Active polypeptide OA-AL14, application and preparation for preventing and treating skin photoaging

Abstract

The invention discloses an active polypeptide OA-AL14, application and a preparation for preventing and treating skin photoaging, and belongs to the technical field of biological medicine. The active polypeptide OA-AL14 has the activity of preventing and treating skin photoaging, the amino acid sequence of the active polypeptide OA-AL14 is ALFWPMKKPWPESC, and the molecular weight of the active polypeptide OA-AL14 is 1720.06 Da; researches show that the active polypeptide has obvious ability to remove ABTS < + > free radicals and DPPH free radicals; in a UVB irradiation model test, the active polypeptide OA-AL14 shows the activity of promoting HaCaT cell proliferation and a cell protection effect, and can effectively relieve the oxidative stress reaction of the HaCaT cell induced by UVB and obviously improve the aging of the HaCaT cell induced by UVB; animal experiments further prove that the active polypeptide OA-AL14 can obviously improve UVB-induced skin photoaging, and has important value for development of novel skin photoaging prevention and treatment drugs.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to an active polypeptide OA-AL14, application and a preparation for preventing and treating skin photoaging. BACKGROUND

[0002] Skin photoaging refers to an accelerated skin aging phenomenon caused by oxidative stress, epigenetic modification and inflammatory response mechanisms after long-term exposure to ultraviolet UVA (320-400 nm) or UVB (280-320 nm) and other environmental light sources. Research data shows that the proportion of photoaging caused by environmental factors is more than 80%; among them, UVB radiation mainly affects the epidermis, and its pathogenic mechanism shows that it can not only directly damage DNA (such as forming cyclobutane pyrimidine dimers), but also can induce keratinocyte apoptosis by activating the p53 signaling pathway, and these pathological changes eventually lead to typical clinical manifestations such as skin erythema, abnormal pigmentation, epidermal thickening and aggravated inflammation. More notably, photoaging not only damages the appearance and barrier function of the skin, but also can induce psychological health problems such as anxiety and depression, thereby causing heavy medical and economic burden to individuals and society.

[0003] At present, the clinical prevention and treatment strategies of skin photoaging are mainly divided into two categories: prevention and treatment. Sunscreen products and nanomaterials are widely used in the prevention aspect, and the treatment methods include drug intervention such as retinoic acid external preparation or antioxidant substances, at the same time, innovative methods such as microneedle introduction, photodynamic therapy and exosome application also gradually show good efficacy; however, the actual protection effect and long-term safety of sunscreen products still need to be improved, retinoic acid external preparation is easy to cause skin adverse reactions, antioxidant substances have problems such as poor stability of effective components and low absorption rate, and the long-term efficacy and potential risks of innovative methods need to be further evaluated; therefore, it has very high practical value and broad application prospect to develop new preparations with high efficiency and better safety for preventing and treating skin photoaging. SUMMARY

[0004] The first object of the present application is to provide an active polypeptide OA-AL14 with the activity of preventing and treating skin photoaging, the second object is to provide an application of the active polypeptide OA-AL14 in preparing a preparation for removing skin free radicals, and the third object is to provide a preparation for preventing and treating skin photoaging.

[0005] The first object of the present application is achieved by providing an active polypeptide OA-AL14 with the activity of preventing and treating skin photoaging, which has an amino acid sequence of ALFWPMKKPWPESC, a molecular weight of 1720.06 Da, and a primary structure of .

[0006] A second object of the present application is achieved in that the active polypeptide OA-AL14 is used in the preparation of a skin ABTS + radical or / and DPPH radical scavenging agent.

[0007] A third object of the present application is achieved in that the active polypeptide OA-AL14 is used in the preparation of a skin photoaging prevention and treatment agent, taking the active polypeptide OA-AL14 as an active ingredient or one of the active ingredients against skin photoaging.

[0008] The present application is screened from a cDNA library of Odorrana guangxiensis Odorrana andersonii in Yunnan, and a new active polypeptide OA-AL14 is obtained, the mature peptide of which has an amino acid sequence of ALFWPMKKPWPESC; researches show that the active polypeptide has obvious ABTS + radical and DPPH radical scavenging ability; in a UVB irradiation model, the active polypeptide OA-AL14 shows promotion of HaCaT cell proliferation activity and cell protection, and can effectively relieve UVB-induced HaCaT cell oxidative stress and obviously improve UVB-induced HaCaT cell senescence; animal experiments further confirm that the active polypeptide OA-AL14 can obviously improve UVB-induced skin photoaging, which is basically consistent with the in vitro test, and shows repair of damaged skin barrier, reduction of erythema and melanin deposition, inhibition of epidermal abnormal hyperplasia, and can significantly enhance the antioxidant defense ability of skin tissue; therefore, the active polypeptide OA-AL14 has important value for the development of a new skin photoaging prevention and treatment drug. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1 Fig. 1 is a primary structure diagram of the active polypeptide OA-AL14; Figure 2 Fig. 2 is an in vitro radical ABTS + , DPPH radical scavenging rate diagram of the active polypeptide OA-AL14, in which A is the ABTS + radical scavenging rate, and B is the DPPH radical scavenging rate; Figure 3 Fig. 3 is a protection effect of the active polypeptide OA-AL14 on UVB-irradiated HaCaT cells, in which A is a scratch condition image of each group, B is a comparison of scratch repair rates of each group involved in UVB, C is a comparison of cell viabilities of each group involved in UVB, and D is a comparison of cell viabilities not involved in UVB, in which UVB corresponds to the UVB+PBS group in the diagram; Figure 4 Fig. 4 is an effect of the active polypeptide OA-AL14 on the oxidative stress level of UVB-irradiated HaCaT cells, in which A and B show the ROS conditions of each group, C is the SOD content of each group, D is the MDA content of each group, and E and F show the effect on Nrf2 protein expression. Figure 5 Effects of active polypeptide OA-AL14 on UVB irradiated HaCaT cell aging, wherein A is the microscope photos of each group, B is the SA-β-Gal condition of each group, C and D show the effects on p53 protein expression; Figure 6 Protective effects of active polypeptide OA-AL14 on UVB irradiated back skin of mice, wherein A is the back photos of each group, B is the skin H&E staining sections of each group, C is the epidermis thickness of each group, D is the corneocyte hydration condition of each group, E is the erythema index condition of each group, F is the melanin deposition condition of each group, and G is the trans-epidermal water loss condition of each group; Figure 7 Effects of active polypeptide OA-AL14 on oxidative stress level and aging of UVB irradiated back skin of mice, wherein A is the SOD content of each group, B is the MDA content of each group, C and D show the effects on Nrf2 protein expression, and E and F show the effects on p53 protein expression. DETAILED DESCRIPTION

[0010] The present application will be further described in detail below with reference to the accompanying drawings and experimental examples, but the present application is not limited in any way by the following description, and any transformation or improvement based on the teaching of the present application falls within the protection scope of the present application.

[0011] The polypeptide OA-AL14 described in the present application has the activity of preventing and treating skin photoaging, and has the amino acid sequence of ALFWPMKKPWPESC, a molecular weight of 1720.06 Da, and a primary structure of: .

[0012] The active polypeptide OA-AL14 is used in the preparation of a preparation for removing ABTS + free radicals or / and DPPH free radicals from skin.

[0013] The preparation for preventing and treating skin photoaging of the active polypeptide OA-AL14 uses the active polypeptide OA-AL14 as an active ingredient or one of the active ingredients for preventing and treating skin photoaging.

[0014] Further, the skin photoaging is the skin photoaging caused by UVB irradiation.

[0015] Further, the effective concentration of the active polypeptide OA-AL14 in the preparation is ≥10 nM.

[0016] Further, the preparation is a skin care cosmetic or a skin external use medicament.

[0017] The present application is derived from the skin of Yunnan Odorrana (Rana Odorrana andersoniiA novel active polypeptide OA-AL14 is screened from a cDNA library of a strain of Bacillus subtilis, the mature polypeptide of which has an amino acid sequence of ALFWPMKKPWPESC, a molecular weight of 1720.06 Da, and a primary structure as shown in Figure 1 The present application is based on the active polypeptide OA-AL14 and the following tests and researches are carried out: Test Example 1: Synthesis of the active polypeptide OA-AL14 The active polypeptide OA-AL14 is synthesized by Wuhan Baiyixin Biotechnology Co., Ltd. according to the primary structure, serving as a test sample A for subsequent tests.

[0018] Test Example 2: In vitro ABTS + , DPPH free radical scavenging rate detection 1. In vitro ABTS + free radical scavenging rate detection, test group 1: 1-1, ddH2O is used as a solvent, 7 mM ABTS + and 2.8 mM K2S2O8 are mixed to prepare an ABTS + stock solution, and ABTS + free radicals are generated by reacting for 6 hours in a dark room at room temperature; 1-2, 6 groups of test sample A are injected into a 96-well plate, respectively, and the ABTS + stock solution is diluted, respectively, so that the final concentration of the test sample A in each group is 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, and 10 μM, respectively; after reacting for 10 min at room temperature in the dark, the absorbance is measured at a wavelength of 415 nm.

[0019] 2. In vitro DPPH free radical scavenging rate detection, test group 2: 2-1, methanol is used as a solvent, and DPPH is prepared into a DPPH stock solution with a concentration of 50×10 -5 M, which is stored at 4°C in the dark for standby use; 2-2, 6 groups of test sample A are injected into a 96-well plate, respectively, and the DPPH stock solution is diluted, respectively, so that the final concentration of the test sample A in each group is 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, and 10 μM, respectively; after reacting for 30 min at room temperature in the dark, the absorbance is measured at a wavelength of 517 nm.

[0020] 3. Negative control group and positive control group: 3-1, the negative control groups of test groups 1 and 2 are set, respectively, 50 μL ddH2O and 50 μL ABTS + stock solution are mixed, 10 μL ddH2O and 190 μL DPPH stock solution are mixed, and the others are the same as test groups 1 and 2. 3-2. Set up positive control groups for experimental groups 1 and 2. Inject 4 groups of vitamin C into 96-well plates, and inject 2 groups with ABTS. + The stock solutions were diluted to final Vc concentrations of 10 μM and 100 μM, respectively. The other two groups were diluted with DPPH stock solutions to final Vc concentrations of 10 μM and 100 μM, respectively. Other details were the same as in experimental groups 1 and 2.

[0021] 4. Test Results: ABTS + The formula for calculating DPPH clearance rate is: (A) blank -A sample )×100 / A blank (%) 4-1, such as Figure 2 As shown in Figure A, experimental group 1 showed that the active peptide OA-AL14 at a concentration of 100 pM had an effect on ABTS. + Free radicals exhibit significant scavenging activity; 4-2, such as Figure 2 As shown in Figure B, experimental group 2 showed that the active peptide OA-AL14 exhibited significant scavenging activity against DPPH free radicals at a concentration of 10 nM. The above results indicate that the active peptide OA-AL1 can be used to scavenge ABTS. + It can eliminate DPPH free radicals and has a relatively ideal scavenging effect.

[0022] Experimental Example 3: Detection of the Proliferative Activity of Active Peptide OA-AL14 on HaCaT Cells Irradiated by UVB 1. Scratch healing test: 1-1, with 2×10 4 HaCaT cells were seeded in 24-well plates at a density of cells / well. After the cells adhered, serum-free DMEM / F12 medium was added. 1-1-1, Control group: No reagents or samples added; 1-1-2, UVB+PBS group, with 0.01M PBS added; Groups 1-1-3 and UVB+OA-AL14 were respectively supplemented with test sample A to make the final concentrations of test sample A 100 pM, 1 nM and 10 nM respectively; 1-1-4, UVB+Vc group, added Vc (Sigma, USA, the same below) and made the final concentration of Vc 10μM; 1-2. Experimental Procedure: Each group was pretreated for 2 hours. Scratches were created using the tip of a 200μL pipette. The solution used was 9W / 01 / 2P, 30mJ / cm². 2UVB lamp (Philips, Holland, same below) irradiation (except Control group), after irradiation, add fresh DMEM / F12 medium and re-add samples or Vc according to the case of each group, and record the scratch healing at 0, 24 and 48 hours respectively; 2、MTS method detects the activity of OA-AL14 on normal and UVB irradiated HaCaT cells: 2-1, the effect of OA-AL14 on the activity of UVB irradiated HaCaT cells: 5×10 3 HaCaT cells were seeded in 96-well plates at a density of 5×10 2 UVB lamp irradiation for 24 hours (except Control group), after irradiation, add fresh DMEM / F12 medium and re-add samples or Vc according to the case of each group, and record the scratch healing at 0, 24 and 48 hours respectively;

[0023] 2-2, the effect of OA-AL14 on the activity of HaCaT cells: according to step 1 in this test group, after HaCaT cells adhere, set Control group and OA-AL14 group (100pM, 1nM, 10nM), incubate the cells at 37° for 24 hours, then add 20μL MTS to each well, continue to incubate at 37℃ for 3-4 hours; finally, use the microplate reader to measure the absorbance at 490nm.

[0024] 3、Test results: 3-1, the active polypeptide OA-AL14 with a concentration of 10nM showed the fastest scratch healing rate, which was almost the same as the scratch healing rate of Vc with a concentration of 10μM at 48h (see Figure 3 A, B); 3-2, MTS detection found that the cell survival rate decreased significantly after UVB irradiation for 24 hours, and the active polypeptide OA-AL14 could effectively improve the cell damage caused by UVB radiation, among which the active polypeptide OA-AL14 with a concentration of 10nM had the best protective effect on UVB irradiated HaCaT cells, and the cell activity was equivalent to that of Vc with a concentration of 10μM (see Figure 3 C); 3-3. MTS assay showed that the active peptide OA-AL14 had significant pro-proliferative activity against normal HaCaT cells, especially at a concentration of 10 nM (see [link to MTS assay]). Figure 3 (D) The above results indicate that the active polypeptide OA-AL14 has good proliferative activity and cell protection effect on HaCaT cells, and an effective concentration of ≥10 nM is sufficient to show excellent results.

[0025] Example 4: Detection of cellular oxidative stress-related indicators by active peptide OA-AL14 1. Using 3×10 5 HaCaT cells were seeded in confocal dishes at a density of cells / well. After cell adhesion, the following groups were set up as described in step 1 of Experiment 3: Control group, UVB+PBS group, UVB+OA-AL14 group (concentration only 10 nM), UVB+Vc group (Vc concentration 10 μM), and UVB group (without PBS). After 2 hours of pretreatment, 9W / 01 / 2P, 30 mJ / cm² was used. 2 Cells were irradiated with UVB lamps for 24 hours (except for the Control group). After irradiation, fresh DMEM / F12 medium was added, and samples or vitamin C were added again according to the situation of each group. Cells were incubated with 10 μM DCFH-DA probe solution and 10 μg / mL Hoechst33242 solution at 37°C for 30 min. After washing with PBS, ROS generation was immediately detected by laser confocal microscopy (Olympus, Japan), and the mean fluorescence intensity (MFI) was analyzed by ImageJ software.

[0026] 2. With 1.2×10 6 HaCaT cells were seeded into 6-well plates at a density of cells / well. After cell adhesion, the following groups were set up according to step 1 of this experimental example: Control group, UVB+PBS group, UVB+OA-AL14 group (concentration only 10 nM), UVB+Vc group (Vc concentration 10 μM), and UVB group (without PBS). After 2 hours of pretreatment, 9W / 01 / 2P, 30 mJ / cm 2 The cells were irradiated with UVB lamps for 24 hours (except for the Control group). After irradiation, fresh DMEM / F12 medium was added, and samples or vitamin C were added again according to the situation of each group. The cells were washed with PBS and lysed for 30-40 minutes with lysis buffer. Then, the SOD activity and MDA content in the cells were measured according to the kit instructions (Nanjing Jiancheng Biotechnology Institute).

[0027] 3、Western Blotting (WB) quantitative analysis was used to evaluate the expression of Nrf2 protein in UVB-irradiated HaCaT cells after different group treatments; in this test example, step 2 used a 9W / 01 / 2P, 30mJ / cm 2 UVB lamp for 24h, then the cell protein was extracted and quantified, and then WB protein detection was performed; the obtained band was analyzed for protein band gray value using Image J software, and the data was statistically analyzed in GraphPad Prism software.

[0028] 4、Test results: 4-1、 Figure 4 A and B in the middle show that UVB irradiation significantly increased the ROS level in HaCaT cells, while the active polypeptide OA-AL14 at a concentration of 10nM completely restored the ROS to normal levels, and its effect was better than that of Vc at a concentration of 10μM; 4-2、 Figure 4 C in the middle shows that the SOD activity in HaCaT cells was significantly reduced after UVB irradiation, while the active polypeptide OA-AL14 at a concentration of 10nM could significantly up-regulate the SOD activity; 4-3、 Figure 4 D in the middle shows that UV radiation caused a significant increase in the accumulation of MDA, while the active polypeptide OA-AL14 at a concentration of 10nM could effectively reduce the accumulation of MDA; 4-4、 Figure 4 E and F in the middle show the results of Western blot analysis, and the active polypeptide OA-AL14 at a concentration of 10nM can significantly up-regulate the expression of the key antioxidant transcription factor Nrf2, indicating that the active polypeptide OA-AL14 can enhance the antioxidant enzyme activity and Nrf2 expression; The above results show that the active polypeptide OA-AL14 can effectively alleviate the oxidative stress response of HaCaT cells induced by UVB.

[0029] Test Example 5 Detection of Cell Level Aging Related Indicators by Active Polypeptide OA-AL14 1、3×10 5HaCaT cells were seeded in 24-well plates at a density of 1.5 x 104cells / well, and the Control group, UVB+PBS group, UVB+OA-AL14 group (only 10 nM concentration was set), UVB+Vc group (Vc concentration was 10 mM), and UVB group (without PBS) were set according to Step 1 of Test Example 3. After 2 hours of pre-treatment, the cells were irradiated with a 9W / 01 / 2P, 30 mJ / cm2UVB lamp for 24 hours (except for the Control group). After irradiation, fresh DMEM / F12 medium was added, and samples or Vc were added according to the group conditions. Then, the SA-β-Gal staining kit (Beyotime, China) was used to detect cell senescence, and the results were observed and photographed under an optical microscope (Zeiss, Germany). The SA-β-Gal positive rate (blue-stained cell number / total cell number x 100%) was calculated using ImageJ software.

[0030] 2. Western Blotting (WB) quantitative analysis was used to evaluate the expression of p53 protein in UVB-irradiated HaCaT cells after different group treatments. In this test example, Step 1 used a 9W / 01 / 2P, 30 mJ / cm2UVB lamp to irradiate the cells for 24 hours. After irradiation, the cell proteins were extracted and quantified, and then WB protein detection was performed. The obtained protein bands were analyzed for their gray scale values using Image J software, and the data were statistically analyzed using GraphPad Prism software. 2

[0031] 3. Test results: 3-1, Figure 5 As shown in Figs. 3A and 3B, UVB irradiation caused a significant increase in the SA-β-Gal positive rate of HaCaT cells, and cell density and morphology abnormalities were also observed. However, after treatment with active polypeptide OA-AL14 at a concentration of 10 nM, the increase in SA-β-Gal activity was significantly inhibited. 3-2, Figure 5 As shown in Figs. 3C and 3D, Western blot analysis showed that active polypeptide OA-AL14 at a concentration of 10 nM could effectively reduce the overexpression of p53 protein induced by UVB. The above results indicate that active polypeptide OA-AL14 has a significant improvement effect on UVB-induced HaCaT cell senescence.

[0032] Test Example 6: Detection of the protective activity of active polypeptide OA-AL14 on the back skin of UVB-irradiated mice ​1. After one week of acclimatization, 22g female Kunming mice were randomly divided into 5 groups (n=6 / group): Control group, UVB group, UVB+PBS group, UVB+OA-AL14 group, and UVB+Vc group. Except for the Control group, the dorsal skin of mice in all other groups was treated with 2000mJ / cm². 2 UVB irradiation (TL20w / 01, Phillips) was performed, with the irradiation time calculated based on the radiation intensity (TM-213, Temars). After irradiation, the Control and UVB groups were given ddH2O, while the remaining groups received local spray administration according to the grouping protocol (i.e., administration of PBS, OA-AL14, and Vc, 1 mL / mouse). 24 hours later, the erythema on the back of the mice was photographed and recorded. Subsequently, the degree of erythema, melanin content, stratum corneum hydration, and epidermal water loss of the mice after hair removal were systematically detected using a Cutometer dual MPA580 skin elasticity tester manufactured by C&K GmbH, Cologne, Germany. After completing the above tests, all mice were anesthetized and euthanized for epidermal protective effect testing (H&E staining). In addition, the skin obtained from each group was used for the determination of oxidative stress and aging-related indicators in Experiment 7.

[0033] 2. Experimental results: Acute UVB radiation (2000mJ / cm²) can cause significant damage to the skin of mice, manifested as typical photoaging symptoms such as characteristic erythema, abnormal melanin deposition, and increased transepidermal water loss (TEWL). 2-1 Figure 6 The results showed that after 24 hours of UVB irradiation, compared with the Control group, the erythema area in the UVB group and the UVB+PBS group was significantly increased, while the erythema severity was significantly reduced with 10 nM of active peptide OA-AL14 and 10 μM of vitamin C. 2-2、 Figure 6 Figures B and C show that the active peptide OA-AL14 at a concentration of 10 nM can effectively inhibit UVB-induced epidermal thickening, and its effect is comparable to that of vitamin C at a concentration of 10 μM. 2-3 Figure 6 The results of D, E, F, and G show that the active peptide OA-AL14 can significantly improve the decrease in stratum corneum hydration, increase in erythema index, melanin deposition, and increase in transepidermal water loss (TEWL) caused by UVB. The above results indicate that the active peptide OA-AL14 can effectively alleviate UVB-induced skin photoaging.

[0034] Experimental Example 7: Detection of Oxidative Stress and Aging-Related Indicators in the Back Skin of Mice Irradiated by UVB Using Active Peptide OA-AL14 1. The skin of the mice after euthanasia in Test Example 6 was washed with pre-cooled normal saline, weighed, cut into pieces, and then ground in a grinder for 15 min in pre-cooled normal saline (the ratio of skin pieces to normal saline was 1 g:9 mL), and then centrifuged at 3000 rpm for 10 min at 4°. The supernatant was used to detect the contents of SOD and MDA using WST-1 and TBA kits, respectively. The two kits and detection schemes were provided by Nanjing Jianshen Biological Engineering Institute (Nanjing, China).

[0035] 2. Western Blotting (WB) quantitative analysis was used to evaluate the expression of Nrf2 protein and p53 protein in the UVB-irradiated skin of mice in different groups. Cell protein extraction and protein quantification were performed, followed by WB protein detection. The obtained protein bands were analyzed for gray scale values using Image J software, and the data were statistically analyzed using GraphPad Prism software.

[0036] 3. Test results: 3-1, Figure 7 As shown in the middle A and B, the active polypeptide OA-AL14 at a concentration of 10 nM can significantly improve the SOD activity and reduce the MDA content of the skin. 3-2, Figure 7 As shown in the middle C and D, the active polypeptide OA-AL14 at a concentration of 10 nM can up-regulate the expression of Nrf2 protein in vivo. 3-3, Figure 7 As shown in the middle E and F, the active polypeptide OA-AL14 at a concentration of 10 nM can reduce the expression of p53 protein associated with aging. The above results are basically consistent with the in vitro tests in Test Examples 4 and 5, indicating that the active polypeptide OA-AL14 can activate antioxidant enzymes and Nrf2, inhibit p53 protein, and effectively improve UVB-induced oxidative stress and photoaging.

[0037] In summary, the active polypeptide OA-AL14 described in the present application has good anti-skin photoaging activity, and has good drug research and development value and clinical application prospects.

Claims

1. An active polypeptide OA-AL14, characterized in that, The active polypeptide OA-AL14 has anti-photoaging activity, and its amino acid sequence is ALFWPMKKPWPESC, its molecular weight is 1720.06 Da, and its primary structure is: 。 2. The active polypeptide OA-AL14 of claim 1 in the preparation of a drug for clearing skin ABTS + Application in free radical and / or DPPH free radical preparations.

3. A skin photoaging prevention and treatment formulation based on the active polypeptide OA-AL14 of claim 1, characterized in that, The active peptide OA-AL14 is used as an active ingredient or one of the active ingredients for anti-skin photoaging.

4. The skin photoaging prevention preparation according to claim 3, characterized in that, The aforementioned photoaging of the skin is caused by UVB irradiation.

5. The skin photoaging prevention preparation according to claim 3 or 4, characterized in that, The effective concentration of the active polypeptide OA-AL14 in the formulation is ≥10 nM.

6. The skin photoaging prevention preparation according to claim 3 or 4, characterized in that, The preparation is a skin care cosmetic or a topical skin medication.

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