, providing an efficient and stable solution for acute photodamage. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 The effect of different UVB irradiation doses on HaCaT cell viability; (⁻) ± SEM, n=6), compared with the control group, ** P <0.01; Figure 2 The results validated the activity of HaCaT cells; Wherein, A represents the effect of different concentrations of GCNY on HaCaT cells; B represents the effect of GCNY on the cell viability of HaCaT cells after UVB irradiation; (⁻ ±SEM, n=6), * indicates comparison with the control group. P <0.05; *** indicates that compared with the control group, P <0.001; # indicates that compared with the UVB group, P <0.05; Figure 3 GCNY inhibits UVB-induced production of reactive oxygen species in HaCaT cells; Where A is a representative ROS fluorescence image, scale bar = 100μm; B is a ROS level statistical plot; (⁻ ±SEM, n=6), *** indicates comparison with the control group, P <0.001; ## indicates that compared with the UVB group, P <0.01; Figure 4 This is a schematic diagram showing the results of GCNY inhibiting the expression of MMP-1 protein in HaCaT cells after UVB irradiation; the upper image shows the results of Western blot analysis of MMP-1, and the lower image shows the results of quantitative analysis of the Western blot results. (⁻) ±SEM, n=4), ** indicates comparison with the control group, P <0.05; ## indicates that compared with the UVB group, P <0.01; Figure 5 Animal experimental design to verify the intervention effects of different treatments on acute photodamaged skin in mice; Figure 6 Images of the skin on the back of mice at 0, 4, 7, 10, and 15 days after UVB irradiation for mice with different treatments; Figure 7 A scoring system for acute photodamage in mice after UVB irradiation under different treatments; From left to right, the images show mouse skin with scores ranging from 0 to 6. Figure 8 Statistical chart of appearance scores of mice after UVB irradiation under different treatments. (⁻) ±SEM, n=4), ** indicates comparison with the control group, P <0.01; ## indicates that compared with the UVB group, P <0.01; Figure 9 The results of HE staining in mice, statistical results of various indicators, and Masson staining results are shown. In this diagram, A shows the HE staining results of KM mice after different pretreatments; statistical results of various indicators of HE staining in mice after different pretreatments; B shows the statistical results of HE epidermal thickness; C shows the statistical results of HE dermal thickness; D shows the statistical results of collagen fiber content; and E shows a representative image of Masson staining of skin tissue. Figure 9 Of B, C, and E, (⁻ ±SEM, n=4), ** indicates comparison with the control group, P <0.01; ## indicates that compared with the UVB group, P <0.01; Figure 10 Results of mouse mast cell staining and Gomori staining; The top row of images shows representative mast cell staining images of the effect of GCNY on mast cell production in mouse skin tissue after UVB irradiation, with black arrows indicating mast cells; the bottom row of images shows the Gomori staining results of mouse skin after different pretreatments. Figure 11 This is a schematic diagram showing the results of GCNY inhibiting the expression of MMP-1 protein after UVB irradiation; The image above shows the results of Western blot analysis of MMP-1, while the image below shows the results of quantitative analysis of the Western blot analysis results. (⁻) ±SEM, n=4), * indicates P<0.05 compared with the control group; ## indicates P<0.01 compared with the UVB group. Detailed Implementation

[0018] This invention provides an application of a traditional Chinese medicine composition in the preparation of sunscreen products, wherein the traditional Chinese medicine composition includes a traditional Chinese medicine composition used in the preparation of Licorice Nourishing Yin Decoction.

[0019] In one embodiment, the herbal composition for preparing the Licorice Nourishing Yin Decoction comprises, by weight, 2-6 parts licorice, 1-5 parts ginseng, 1-3 parts dried ginger, 1-3 parts dandelion, 2-6 parts monk fruit, 1-5 parts Solomon's seal, and 2-6 parts jujube. In another embodiment, the herbal composition includes Licorice Nourishing Yin Decoction. In another embodiment, the raw material ratio of the Licorice Nourishing Yin Decoction is, by weight, licorice: ginseng slices: Solomon's seal: dandelion: dried ginger: monk fruit: jujube = 5:2:3:3:3:4:2. In another embodiment, the preparation method of the Licorice Nourishing Yin Decoction includes: mixing the raw materials according to the ratio, decocting in water for at least 1 hour to obtain the Licorice Nourishing Yin Decoction. In another embodiment, the concentration of the Licorice Nourishing Yin Decoction is 1.32 g / ml.

[0020] In one embodiment, the sun protection includes preventing and treating acute photodamage to the skin induced by ultraviolet radiation; in one embodiment, the ultraviolet radiation includes UVB (medium-wave ultraviolet radiation). In one embodiment, the wavelength of the UVB is 280-315 nm. In one embodiment, the symptoms of acute photodamage to the skin include any one or more of the following: 1) erythema of the skin; 2) wrinkles of the skin; 3) increased thickness of the epidermis; 4) increased thickness of the dermis; 5) increased number of mast cells; 6) the elastic fiber network is flocculent, damaged, thickened, curled, and / or disordered, accompanied by breakage, twisting, and / or hyperplasia; 7) increased MMP-1 protein content.

[0021] In one embodiment, the product includes a drug and / or health supplement. In one embodiment, when the drug is applicable to mice, the dosage of the licorice-nourishing yin decoction is 2.5 g / kg to 10 g / kg of mouse body weight. In one embodiment, the drug is administered orally. In one embodiment, the drug is in the form of a liquid preparation, capsule, or powder. In one embodiment, the drug further includes pharmaceutically acceptable excipients.

[0022] To further illustrate the present invention, the application of the licorice yin-nourishing decoction provided by the present invention in the preparation of sunscreen products is described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0023] Example 1: Verification Experiment of the Effect of Licorice Yin-Nourishing Decoction at the Cellular Level 1.1 Main Experimental Materials 1.1.1 Experimental Cells Human immortalized keratinocytes (HaCaT) were purchased from Shanghai Gaining Biotechnology Co., Ltd.

[0024] 1.2 Experimental Apparatus The UV crosslinker was purchased from Vilber Lourmat (France), the CO2 incubator from Thermo Fisher Scientific (USA), the clean bench from Suzhou Purification Equipment Factory, the low-speed centrifuge from Anhui Zhongke Zhongjia Scientific Instruments Co., Ltd., the digital display constant temperature water bath from Shanghai Lichen Bangxi Instrument Technology Co., Ltd., the cell counter from Shanghai Ruiyu Biotechnology Co., Ltd., the Mini-Protean® Tetra electrophoresis tank from Bio-Rad Scientific (USA), and the Bio-Rad Tran-Blot SD from Bio-Rad Scientific (USA).

[0025] 1.3 Experimental Reagents Cell Counting Kit-8 was purchased from Dojindo, Japan (catalog number TR688); DMEM culture medium was purchased from Gibco, USA (catalog number 8122500); penicillin-streptomycin-amphotericidal B mixed solution (100×) was purchased from Beijing Solarbio Science & Technology Co., Ltd. (catalog number P7630); trypsin was purchased from Gibco, USA (catalog number 8122504); fetal bovine serum was purchased from Adamas Life Shanghai Titan Technology Co., Ltd. (catalog number C8010); PBS was purchased from Wuhan Saive Biotechnology Co., Ltd. (catalog number G4202); and ROS detection kit was purchased from Beijing Solarbio Science & Technology Co., Ltd. (catalog number CA1410).

[0026] 2.1 Culture of Immortalized Human Keratinocytes (HaCaT) 2.1.1 Cell resuscitation Remove HaCaT cells from liquid nitrogen or a -80°C freezer, thaw them rapidly in a 37°C water bath, and immediately transfer the cell suspension to a 10mL centrifuge tube containing 3mL of complete culture medium (DMEM medium containing 10% FBS and 1% antibiotics). Centrifuge at 1000rpm for 5min, discard the supernatant, add 8mL of complete culture medium to resuspend the cells, and transfer them to a 100mm culture dish. Incubate in a 5% CO2, 37°C incubator. Once the cells have adhered to the culture dish, discard the old culture medium and add 10mL of complete culture medium.

[0027] 2.1.2 Cell passage When the cells have grown to approximately 80% confluence, aspirate the culture medium from the original culture dish, add 2 mL of PBS, gently shake and aspirate, add 2 mL of 0.25% trypsin, gently pipette, and incubate at 37°C, 5% CO2 for 5 minutes. Observe under a microscope after the cells have detached from the cell wall, add 2 mL of complete culture medium, gently pipette to mix, and stop digestion. Transfer the cell suspension to a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, aspirate the supernatant, resuspend the cells in 20 mL of complete culture medium, and divide the cells in half into 100 mm culture dishes for further culture.

[0028] 2.1.3 Cell cryopreservation When the cells have grown to over 80%, aspirate the culture medium from the original culture dish, add 2 mL of PBS, gently shake and aspirate, add 2 mL of 0.25% trypsin, gently pipette, and incubate at 37°C with 5% CO2 for 5 min. After observing the cells detach from the cell wall under a microscope, add 2 mL of complete culture medium and gently pipette to mix, stopping the digestion. Transfer the cell suspension to a centrifuge tube, centrifuge at 1000 rpm for 5 min, aspirate the supernatant, add 1.5 mL of cell cryopreservation medium (90% FBS + 10% DMSO) and resuspend the cells, transfer to a cell cryopreservation tube, seal with sealing film, transfer to a programmed cooling box, and incubate at -80°C overnight before transferring to liquid nitrogen for storage.

[0029] 2.2 Screening of effective and safe concentrations of GCNY Cells in the logarithmic growth phase were taken, digested, and counted at a ratio of 1 × 10⁻⁶ cells. 4 Cells were seeded at a density of 1 cell / well in 96-well plates. An experimental control group and different concentrations of GCNY (0.125 mg / mL, 0.5 mg / mL, 2 mg / mL, 8 mg / mL, 16 mg / mL) were set up, with 6 replicates per group. When cell confluence reached 80% or higher, waste liquid was aspirated, and the cells were washed with PBS. 100 μL of DMEM (containing 1% antibiotics) medium was added to each well of the control group, and the same volume of GCNY at the corresponding concentration was added to each of the other groups. The cells were incubated at 37°C and 5% CO2 for 24 h. Subsequently, a solution was prepared by mixing CCK-8 and DMEM (containing 1% antibiotics) medium at a ratio of 1:10. Waste liquid was aspirated, and the cells were washed with PBS. 100 μL of this solution was added to each well, and the cells were incubated at 37°C and 5% CO2 for another 1.5 h. The absorbance of each well was measured at 450 nm using a microplate reader.

[0030] 2.3 Establishing an acute photodamage model of HaCaT cells 2.3.1 Establishment of a UVB-induced acute photodamage model of HaCaT cells Cells in the logarithmic growth phase were harvested, digested, counted, and seeded at an appropriate cell density into well plates or cell culture dishes. The experiment consisted of five groups: control group, UVB group, UVB + low-dose (0.125 mg / mL) GCNY group, UVB + medium-dose (0.5 mg / mL) GCNY group, and UVB + high-dose (2 mg / mL) GCNY group. When cell confluence reached 80% or higher, waste liquid was aspirated, and the cells were washed with PBS. The control group was treated with DMEM (containing 1% penicillin and antibiotic-containing medium), and the UVB + GCNY group was given the appropriate concentration of GCNY. The cells were incubated at 37°C in a 5% CO2 incubator for 24 hours. Waste liquid was aspirated, and the cells were washed with PBS. PBS was then added to fully cover the cells, with the PBS volume calculated at 1 mL per 6-well plate. UVB irradiation was then performed using a UV crosslinker with the appropriate irradiation dose set, and the lids of the well plates or cell culture dishes were kept open during irradiation. When using cell culture plates, aluminum foil was used to cover the groups that did not require UVB irradiation, while only the culture dishes that required UVB irradiation were placed in the plates. After irradiation, all containers were replaced with DMEM (containing 1% antibiotics) medium, and cells were collected at different time points after UVB irradiation for subsequent experiments.

[0031] 2.3.2 Effects of UVB on HaCaT cell viability Cells in the logarithmic growth phase were taken, digested, and counted at a ratio of 1 × 10⁻⁶ cells. 4 The cells were seeded at a density of cells / well in 96-well plates. Experimental setups included a control group and groups receiving different UVB irradiation doses (30, 60, and 90 mJ / cm²). 2 Each group was configured with 6 replicates. When cell confluence reached 80% or higher, the waste liquid was aspirated, and the cells were washed with PBS. 100 μL of PBS was added to each well to cover the cells. The caps were opened, and irradiation was performed using a UV crosslinker with different irradiation doses. For each irradiation dose, the cells in all other groups were covered with aluminum foil. After irradiation, the cells were incubated at 37°C and 5% CO2 for 24 hours. Subsequently, a solution was prepared using CCK-8 and DMEM (containing 1% antibiotics) medium at a ratio of 1:10. The waste liquid was aspirated, and the cells were washed with PBS. 100 μL of this solution was added to each well, and the cells were incubated at 37°C and 5% CO2 for another 1.5 hours. The absorbance of each well was then measured at 450 nm using a microplate reader.

[0032] 2.4 Effect of GCNY on HaCaT cell viability after UVB irradiation Cells in the logarithmic growth phase were taken, digested, and counted at a ratio of 1 × 10⁻⁶ cells. 4Cells were seeded at a density of 1 cell / well in 96-well plates. When the cell confluence reached approximately 80%, the experimental procedure was performed as described in 2.3.1. Each well was covered with 100 μL of PBS and irradiated. After irradiation, the cells were incubated at 37°C and 5% CO2 for 24 hours. Subsequently, a solution was prepared by mixing CCK-8 and DMEM (containing 1% antibiotics) at a ratio of 1:10. The waste solution was removed, and the cells were washed with PBS. 100 μL of the solution was added to each well, and the cells were incubated at 37°C and 5% CO2 for another 1.5 hours. The absorbance of each well was then measured at 450 nm using a microplate reader.

[0033] 2.5 Measurement of Reactive Oxygen Species (ROS) Content in Cells Cells in the logarithmic growth phase were harvested and treated with 2.5 × 10⁻⁶ cells. 5 Cells were seeded at a density of 1 cell / well in 6-well plates. When the cell confluence reached approximately 80%, the experimental procedure was performed as described in 2.3.1. After UVB irradiation for 15 min, the waste liquid was aspirated, and the cells were washed once with PBS. 1 mL of DCFH-DA (diluted 1:1000 with serum-free medium) to a final concentration of 10 μmol / L was added under light-protected conditions, and the plates were incubated at 37°C for 30 min. Subsequently, the cells were washed three times with PBS for 3 min each time, and observed and imaged under an inverted fluorescence microscope.

[0034] 2.6 Western Blot analysis of MMP-1 protein expression HaCaT cells were seeded at a density of 5 × 10⁵ cells / dish on 60 mm culture dishes. The experimental procedure was performed according to 2.3.1. Waste liquid was removed at different time points of UVB irradiation and the cells were washed with PBS. 150 μL of RIPA lysis buffer (RIPA:PMSF:phosphatase inhibitor = 100:1:1) was added and the cells were lysed on ice for 30 min. Samples were collected for protein concentration determination and subsequent Western blotting experiments were completed.

[0035] The specific experimental method is as follows: (1) Protein extraction: Weigh 30 mg of mouse skin tissue into an EP tube, add 300 μL of RIPA lysis buffer (RIPA: PMSF: phosphatase mixed inhibitor = 100:1:1), add two 1 mm grinding beads and one 2 mm grinding bead to each tube, set the grinder to a low temperature program of 7 m / s for 6 min, place on ice for lysis for 30 min, centrifuge at 12000 g for 10 min, and take the supernatant for analysis.

[0036] (2) Protein concentration determination using the BCA kit: Add 1.6 μL and 78.4 μL of PBS to each sample tube. After mixing the liquids in each tube, add 200 μL to each well of a 96-well plate. Prepare the BCA working solution according to the instructions at a ratio of A:B=50:1 and mix well. Add 200 μL of BCA working solution to each well and incubate in a 37°C oven for 30 min. Measure the absorbance of each well at 562 nm using a microplate reader. After plotting the standard curve, calculate the sample protein concentration and the loading system.

[0037] (4) Protein denaturation: Add the calculated protein sample solution, PBS, and 5× loading buffer to the EP tube respectively. Heat at 100℃ for 10 min in a dry heat denaturer and then store in a -20℃ refrigerator.

[0038] (5) Gel preparation: Select different concentrations of separating gel and stacking gel according to the measured protein molecular weight. The specific ratio is shown in Table 1.

[0039] Table 1 Raw material ratios for separating gel and stacking gel

[0040] (6) Electrophoresis: Place the prepared gel glass plates into the electrophoresis tank, with the shorter plates facing inwards and the longer plates facing outwards. Add freshly prepared 1× electrophoresis buffer between the glass plates until it overflows the gaps between the glass plates. Recycled electrophoresis buffer can be added to both sides. Gently remove the comb to remove air bubbles from the wells. After warming the protein sample, vortex to mix and centrifuge at low speed. Add 1.5 μL of Pageruler™ Prestained Protein Ladder to the wells on both sides of the sample to be tested, and add 5 μL of the sample to be tested in sequence in the middle. Then connect the power supply and electrophoresis at 70V for about 30 minutes until the Pageruler™ Prestained Protein Ladder is clearly separated. Then increase the voltage to 110V and stop electrophoresis when the target protein molecular weight corresponds to the marker position clearly separated.

[0041] (7) Semi-dry electroporation: Cut the gel to the corresponding molecular weight and cover it on the PVDF membrane. Use a roller to gently remove the air bubbles between the PVDF, filter paper and gel. Adjust the position of the gel and finally cover it with the upper filter paper. After loading the electroporation box, perform the electroporation under constant voltage of 25V and 1.3A. The electroporation time is adjusted according to the different molecular weights of the proteins.

[0042] (8) Sealing: Use rapid sealing solution to seal on a shaker at room temperature for 10-20 min, then wash with TBST solution 3 times, 10 min each time.

[0043] (9) Primary antibody incubation: Prepare the corresponding primary antibody with antibody diluent according to the antibody concentration recommended in the instructions, place the strip in the corresponding primary antibody back to back in the antibody incubation box, and incubate at 4°C on a shaker for more than 16 hours.

[0044] (10) Secondary antibody incubation: Wash 3 times with TBST solution, 10 min each time. Place the band in the secondary antibody of the corresponding species as the primary antibody and incubate at 4℃ on a shaker for 30 min.

[0045] (11) Development: Wash with TBST solution 3 times, 10 min each time. Prepare ECL colorimetric solution according to the ratio of solution A: solution B = 1:1, add it evenly to the strip, develop it under a chemiluminescent gel imaging system, export the image and perform statistical analysis on the image using ImageLab software.

[0046] 2.12 Statistical Analysis All experiments were repeated at least three times. Data from each group were expressed as mean ± standard error (⁻). ±SEM) indicates the statistical significance of changes among groups using one-way ANOVA performed with SPSS 26 statistical software. For homogeneous variance analysis, the Bonferroni method was used; for unequal variances, Dunnett's T3 method was used. P <0.05 indicates that the difference is significant.

[0047] 3. Results 3.1 Protective effect of GCNY against UVB-induced HaCaT cell damage This application investigated the effects of different doses of UVB irradiation on HaCaT cell viability using the CCK-8 assay. A range of 30-150 mJ / cm² was selected. 2 To test the dose range, the results Figure 1 As shown, cell viability decreases with increasing UVB radiation dose. P <0.01). When HaCaT cells are at a concentration greater than 30 mJ / cm², 2 After 24 hours of irradiation at a UVB dose, cell viability significantly decreased. Therefore, this application selected 30 mJ / cm². 2 The UVB radiation dose was used to complete most of the subsequent experiments. In addition, this application also observed the effects of different concentrations of GCNY on HaCaT cell activity, such as... Figure 2 As shown in Figure A, HaCaT cells incubated with GCNY at concentrations ranging from 0 to 16 mg / mL for 24 hours did not exhibit cytotoxicity; in fact, cell viability in some concentration groups (such as the 2 mg / mL group) was significantly higher than that in the control group. Finally, this application investigated the effect of GCNY pretreatment on the cell viability of UVB-irradiated cells. Figure 2As shown in B, incubating GCNY can significantly restore the viability of HaCaT cells before UVB irradiation. The above results indicate that GCNY plays a key role in improving the cell viability of HaCaT cells after UVB radiation.

[0048] 3.2 Effect of GCNY on the ROS content in HaCaT cells after UVB irradiation As Figure 3 can be seen, compared with the control group, the intracellular ROS level in the UVB group was significantly increased, and the fluorescence intensity in the UVB group was about more than twice that of the control group. After treatment with GCNY, the ROS level induced by UVB irradiation was significantly reduced. Among them, the ROS levels of UVB+GCNY-L, UVB+GCNY-M, and UVB+GCNY-H decreased successively, and the decrease in the ROS level in the high-dose group (UVB+GCNY-H) was more significant (the fluorescence intensity was close to the control group level).

[0049] 3.3 Effect of GCNY on the expression level of MMP-1 protein in HaCaT cells after UVB irradiation As Figure 4 can be seen, MMP1 is a key enzyme that can directly degrade collagen. Compared with the treatment with GCNY alone, the expression of MMP1 in the group irradiated only with UVB (30 mJ / cm²) was significantly increased; after treatment with GCNY, the expression of MMP1 induced by UVB was significantly inhibited, and the inhibitory effect on the expression of MMP1 in the UVB+medium-concentration GCNY group was more significant, suggesting that GCNY can down-regulate the overexpression of MMP1 induced by UVB.

[0050] Example 2 Verification test on the prevention and treatment effect of Gancao Yangyin Decoction on acute light damage in mice I. Experimental preparation 1. Experimental materials and instruments 1.1 Experimental animals SPF-grade female Kunming (KM) mice, 6 weeks old, weighing 18 - 22 g, were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. (License number: SCXK (Xiang) 2021-0002). All animals were housed in the SPF-grade animal house of the Key Laboratory of Basic Pharmacology of the Ministry of Education, Zunyi Medical University. The feeding conditions were: environmental temperature 18 - 25 °C, humidity 40% - 70%, 12-h light-dark cycle, and free access to food and water.

[0051] 1.2 Main instrument equipment The UVB lamp (TL20W / 01) was purchased from Philips GmbH, Germany; the digital camera was purchased from Sony GmbH, Japan; the TP1020 tissue dehydrator was purchased from Leica GmbH, Germany; the EG1150 paraffin embedding machine, the RM2245 rotary microtome, and the TKY-TKA slide spreader / batter were purchased from Hubei Taikang Company, China; the Milli QA pure water processor was purchased from Millipore, France; and the ultrasonic cleaner was purchased from Shanghai Yijing Ultrasonic Instrument Co., Ltd., China.

[0052] 1.3 Main Experimental Reagents Licorice Yin-Nourishing Decoction: The following ingredients were weighed according to the following ratio: licorice: ginseng slices: Solomon's seal: dandelion: dried ginger: monk fruit: jujube = 5:2:3:3:3:4:2. The ingredients were mixed in proportion and then decocted in water for at least 1 hour to obtain the Licorice Yin-Nourishing Decoction. The concentration of the decoction was 1.32 g / ml. Hematoxylin was purchased from Wuhan Sewell Biotechnology Co., Ltd., and the elastic fiber staining kit (modified Gomori aldehyde fuchsin method), modified Masson trichrome staining kit, and G1346 mast cell staining solution (toluidine blue method) were purchased from Beijing Solarbio Technology Co., Ltd.

[0053] II. Experimental Methods 2.1 Experimental Grouping Six-week-old female Kunming mice were selected and acclimatized under normal feeding conditions for one week. A hairless area of ​​approximately 3cm × 5cm was created on the back of the experimental animals, and the hair was shaved every 3 days. After hair removal, the mice were randomly divided into five groups: control group, UVB group, UVB + vitamin E group (UVB + VE, 170mg / kg), UVB + low-dose GCNY group (UVB + GCNY-L, 2.5g / kg), and UVB + high-dose GCNY group (UVB + GCNY-H, 10g / kg).

[0054] 2.2 Establishment and administration of an acute photodamage model of mouse skin 2.2.1 UVB Modeling Irradiation chamber preparation: Stainless steel front-opening box (67cm×54.5cm×54cm), with 4 312nm UVB lamps installed at 3cm intervals on the top of the box; a corrugated cardboard box is placed in the center of the bottom of the box and the position of the fixation device is marked. The distance between the fixation device and the lamp is 28cm. The mouse is placed perpendicular to the lamp. During irradiation, a green cloth is used to shield the light and prevent leakage.

[0055] Preparation before irradiation: Preheat the UVB lamp for 15 minutes, measure the intensity of the UV irradiator (retest periodically), and adjust the time according to "irradiation dose (mJ / cm²) = irradiation intensity (mW / cm²) × time (s)" to ensure consistent dose; fix the mice in a self-made restraint, cover their heads with black cloth and expose their backs.

[0056] MED (minimum dose that still produces erythema after 24 hours) determination: 80 mJ / cm² was selected as the minimum erythema dose in the preliminary experiment.

[0057] Modeling protocol: UVB induced acute photodamage in mice, with continuous irradiation for 14 days (1 time / day): starting with 2 MED, increasing by 2 MED every 3 days, and maintaining 8 MED for the last 5 days; the control group did not receive irradiation.

[0058] 2.2.2 Administration method The medication was administered via gavage once daily, followed by ultraviolet irradiation 30 minutes after administration. The Control and UVB groups were administered 0.9% saline via gavage, the UVB+VE group was administered vitamin E (170 mg / kg) via gavage, and the UVB+GCNY-L group was administered (2.5 g / kg) via gavage. The UVB+GCNY-L group was administered (10 g / kg) via gavage.

[0059] 2.3 Sampling and Specimen Preparation On day 15 post-irradiation, mice were intraperitoneally injected with 100 mg / kg sodium pentobarbital. Immediately after anesthesia, images of the mouse's back skin were recorded using a camera. The back skin was wiped with 75% alcohol, and the skin was harvested parallel to the spine at the injection site. A portion of the skin was trimmed into 1 cm wide strips and fixed in 4% neutral paraformaldehyde solution for 24–36 h. The remaining skin was cut into pieces weighing approximately 20 mg each and placed in EP tubes for storage at -80°C for later use.

[0060] 2.4H&E staining: Raw materials for eosin staining solution: 0.5-1g of water-soluble eosin powder, 100ml of distilled water, and 1-2 drops of glacial acetic acid.

[0061] Preparation method of eosin dye: (1) Dissolve eosin powder directly in distilled water and stir until completely dissolved.

[0062] (2) Add glacial acetic acid (the purpose is to fix the staining effect and prevent fading), mix well and filter before use. No special storage is required.

[0063] Specific steps for H&E staining: (1) Transparent: Place xylene I and xylene II in the solution for 10 minutes each; (2) Dewaxing: Dewaxing with a gradient of ethanol (anhydrous ethanol, 95% ethanol, 80% ethanol, 75% ethanol) followed by double-distilled water washing; (3) Staining: Stain with hematoxylin solution for 5 min, then rinse with running water for 60 s; (4) Differentiation: Differentiate with 1% hydrochloric acid ethanol for 3-5 seconds, then rinse with running water for 5 minutes; (5) Staining: Counterstain with eosin for 5 min, then rinse off excess stain with running water for 30 s; (6) Dehydration: 70% ethanol 2s → 80% ethanol 2s → 95% ethanol 10s → anhydrous ethanol 15s; (7) Mounting: After the sections have air-dried, mount them with a neutral resin containing xylene.

[0064] 2.5 Gomori Aldehyde Fuchsin Staining: Performed according to the method described in the instructions of the Elastic Fiber Staining Kit (Modified Gomori Aldehyde Fuchsin Method).

[0065] 2.6 Masson staining: Performed according to the instructions of the modified Masson trichrome staining kit.

[0066] 2.7 Mast cell staining: Performed according to the method described in the instructions for G1346 mast cell staining solution (toluidine blue method).

[0067] 2.8 Western blot assay for proteins: Refer to the experimental content in section 2.6 of Example 1. 2.9 Statistical Analysis of Data Experimental data were analyzed using SPSS 21.0 statistical software. All data are expressed as (Mean ± SEM). One-way ANOVA was used to compare group differences in means. The LSD test was used to test homogeneity of variance. Dunnett's T3 was used to compare group differences in means. P < 0.05 was considered statistically significant.

[0068] III. Experimental Results 3.1 Results of the intervention effect test of different treatments on acute photodamaged skin in mice To evaluate the intervention effect of GCNY pretreatment on acute photodamaged skin in mice, this embodiment treated KM mice according to the modeling and drug administration methods described in Example 1. Skin images of the backs of mice with different treatments were collected at different time points after UVB irradiation. The experimental design is as follows: Figure 5 As shown, the results are as follows Figure 6 As shown.

[0069] Depend on Figure 6It is evident that, throughout the experimental period, the skin of mice in the control group that did not receive UVB irradiation maintained normal texture, with a smooth and delicate consistency, and no wrinkles or erythema appeared. However, the UVB group mice developed skin wrinkles starting from the 4th day of irradiation. As the irradiation time increased, the degree of erythema and the depth of wrinkles progressively worsened. By the 15th day after the first irradiation, their skin thickness had significantly increased, exhibiting deep wrinkles and a leathery appearance, with some areas accompanied by local ulceration. These characteristics confirm the successful establishment of the acute photodamage model of mouse skin. The positive control groups UVB+VE, UVB+GCNY-L, and UVB+GCNY-H all significantly alleviated the above-mentioned acute photodamage phenotypes. Specifically, although a few wrinkles and erythema were still visible in the UVB+VE group, the symptoms were significantly reduced compared to the UVB group. The skin protection effect of GCNY showed a dose-dependent increasing trend—the mice in the low-dose UVB+GCNY-L group had no obvious skin damage, only mild desquamation, and the erythema area was significantly reduced compared to the UVB group. The protective effect of the high-dose UVB+GCNY-H group was similar to that of the UVB+VE group, and it could further reduce skin roughness, with a more significant reduction in erythema area.

[0070] To quantify the degree of photodamage to the skin, this study introduced a macroscopic skin scoring system. The scoring criteria were referenced from "Bissett DL, Chatterjee R, Hannon DP. Photoprotective effect of topical anti-inflammatory agents against ultraviolet radiation-induced chronic skindamage in the hairless mouse [J]. Photodermatol Photoimmunol Photomed, 1990, 7(4): 153-158.", as detailed in Table 2 and... Figure 7 As shown, a higher score indicates more severe light damage.

[0071] Table 2 Evaluation criteria for acute photodamage to the skin

[0072] Evaluation results as follows Figure 8 As can be seen, compared with the control group, the macroscopic skin score of mice in the UVB group was significantly higher; while the macroscopic scores of each pretreatment group (UVB+VE group, UVB+low-dose GCNY group, UVB+high-dose GCNY group) were significantly lower than those of the UVB group. This quantitative result is consistent with the qualitative observation conclusion of skin appearance, further verifying that GCNY has a protective effect against UVB-induced acute photodamage to the skin in mice.

[0073] 3.2 H&E staining results: After ultraviolet radiation, the thickness of the epidermis and dermis changes significantly histologically. Therefore, the degree of photodamage to mouse skin can be characterized by quantifying these indicators in HE staining. Epidermal hyperplasia is one of the important characteristics of acute photodamage. The H&E staining results and statistical results of each indicator are shown below. Figure 9 As shown in Figures A through D, the epidermal thickness of the UVB group increased compared to the control group. However, pretreatment with low and high doses of GCNY and VE before UVB irradiation significantly reduced the degree of epidermal thickening. Figure 9 (B)

[0074] A similar pattern of change was observed in dermal thickness. Compared to the control group, the dermal thickness of the UVB group increased sharply, while the dermal thickness of mice in all pretreatment groups (low-dose GCNY pretreatment group, high-dose GCNY pretreatment group, and VE pretreatment group) decreased significantly. Figure 9 (C)

[0075] The above results indicate that GCNY can inhibit UVB-induced increases in epidermal and dermal thickness in mouse skin. 3.3 Masson staining results: Masson staining results are as follows: Figure 9 As shown in Figure E, histologically, the collagen fibers in the control group were wavy and relatively evenly and orderly distributed. In contrast, the collagen fiber content in the UVB group was significantly reduced, with disordered and irregular dermal fiber arrangement, and some areas showing signs of photodamage such as breakage and sparseness. However, the low-dose GCNY pretreatment group, the high-dose GCNY pretreatment group, and the VE pretreatment group all significantly improved the reduction of collagen fibers, increasing the density and number of collagen fibers. This indicates that both GCNY and VE can inhibit the reduction of collagen fibers in skin tissue caused by UVB.

[0076] 3.4 Mast cell staining results Mast cell staining results as follows Figure 10 As shown in Figure A, the number of mast cells in the UVB group was significantly increased compared to the control group, indicating that UVB irradiation stimulated mast cell production, leading to an inflammatory response. Compared to the UVB group, the number of mast cells in mice in the UVB+VE group, UVB+GCNY-L group, and UVB+GCNY-H group was significantly reduced.

[0077] 3.5 Gomori staining results The extracellular matrix constitutes the skin's framework, with collagen and elastic fibers being its most important components and fundamental to maintaining the skin's structural integrity. Gomori staining results are as follows... Figure 10As shown in Figure B, changes in the elastic fibers of mouse skin can be characterized by Gomori staining.

[0078] As can be seen, the elastic fibers in the Control group exhibit a network structure, with slender and orderly arrangement, without curling or twisting. In contrast, the UVB group shows a flocculent network of elastic fibers, with obvious damage to the elastic fibers, exhibiting characteristic changes of photodamage such as thickening, curling, and disordered arrangement, accompanied by breakage, twisting, and proliferation; some elastic fibers are even intertwined.

[0079] Mice in the UVB+VE, UVB+GCNY-L, and UVB+GCNY-H groups showed varying degrees of repair of elastic fibers compared to the UVB group. The number of elastic fibers in the UVB group decreased sharply, while the UVB+GCNY-H group showed significant improvement in skin elastic fibers, exceeding that of the UVB+VE group. The number of elastic fibers increased significantly, exhibiting a fine, interwoven pattern with an orderly arrangement, and some fibers had recovered their reticular structure. These results suggest that GCNY can resist elastic fiber damage caused by ultraviolet radiation and maintain its normal physiological structure.

[0080] 3.6 Results of Western blot assay for protein immunoblotting The results of the Western blot experiment are as follows: Figure 11 As shown, MMP-1 is an important class of enzymes that can directly degrade collagen. (From...) Figure 11 As can be seen, compared with the control group, MMP-1 expression was significantly increased in the UVB group. Conversely, the UVB+GCNY-H group showed the highest inhibition rate of MMP-1 expression, slightly better than the UVB+VE group. These results indicate that GCNY can downregulate the overexpression of MMP-1 in UVB-induced acute photodamaged skin.

[0081] In summary, this invention provides a novel sun protection approach. Through mouse modeling and gavage treatment, this invention verified the feasibility of using licorice-based yin-nourishing decoction for sun protection, showing effects comparable to or even better than vitamin E. This invention provides a highly effective and stable treatment method for sun protection.

[0082] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments without creative effort, as shown in these embodiments, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of a traditional Chinese medicine composition in the preparation of sunscreen products, characterized in that, The traditional Chinese medicine composition includes a traditional Chinese medicine composition used in the preparation of Licorice Nourishing Yin Decoction.

2. The application according to claim 1, characterized in that, The components of the traditional Chinese medicine composition used to prepare the Licorice Nourishing Yin Decoction include 2-6 parts of licorice, 1-5 parts of ginseng, 1-3 parts of dried ginger, 1-3 parts of dandelion, 2-6 parts of monk fruit, 1-5 parts of Solomon's seal, and 2-6 parts of jujube.

3. The application according to claim 1 or 2, characterized in that, The traditional Chinese medicine composition includes Licorice Nourishing Yin Decoction.

4. The application as described in claim 1, characterized in that, The sun protection includes protection against ultraviolet-induced acute photodamage to the skin.

5. The application as described in claim 4, characterized in that, The ultraviolet radiation includes medium-wave ultraviolet radiation.

6. The application as described in claim 4 or 5, characterized in that, The symptoms of acute photodamage to the skin include any one or more of the following: 1) Skin develops erythema; 2) Wrinkles appear on the skin; 3) Increased thickness of the upper epidermis; 4) Increased thickness of the dermis; 5) Increased number of mast cells; 6) The elastic fiber network is flocculent and damaged, with thickening, curling and / or disordered arrangement, accompanied by breakage, twisting and / or hyperplasia; 7) Increased MMP-1 protein content.

7. The application as described in claim 1, characterized in that, The products include pharmaceuticals.

8. The application as described in claim 7, characterized in that, When the drug is applicable to mice, the dosage of licorice-nourishing yin decoction is 2.5g / kg to 10g / kg of mouse body weight.

9. The application as described in claim 1, characterized in that, The product can be taken orally.

10. The application as described in claim 1, characterized in that, The dosage form of the product includes liquid formulations.

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