Application of isodecursorlactone in preparation of medicine for preventing or treating skin photoaging injury

Abstract

The invention belongs to the technical field of biomedical treatment, and particularly discloses application of isodecursolactone in preparation of a medicine for preventing or treating skin photoaging injury. The invention develops the protective effect of the marmeisn on the skin photoaging injury caused by UVB, the action mechanism and association of the marmeisn are determined from the perspectives of oxidative stress, DNA injury, cell cycle, apoptosis and the like, potential key targets and pathways of the marmeisn are known based on transcriptomics, the skin photoaging injury resisting effect of the marmeisn component is clarified, and the application of the marmeisn to the skin photoaging injury caused by UVB is developed. The invention provides a credible basis for developing an anti-photoaging injury protective agent derived from plant active components, and provides an applicable component for preventing and treating skin photoaging injury caused by UVB.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of isopimpinellin in the preparation of a drug for preventing or treating skin photoaging damage. Background Art

[0002] Skin aging is a complex pathological process, including intrinsic aging and extrinsic aging. The former is mainly affected by genetic factors, while the latter depends on environmental factors such as chemical pollution, ionizing radiation, light, etc. Among them, ultraviolet (UV) irradiation, as the main factor of skin extrinsic aging damage, is called photoaging, which was first elaborated by Kligman et al. in 1986. UV is divided into: UVA (320 - 400 nm), UVB (280 - 320 nm), and UVC (100 - 280 nm) according to wavelength. For the skin, the damage caused by UVB is the greatest, which is 800 - 1000 times that of UVA.

[0003] Common clinical manifestations of skin photoaging include wrinkles, increased spider veins, pigmentation, and sunburn, etc. In terms of the mechanism, current research shows that the mechanism of skin photoaging is mainly related to increased cellular DNA damage, ROS levels and oxidative stress, elevated inflammation levels, non - enzymatic glycosylation, and decreased collagen levels. Excessive external ultraviolet irradiation can ultimately cause changes in the internal microenvironment such as inflammation and glycosylation, and its key mechanism is still related to the up - regulation of skin cell ROS and increased oxidative stress. Therefore, reducing the production of skin cell ROS is the top - priority strategy for alleviating skin photoaging damage.

[0004] Recently, considering the impact of UVB on skin beauty and health, photoaging damage has occupied an important position in the beauty industry and clinical treatment. Therefore, finding suitable therapeutic drugs has become an urgent task. Currently, the first - line therapeutic drugs for skin photoaging damage are mainly topical retinoid drugs (such as tretinoin, tazarotene, adapalene, etc.). Although such drugs have provided more choices with optimization and iteration, many patients still stop treatment due to adverse reactions such as skin irritation, redness, desquamation, dryness, burning, stinging, and peeling. Therefore, researchers and the industry are looking forward to the birth and application of drugs with better efficacy and no side effects, which has shifted their attention to natural plant ingredients. How to obtain plant - derived drugs that can improve UVB - induced skin photoaging damage has become a problem to be solved. Summary of the Invention

[0005] The present invention aims to explore the anti-skin photoaging damage effect of a novel plant-derived active ingredient, isopimpinellin. It is to solve the adverse reactions of existing drugs for skin photoaging damage, such as skin irritation, redness, desquamation, dryness, burning, stinging, and peeling, etc., in order to provide a drug ingredient with better efficacy and no side effects in the field of beauty and skin care treatment, and to provide an applicable ingredient for preventing and treating UVB-induced skin photoaging damage.

[0006] To achieve the above object, the specific technical solutions adopted by the present invention are as follows:

[0007] The present invention provides the application of isopimpinellin in the preparation of a drug for preventing or treating skin photoaging damage. The isopimpinellin is a kind of furanocoumarin, which has been isolated from different medicinal plants and can be directly purchased. Its structural formula is shown as follows:

[0008]

[0009] Furthermore, the drug includes an active ingredient, and the active ingredient is isopimpinellin.

[0010] Furthermore, in some embodiments, the concentration of isopimpinellin in the drug is 1 - 100 μM.

[0011] Furthermore, the drug also includes a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" means that when the carrier is appropriately administered to animals or humans, they do not produce adverse, allergic, or other adverse reactions. The pharmaceutically acceptable carrier can be selected from one or more of solvents, diluents, fillers, surfactants, absorption promoters, disintegrants, wetting agents, dispersants, etc. In one embodiment, the pharmaceutically acceptable carrier is the solvent ethanol.

[0012] Furthermore, the dosage form of the drug includes but is not limited to any one of injections, emulsions, tablets, powders, granules, gels, ointments, capsules, oral liquids.

[0013] Furthermore, the skin photoaging damage is UVB-induced skin photoaging damage, specifically one or more of wrinkles, erythema, dryness, increased spider veins, pigmentation, etc.; the drug is used before light (including UVB) irradiation, and the effect is better than that after irradiation.

[0014] Furthermore, the isopimpinellin prevents or treats skin photoaging damage through the following pathways:

[0015] 1) Oxidative stress: including reducing the ROS level, increasing the mitochondrial membrane potential, increasing the activity of SOD, and reducing the MDA content;

[0016] 2) DNA damage and apoptosis: including improving the accumulation and arrest of cells in the S phase and G2 / M phase, reducing apoptosis, and restoring the cell proliferation level.

[0017] Furthermore, the prevention or treatment of skin photoaging damage by imperatorin involves the following target and pathway gene regulations:

[0018] 1) The expression of the SPRR1B gene related to the epidermal barrier was significantly upregulated;

[0019] 2) The expression of the IFI6 gene related to anti-cell apoptosis was significantly upregulated;

[0020] 3) The expressions of the MAPK, NF-κB, HO-1, and mTOR genes related to epidermal inflammation were significantly downregulated.

[0021] The present invention has the following beneficial effects:

[0022] 1. The present invention confirmed the improvement effect of imperatorin on UVB-induced skin photoaging damage and discovered a new indication for imperatorin.

[0023] 2. Through the research on the mechanism of action, it was found that imperatorin exerts its effect by affecting mitochondrial-related oxidative stress, apoptosis, and metabolism, and improving skin photoaging cell damage manifestations such as DNA damage and cell proliferation activity damage in skin cells. Description of the Drawings

[0024] Figure 1 : Flow chart of the overall method of the cell experiment (Examples 1-3) of the present invention.

[0025] Figure 2 : Detection results of cell viability in Example 1. In the figure, A is the viability of HaCaT cells treated with different concentrations of imperatorin, B is the viability of HaCaT cells irradiated with different doses of UVB, C is the microscopic picture of HaCaT cells treated with different drug addition times, and D is the viability of HaCaT cells treated with different drug addition times.

[0026] Figure 3 : Detection results of ROS level in Example 1.

[0027] Figure 4 : Detection results of mitochondrial membrane potential in Example 1. In the figure, from top to bottom are the polymer (J-aggregates), monomer (monomer), and merge views in turn.

[0028] Figure 5 : Detection results of antioxidant enzyme activity (A) and oxidative damage level (B) in Example 1.

[0029] Figure 6 : Results of detecting cell apoptosis by Hoechst staining method in Example 2.

[0030] Figure 7 : Results of detecting cell cycle by propidium iodide (PI) staining method in Example 2.

[0031] Figure 8 : Results of detecting DNA synthesis and cell proliferation by EdU method in Example 2.

[0032] Figure 9 : Results of screening differentially expressed genes in transcriptomics in RNA-seq data analysis of Example 3. In the figure, A and B are the volcano plot and heat map of differentially expressed genes between the UVB group and the marmesin group, respectively.

[0033] Figure 10 : Results of verifying differentially expressed genes by qRT-PCR in Example 3.

[0034] Figure 11 : Results of detecting genes related to photoaging damage by qRT-PCR in Example 3.

[0035] Figure 12 : Results of animal experiments in Example 4. Specific implementation manners

[0036] In this specific implementation manner, through cell experiments, fluorescence microscopy, laser confocal microscopy, flow cytometry, qRT-PCR and other technologies are used to explore the protective effect of the plant-derived active ingredient - marmesin on UVB-induced skin photoaging damage, and clarify its mechanism of action and association from the perspectives of oxidative stress, DNA damage, cell cycle and apoptosis. Based on transcriptomics, its potential key targets and pathways are understood, and the anti-skin photoaging damage effect of the marmesin component is clarified, providing a reliable basis for the development of an anti-photoaging damage protectant derived from plant active ingredients. In addition, this specific implementation manner also more intuitively reflects the anti-skin photoaging damage effect of marmesin through animal experiments.

[0037] The following is a detailed description in combination with the accompanying drawings and specific examples. Among them: Examples 1-3 are cell experiments, and the overall method process is as Figure 1 shown. It mainly uses the classical in vitro cell model of skin photoaging - HaCaT cell model to simulate the reaction of skin keratinocytes under UVB irradiation, and studies the damage mechanism of photoaging to cells, including oxidative stress, DNA damage, and target pathways. Example 4 is an animal experiment.

[0038] Example 1: Experiments related to the protective effect of marmesin on UVB-induced oxidative stress in HaCaT cells

[0039] 1. Cell Viability Detection

[0040] 1.1 UVB irradiation dose and drug dose screening

[0041] 3×10 3 HaCaT cells (immortalized human keratinocytes) were quantitatively inoculated into 96-well cell culture plates at a density of 100 cells / well. After 24 or 48 hours in the cell culture incubator, the HaCaT cells were irradiated with different doses using a UVB irradiator; or different concentrations of peucedanum praeruptolide diluted with cell culture medium were added (before the experiment, the peucedanum praeruptolide powder was first prepared into a high-concentration mother solution with a small amount of anhydrous ethanol; in the cell experiment, the mother solution was further diluted to various experimental concentrations using cell culture medium, i.e., DMEM high-glucose complete medium containing 10% special fetal bovine serum and 1% penicillin-streptomycin double antibody). Each group was set up with gradient concentration / irradiation dose, and each group had 6 parallels. PBS buffer or culture medium was used to supplement the edge wells around the 96-well cell culture plate to avoid experimental errors caused by edge effects. After culturing for 24 or 48 hours in a 37°C, 5.0% CO2 constant temperature cell culture incubator, discard the culture medium in each well and add 100 μL of premixed complete medium + 10 μL of CCK-8 solution to each well, 110 μL each. Incubate at 37°C for 1-2 hours. Place in a microplate reader to detect the absorbance of each well of the 96-well plate at 450 nm. Cell activity (%) = (OD experimental group - OD blank control group) / (OD normal control group - OD blank control group) × 100%.

[0042] The results are as follows Figure 2 As shown in AB, Figure 2 A shows that the cell activity of the experimental groups with different concentrations of purpurogenol did not decrease compared with the normal control group, and the cell activity of each experimental group was above 80% of the safe concentration, indicating that 1-100μM purpurogenol was non-cytotoxic and had better safety; but since the 100μM concentration group showed a certain degree of cell activity decrease compared with the previous groups, 50μM was used as the highest concentration in subsequent experiments in this case. Figure 2 B shows 1-300mJ / cm 2 The cell proliferation activity of the UVB-irradiated group was significantly lower than that of the blank group, indicating that UVB can clearly damage HaCaT cells.

[0043] 1.2 Timing of drug dosage addition and establishment of effect

[0044] At 30mJ / cm 2HaCaT cells were treated with a UVB irradiation dose of 50 μM and an addition concentration of marmesin. Marmesin was administered before and after UVB irradiation (denoted as M50+UVB and UVB+M50 groups, respectively), and control groups of blank, single UVB irradiation, and single marmesin treatment were set up (denoted as Control, UVB, and M50 groups, respectively). The cell viability of HaCaT cells in each group was detected.

[0045] The results are as Figure 2 shown in C-D. The cell proliferation activities of the UVB+M50 group and the M50+UVB group were increased to varying degrees compared with the UVB group, and the cell proliferation activity of the M50+UVB group was higher than that of the UVB+M50 group. This indicates that intervention with marmesin before or after UVB irradiation can protect HaCaT cells, but the protective effect is better when marmesin is administered before UVB irradiation.

[0046] 2. Detection of reactive oxygen species (ROS) level

[0047] Cell slides were pre-placed in a 6-well cell culture plate. HaCaT cells were seeded onto the cell slides in the 6-well cell culture plate at a density of 5×10 3 cells / well and cultured in a constant temperature cell incubator at 37 °C and 5.0% CO2. After 24 h, they were treated with different concentrations (10 μM, 20 μM, 50 μM) of marmesin, and after 48 h, they were irradiated with a UVB irradiator at a dose of 30 mJ / cm 2 . The DCFH-DA probe was diluted 1:1000 with serum-free culture medium. The original cell culture medium in each well was discarded, and 1 mL of the diluted DCFH-DA probe was added to each well. Incubate at 37 °C for 20 min, wash 3 times with serum-free culture medium, take out the cell slides, mount them on glass slides, and observe the cell fluorescence under a fluorescence microscope.

[0048] The results are as Figure 3 shown. The ROS level increased after UVB irradiation, while marmesin could dose-dependently reduce the ROS level, and intervention with medium and high concentrations (20 μM, 50 μM) of marmesin was significant.

[0049] 3. Detection of mitochondrial membrane potential

[0050] Cell slides were pre-placed in a 6-well cell culture plate. HaCaT cells were seeded onto the cell slides in the 6-well cell culture plate at a density of 5×10 3Inoculate HaCaT cells onto the cell culture slides in a 6-well cell culture plate at a density of [number] cells / well, and culture them in a constant temperature cell incubator at 37°C and 5.0% CO₂. After 24 hours, treat them with imperatorin at different concentrations (10 μM, 20 μM, 50 μM). After 48 hours, irradiate them with a UVB irradiator, and the irradiation dose is 30 mJ / cm 2 。Aspirate and discard the culture medium, and add 1 mL of complete culture medium. Add 1 mL of JC-1 staining working solution to each well, and mix well by shaking in a cross / figure-eight pattern. Incubate at 37°C for 20 minutes. Discard the supernatant, and wash each well twice with 1 mL of JC-1 staining buffer. Take out the cell culture slides, place them on glass slides for mounting, and observe the cell fluorescence under a confocal microscope. When the mitochondrial membrane potential is high, JC-1 forms polymers (J-aggregates) in mitochondria, showing red fluorescence; when the mitochondrial membrane potential is low, JC-1 is a monomer in mitochondria, showing green fluorescence.

[0051] The results are as Figure 4 shown. After UVB irradiation, HaCaT cells showed strong green fluorescence and weak red fluorescence, indicating a decrease in mitochondrial membrane potential and the cells being in the early apoptotic stage. Intervention with marmesin increased the red fluorescence intensity and decreased the green fluorescence intensity of HaCaT cells after UVB irradiation. The results showed that marmesin could restore the decrease in mitochondrial membrane potential of HaCaT cells induced by UVB.

[0052] 4. Detection of antioxidant enzyme activity and level of oxidative damage

[0053] 4.1 Detection of superoxide dismutase (SOD) activity

[0054] Aspirate the cell culture medium completely, and wash the cells once with pre-cooled PBS. Add 200 μL of SOD sample preparation solution to each well, and pipette to lyse the cells thoroughly. Centrifuge at 4°C and 12,000 rcf for 3 - 5 minutes, and take the supernatant. Add it to a 96-well cell culture plate, and then add each detection working solution in sequence, and mix well by shaking in a cross / figure-eight pattern. Incubate at 37°C for 30 minutes. Place it in an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance value of each well in the 96-well cell culture plate at 450 nm. Among them, superoxide dismutase (SOD) can catalyze the dismutation of superoxide anions to generate hydrogen peroxide (H₂O₂) and oxygen (O₂), and it is an important antioxidant enzyme in organisms.

[0055] 4.2 Detection of lipid oxidation (MDA) level

[0056] Use cell lysate to fully lyse cells on ice. Centrifuge at 12,000 rcf for 10 min and take the supernatant. Determine the protein concentration using a BCA protein concentration assay kit. Add 100 μL of the supernatant from each group and 200 μL of the MDA detection working solution to a 1.5 mL Eppendorf tube. After mixing, heat at 100 °C for 15 min. Cool in a water bath, centrifuge at 1000 rpm for 10 min, and take the supernatant. Add 200 μL to each well of a 96-well plate and measure the absorbance at 532 nm for each well of the 96-well cell culture plate using an enzyme-linked immunosorbent assay (ELISA) reader. Among them, malondialdehyde (MDA) is a natural product of lipid oxidation in organisms, and the level of lipid oxidation can be understood by detecting the level of MDA.

[0057] The results are as Figure 5 shown. Marmesin can reduce UVB-induced oxidative stress by increasing the activity of SOD and enhancing antioxidant capacity. Marmesin can significantly reduce the increase in MDA content and has a certain effect on improving the oxidative environment in UVB-induced HaCaT cells.

[0058] The experimental results of this example show that isopimpinellin at 1 - 50 μM has no cytotoxicity; after UVB irradiation damages HaCaT cells, pretreatment with isopimpinellin increases the proliferation activity of HaCaT cells and alleviates the damage caused by UVB irradiation of HaCaT cells, thus playing a certain protective role against UVB-induced damage to HaCaT cells. In addition, the detection results of intracellular ROS levels, mitochondrial membrane potential, and various oxidative damage indicators prove that the anti-photoaging damage effect of isopimpinellin is at least partially due to its antioxidant properties.

[0059] Example 2: Experiments related to the protective effect of isopimpinellin on DNA damage and apoptosis in UVB-induced HaCaT cells

[0060] 1. Detection of cell apoptosis by Hoechst staining

[0061] Pre-place cell slides in a 6-well cell culture plate. Seed HaCaT cells onto the cell slides in the 6-well cell culture plate at a density of 5×10 3 cells / well. After 24 h, treat with isopimpinellin at different concentrations (10 μM, 20 μM, 50 μM), and irradiate with a UVB irradiator after 48 h. The irradiation dose is 30 mJ / cm 2 to stimulate cell apoptosis. After 24 h, wash the cells twice with PBS buffer. Add 0.5 mL of Hoechst 33342 staining solution to each well, incubate in the dark for 5 min, and wash the cells twice with PBS buffer. Take out the cell slides, mount them with an anti-fluorescence quenching agent, and observe under a fluorescence microscope.

[0062] The results are as Figure 6As shown, in the UVB group, apoptosis of HaCaT cells was visible, with abnormally intense nuclear staining, and the number of apoptotic nuclei was significantly increased compared with the Control group. The number of apoptotic nuclei in each concentration of marmesin group was decreased compared with the UVB group, and with the increase of marmesin concentration, the apoptotic condition improved more significantly.

[0063] 2. Detection of cell cycle by propidium iodide (PI) staining

[0064] Collect the cells of each group into 15 mL centrifuge tubes, add 1 mL of pre-cooled 70% ethanol to each tube, and fix them at 4°C for 24 h. Centrifuge at 1000 rpm for 5 min to collect the cell precipitate. Resuspend each tube with 1 mL of pre-cooled PBS buffer. Centrifuge again at 1000 rpm for 5 min to collect the cell precipitate. Add 0.5 mL of PI staining solution to each tube, gently pipette and mix well, and incubate at 37°C in the dark for 30 min. Place it in a flow cytometer to detect the light scattering and fluorescence of cells in each group at 488 nm. Analyze the data with Flowjo software.

[0065] The results are as Figure 7 shown. In the UVB group, the accumulation and arrest of cells in the S phase and G2 / M phase were significant. It indicates that after UVB irradiation, cell growth stops during DNA synthesis, which can lead to a decrease in cell proliferation. In each concentration of marmesin group, the accumulation and arrest of cells in the S phase and G2 / M phase were significantly improved compared with the UVB group.

[0066] 3. Detection of DNA synthesis and cell proliferation by EdU

[0067] In a 6-well cell culture plate, add 2.5 mL of 20 μM EdU working solution to each well and incubate at 37°C for 2 h. Discard the liquid in each well, add 1 mL of fixative to each well, and incubate for 15 min. Discard the fixative in each well, add 1 mL of washing solution to each well to wash the cells, and repeat 3 times. Discard the washing solution in each well, add 1 mL of permeabilization solution to each well, and incubate for 15 min. Discard the permeabilization solution in each well, add 1 mL of washing solution to each well to wash the cells, and repeat 2 times, 3 - 5 min each time. Discard the washing solution in each well, add 0.5 mL of Click reaction solution to each well, cross / 8-shaped shake to mix the reaction solution evenly, and cover the cells evenly. Incubate in the dark for 30 min. Discard the Click reaction solution, add 1 mL of washing solution to each well to wash the cells, and repeat 3 times. Observe under a fluorescence microscope. The maximum excitation wavelength of Azide 488 is 495 nm, and the maximum emission wavelength is 519 nm.

[0068] The results are as Figure 8 shown. The green fluorescence in the UVB group decreased. After pretreatment with each concentration of marmeisn, the green fluorescence increased in a gradient. It indicates that the cell proliferation condition recovered to near the normal level, and its protective effect enhanced with the increase of concentration, showing a dose-dependent relationship.

[0069] The experimental results of this example show that after pretreatment with nodakenetin, the nuclear staining morphology is round and clear, showing a full and uniform blue fluorescence, and the apoptosis of cells is significantly improved compared with the UVB group; at the same time, the cell cycle results obtained by PI staining combined with flow cytometry analysis show that the accumulation and arrest of cells in the S phase and G2 / M phase caused by UVB are significantly improved after pretreatment with nodakenetin at various concentrations; in addition, the results of labeling newly synthesized DNA with EdU fluorescent probe also prove again the protective effect of nodakenetin on the proliferation of UVB-induced photoaged damaged cells.

[0070] Example 3: Exploring the targets and pathways of nodakenetin on UVB-induced skin photoaging damage based on transcriptomics

[0071] 1. Extraction and quality detection of total RNA

[0072] (1) Take out freshly cultured HaCaT cells, observe the cells under a microscope, and determine that the growth state is good.

[0073] (2) Discard the culture medium, add 5 mL of pre-cooled 1X PBS to the cell culture dish, and wash twice.

[0074] (3) Discard the PBS, and use a cell scraper to collect the cells into a centrifuge tube.

[0075] (4) Centrifuge at 1000 rpm for 5 min, obtain the cell pellet, and discard the culture medium.

[0076] (5) Add 3 mL of 1X PBS (prepared with RNase-free water) to resuspend the cell pellet, centrifuge at 1000 rpm for 5 min, discard the PBS, and wash twice.

[0077] (6) Add 1 mL of trizol and pipette repeatedly to fully lyse the cells until the mixture of cells and lysis solution forms a clear and non-viscous liquid without residual cell clumps.

[0078] (7) Transfer to a 1.5 mL centrifuge tube and store at -80 °C.

[0079] (8) Use a Qubit4 Fluorometer nucleic acid and protein fluorescence quantifier and agarose gel electrophoresis to detect the purity, concentration, and integrity of RNA.

[0080] 2. Construction of RNA-seq library

[0081] (1) Extract RNA

[0082] (2) Reverse transcribe cDNA

[0083] (3) Purify cDNA with magnetic beads

[0084] (4) End filling

[0085] (5) Ligation of adaptor

[0086] (6) PCR amplification

[0087] (7) Magnetic bead purification of DNA

[0088] (8) Sequencing on the machine

[0089] 3. RNA-seq data analysis

[0090] The details of data sequencing are shown in Table 1 below. First, the Fastqc software was used to perform quality assessment (QC, quality control) on the RNA-Seq raw data. Then, the cutadapt software was used to remove the adapter sequences to obtain the processed clean data. Next, the STAR software was used to align the data with the reference genome sequence. Then, the cufflinks software was used to calculate the expression levels of each sample, annotate and analyze the differentially expressed genes (diffGene) among different groups of samples. Finally, through the R package, the differentially expressed gene data was further analyzed to draw the corresponding heatmap and volcano plot.

[0091] Table 1 Details of sequencing

[0092]

[0093] The HaCaT cells treated with UVB (UVB group) and those treated with marmesin + UVB (marmesin group) were operated according to the above process, and the results are as Figure 9 shown. A total of 482 differentially expressed genes were screened between the UVB group and the marmesin group, including 261 down-regulated genes and 221 up-regulated genes (9A); genes such as KRT15 related to keratin and squamous cell carcinoma, S100P related to epidermal inflammatory diseases, and matrix metalloproteinase MMP12 related to matrix degradation were significantly down-regulated. At the same time, genes such as SPRR1B related to the epidermal barrier, IFI6 related to anti-cell apoptosis, and HMOX1 related to iron metabolism and keratinocyte differentiation were significantly up-regulated (9B).

[0094] 4. Verification of differentially expressed genes by qRT-PCR

[0095] The mRNA expression levels of SPRR1B, IFI6, TGF-β, HO-1, KRT15, S100P, mTOR, NF-κB, and MAPK genes in HaCaT cells were detected by real-time fluorescence quantitative PCR (qRT-PCR). Primers were designed according to the following steps: Obtain the Gene ID of the target gene from the NCBI database. Based on the Gene ID of the target gene, screen the optimal primer sequences of the target gene in PrimerBank according to primer length, annealing temperature, repeat sequence conditions, etc. After homology comparison and analysis in the BLAST database, determine the specific primers. The primer names and their sequences are shown in Table 2 below. After primer synthesis, store them at -20°C for long-term preservation.

[0096] Table 2 Real-time fluorescence quantitative PCR primer sequences

[0097]

[0098] After administering drugs and establishing the model in HaCaT cells, RNA was extracted. The total RNA of the samples was extracted using the EZ-press RNA Purification Kit (EZBioscience, USA). Reverse transcription reaction was performed using 4×Reverse Transcription Master Mix (EZBioscience) at 42°C for 15 minutes and 95°C for 30 seconds. qPCR was performed using 2×SYBR Green qPCR Master Mix (EZBioscience) according to the following protocol: Denaturation (5 minutes, 95°C), and analysis was performed using Strata Gene Mx3000p (Agilent Technologies, Inc., Santa Clara, CA). Each relevant gene was normalized to GAPDH, and the fold change relative to the control sample was compared.

[0099] The results are as Figures 10 - 11 shown. The gene expressions of IFI6, SPRR1B, S100P, and KRT15 were all significantly upregulated ( Figure 10 ). Among them, the results of IFI6 and SPRR1B were consistent with transcriptomics, indicating that the sequencing results of this part were accurate and reliable. However, the results of S100P and KRT15 were inconsistent with the sequencing, and there was no statistical significance between some low- and medium-concentration marmesin intervention groups and the UVB group. qRT-PCR analysis was performed on genes related to skin photoaging damage, metabolism, oxidative stress, etc. The results showed that the expressions of MAPK, NF-κB, HO-1, and mTOR genes were all significantly downregulated ( Figure 11 ), suggesting that the photoprotective effect of marmesin may be related to the above gene targets and their pathways.

[0100] The experimental results of this example show that SPRR1B related to the epidermal barrier and IFI6 related to anti-cell apoptosis are significantly up-regulated, suggesting the great potential of imperatorin in barrier protection and anti-apoptosis. While MAPK, NF-κB, HO-1 and mTOR related to epidermal inflammation are significantly down-regulated after pretreatment with imperatorin, suggesting that imperatorin exerts its effect by inhibiting related pathways.

[0101] Example 4: Animal experiments confirm that imperatorin can resist skin photoaging damage

[0102] Six-week-old female SKH-1 hairless mice were randomly assigned to 2 groups: UVB treatment (UVB group), marmesin + UVB treatment (marmesin group). The UVB lamp was preheated for 10 min before irradiation, and the UVB irradiator showed that the UVB intensity was stable at (2.0 ± 0.2) mW. Both groups were irradiated with 200 mJ / cm 2 of UVB 3 times a week for 8 consecutive weeks. The marmesin group was given 2% marmesin application before each irradiation. Observed continuously for 8 weeks, and the appearance was photographed and recorded with a camera.

[0103] The results are as Figure 12 shown. Compared with the control group treated with UVB, the pretreatment group with marmesin can reduce the occurrence of skin photoaging damage such as wrinkles, dryness, and erythema. It can be seen that imperatorin has a protective effect on the skin of the photoaging mouse model.

[0104] This specific implementation manner is only an interpretation of the present invention and not a limitation thereto. Any changes made by those skilled in the art after reading the specification of the present invention will be protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. Application of purpurogenol in the preparation of drugs for preventing or treating skin photoaging damage.

2. The use according to claim 1, characterized in that: The medicine comprises an active ingredient, and the active ingredient is peucedanum lactone.

3. The use according to claim 2, characterized in that: The medicament further includes a pharmaceutically acceptable carrier.

4. The use according to claim 3, characterized in that: The pharmaceutically acceptable carrier is ethanol.

5. The use according to claim 1, characterized in that: The dosage form of the drug is any one of injection, emulsion, tablet, powder, granule, gel, ointment, capsule and oral solution.

6. The use according to claim 1, characterized in that: The skin photoaging damage is skin photoaging damage caused by UVB.

7. The use according to claim 6, characterized in that: The skin photoaging damage caused by UVB is specifically one or more of wrinkles, erythema, dryness, increased spider veins, and pigmentation.

8. The use according to claim 6, characterized in that: The drug is used before light irradiation.

9. The use according to claim 1, characterized in that: The isopructicolaline prevents or treats skin photoaging damage through the following pathways: 1) Oxidative stress: including reducing ROS levels, increasing mitochondrial membrane potential, increasing SOD activity, and reducing MDA content; 2) DNA damage and apoptosis: including improving cell accumulation and arrest in the S phase and G2 / M phase, reducing cell apoptosis, and restoring cell proliferation levels.

10. The use according to claim 1, characterized in that: The prevention or treatment of skin photoaging damage by isopructicola lactone involves the following target and pathway gene regulation: 1) The expression of SPRR1B gene related to epidermal barrier was significantly upregulated; 2) The expression of IFI6 gene, which is related to anti-apoptosis, was significantly upregulated; 3) The expression of MAPK, NF-κB, HO-1 and mTOR genes associated with epidermal inflammation was significantly downregulated.

Images