Application of 18 beta-glycyrrhetinic acid in preparation of medicines or cosmetics for treating or repairing skin light injury

Abstract

The invention discloses application of 18 beta-glycyrrhetinic acid in preparation of a medicine or cosmetic for treating or repairing skin light injury, and belongs to the technical field of medicines.The medicine or cosmetic prepared from 18 beta-glycyrrhetinic acid for treating or repairing skin light injury can be used for protecting or repairing skin barrier and enhancing skin hydration; epidermis thickening is relieved, keratinization is improved, and inflammatory cell infiltration and corium layer collagenous fiber arrangement are improved; the catalase activity is improved, and the active oxygen level is reduced; fibroblast proliferation and survival are promoted; the expression level of the III type collagen is improved; and the expression levels of MMP2, p53 and p21 are reduced. Besides, animal experiment observation finds that the medicine or the cosmetic prepared from the 18 beta-glycyrrhetinic acid or the extract thereof can be used for improving erythema, induration, scales and pigmentation of skin light injury.

Description

Technical Field

[0001] The invention belongs to the field of medical technology and relates to the application of 18β-glycyrrhetinic acid in preparing medicines or cosmetics for treating or repairing skin photodamage. Background Art

[0002] Glycyrrhetinic acid (GA) is a pentacyclic triterpenoid compound mainly extracted from licorice. 18β-Glycyrrhetinic acid (18β-GA) is one of the configurations of glycyrrhetinic acid. In the prior art, the research on the active effects of 18β-Glycyrrhetinic acid mainly focuses on antioxidant, immune inflammation regulation, anti-tumor and other aspects.

[0003] Skin photodamage refers to the adverse changes and damage to the skin due to long-term or excessive exposure to ultraviolet light, visible light and infrared light radiation. Ultraviolet B (UVB) is a type of ultraviolet light with a wavelength range of 280nm to 320nm. UVB mainly acts on the epidermis of the skin, causing sunburn, erythema, pigmentation and other problems. In addition, UVB exposure to the skin will increase the decomposition of collagen and promote the breakage of elastic fibers, resulting in decreased skin elasticity, sagging and wrinkles, and accelerated skin aging.

[0004] At present, the research reports on the potential biological activity of 18β-glycyrrhetinic acid in anti-skin aging are not comprehensive. Therefore, studying the biological activity of 18β-glycyrrhetinic acid in the treatment or repair of skin photodamage is of great significance and value in promoting the development of corresponding drugs or cosmetics. Summary of the invention

[0005] According to a first aspect of the present invention, there is provided use of 18β-glycyrrhetinic acid in the preparation of a drug or cosmetic for treating or repairing photodamage of the skin.

[0006] In some embodiments, the medicine or cosmetic is a substance used to protect or repair the skin barrier and enhance skin hydration.

[0007] In some embodiments, protecting or repairing the skin barrier is reducing the transepidermal water loss score and improving the skin barrier score, and enhancing skin hydration is improving the skin hydration score.

[0008] In some embodiments, the medicine or cosmetic is a substance used to reduce epidermal thickening, improve keratinization, improve inflammatory cell infiltration, and improve the arrangement of collagen fibers in the dermis.

[0009] In some embodiments, the medicine or cosmetic is a substance for increasing the activity of catalase (CAT) and reducing the level of reactive oxygen species (ROS).

[0010] In some embodiments, the medicine or cosmetic is a substance for promoting the proliferation and survival of fibroblasts.

[0011] In other embodiments, the fibroblasts may be human dermal fibroblast (HSF) cells.

[0012] In some embodiments, the medicine or cosmetic is a substance for increasing the expression level of type III collagen.

[0013] In some embodiments, the medicine or cosmetic is a substance for reducing the expression levels of MMP2, p53 and p21.

[0014] In some embodiments, 18β-glycyrrhetinic acid is used in the preparation of a drug or cosmetic for treating or repairing skin photodamage having one or more of the following effects: improving skin erythema, nodules, scaling, skin gloss, moisture and pigmentation.

[0015] In some embodiments, the structural formula of 18β-glycyrrhetinic acid is as follows:

[0016]

[0017] In some embodiments, the skin photodamage is skin damage caused by ultraviolet light.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The medicine or cosmetic for treating or repairing skin photodamage prepared from 18β-glycyrrhetinic acid or an extract containing 18β-glycyrrhetinic acid of the present invention can be used to protect or repair the skin barrier, enhance skin hydration; reduce epidermal thickening, improve keratinization, improve inflammatory cell infiltration and dermal collagen fiber arrangement; increase catalase activity, reduce reactive oxygen levels; promote fibroblast proliferation and survival, increase the expression level of type III collagen, and reduce the expression levels of MMP2, p53 and p21;

[0020] Animal experimental observations have shown that drugs prepared from 18β-glycyrrhetinic acid or extracts containing 18β-glycyrrhetinic acid can be used to improve erythema, nodules, scales and pigmentation caused by photodamage to the skin. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 These are photos of damaged skin on the backs of mice in different groups in the experiment of the present invention;

[0022] Figure 2 This is a graph showing the scores of the GPSkin Barrier test on the skin of mice in different groups in the experiment of the present invention;

[0023] Figure 3 The results of HE staining of skin tissues of mice in different groups in the experiment of the present invention are shown in FIG.

[0024] Figure 4 The figure is a test result diagram of the skin tissue of different groups of mice in the experiment of the present invention by Masson staining;

[0025] Figure 5 The figure is a test result diagram of the skin tissue of different groups of mice in the experiment of the present invention stained with Sirius red;

[0026] Figure 6 The figure is a test result diagram of CAT activity, SOD activity and HYP content of skin tissue protein of different groups of mice in the experiment of the present invention;

[0027] Figure 7 Results of different UVB irradiation on HSF cells in the experiment of the present invention;

[0028] Figure 8 This is the staining display of senescence-related β-galactosidase in the experiment of the present invention;

[0029] Fig. 9 This is a comparison chart of the percentage of positively stained cells in the normal group and the model group in the experiment of the present invention;

[0030] Fig.10 , Fig.11 The results of flow cytometry analysis of cell cycle in the normal group and model group in the experiment of the present invention;

[0031] Fig.12 , Fig.13 The expression of aging markers type III collagen, MMP2, p53 and p21 in the normal group and the model group in the experiment of the present invention;

[0032] Fig.14 The effect of different concentrations of 18β-GA on the viability of HSF cells after UVB treatment in the experiment of the present invention;

[0033] Fig.15 , Fig.16 This is the effect of different groups on UVB-induced ROS in HSF cells in the experiment of the present invention;

[0034] Fig.17 This is the effect of 60 μmol / L 18β-glycyrrhetinic acid on the number of HSF cells damaged by UVB in the experiment of the present invention;

[0035] Fig.18 This is the expression of aging markers type III collagen, MMP2, p53 and p21 in different groups in the experiment of the present invention. DETAILED DESCRIPTION

[0036] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0037] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially. Among them, the molecular formula of 18β-glycyrrhetinic acid (CAS: 471-53-4) used in the present invention is C 30 H 46 O4, molecular weight is 470.68, structural formula is:

[0038]

[0039] 1. Experiment on the repair of UVB-damaged mouse skin model by 18β-glycyrrhetinic acid

[0040] 1. Main instruments, materials and reagents

[0041] UV lamp, GPSkin Barrier detector, tissue embedding machine (Leica Biosystems, Germany), paraffin slicer (Leica Biosystems, Germany), optical microscope, OLYPUMS image acquisition system.

[0042] 0.9% saline, vitamin E (VE), 18β-glycyrrhetinic acid (18β-GA), glycerol, propylene glycol, 4% paraformaldehyde, paraffin, xylene, anhydrous ethanol, 95% ethanol, 90% ethanol, 85% ethanol, 75% ethanol, staining jar, slide, cover slip, immunohistochemistry pen, wet box, 1mL syringe.

[0043] Minute TM The skin tissue total protein extraction kit was purchased from Beijing Yingwente Biotechnology Co., Ltd.; the hematoxylin-eosin staining kit, Masson trichrome staining kit, catalase (CAT) activity detection kit, superoxide dismutase (SOD) activity detection kit, and hydroxyproline (HYP) content detection kit were all purchased from Beijing Solebow Technology Co., Ltd.

[0044] 2. Experimental animals: Healthy female BALB / c mice aged 8 weeks and weighing 20-24 g were fed adaptively for 7 days.

[0045] 3. Modeling and drug administration of UVB-damaged mouse skin model

[0046] The back skin of BALB / c mice was depilated in an area of ​​about 2cm×5cm and randomly divided into a normal control group (Blank), a model group (UVB), a positive drug group (UVB+VE), a low-dose 18β-glycyrrhetinic acid group (UVB+90mg / kg 18β-GA), a medium-dose 18β-glycyrrhetinic acid group (UVB+180mg / kg 18β-GA) and a high-dose 18β-glycyrrhetinic acid group (UVB+320mg / kg18β-GA), and the mice were raised normally.

[0047] The normal control group was injected with 100 μL of 0.9% saline every day starting from the 3rd day, once a day for 9 consecutive days;

[0048] The model group was irradiated with 280-320nm medium-wave ultraviolet UVB at a distance of 15cm from the back of the mice for 2min every day from the 3rd day, and then 100μL of 0.9% saline was applied to the hair loss of the mice once a day for 9 consecutive days.

[0049] The positive drug group was irradiated with 280-320nm medium-wave ultraviolet UVB at a distance of 15cm from the back of the mouse for 2min every day from the 3rd day, and then 100μL vitamin E was applied to the mouse hair loss area once a day for 9 consecutive days.

[0050] In the low-dose 18β-glycyrrhetinic acid group, starting from the third day, 280-320 nm medium-wave ultraviolet UVB was used to irradiate the hair loss area of ​​the mice at a distance of 15 cm from the back of the mice for 2 minutes every day, once a day, and then 56 μL of 18β-glycyrrhetinic acid gel preparation (90 mg / kg / d) was applied to the hair loss area of ​​the mice, once a day, for 9 consecutive days;

[0051] The 18β-glycyrrhetinic acid medium-dose group was irradiated with 280-320nm medium-wave ultraviolet UVB at a distance of 15cm from the back of the mice for 2min every day starting from the 3rd day, once a day, and then 113μL of 18β-glycyrrhetinic acid gel preparation (180mg / kg / d) was applied to the depilatory area of ​​the mice, once a day, for 9 consecutive days;

[0052] Starting from the third day, the 18β-glycyrrhetinic acid high-dose group was irradiated with 280-320nm medium-wave ultraviolet UVB at a distance of 15cm from the back of the mice for 2min every day, once a day, and then 225μL of 18β-glycyrrhetinic acid gel preparation (320mg / kg / d) was applied to the depilatory area of ​​the mice, once a day, for 9 consecutive days.

[0053] The 18β-glycyrrhetinic acid gel preparation was prepared by mixing 20% ​​glycerol (v / v), 1.5% propylene glycol (v / v) and 2 g of 18β-glycyrrhetinic acid, and then adding deionized water until the volume of the solution was 50 mL.

[0054] 4. Effect of 18β-glycyrrhetinic acid on symptoms of UVB-damaged back skin in mice

[0055] After the 9th day of drug treatment, a digital camera was used to record and observe the skin lesions on the back of mice treated with different drugs, and the skin barrier, transepidermal water loss and skin hydration of the back skin of mice were evaluated using a GPSkin Barrier detector. The results are as follows: Figure 1 and Figure 2 shown.

[0056] The hydration score is measured using the GPSkin Barrier detector to detect changes in skin moisture content. It is assessed based on skin surface moisture content, skin elasticity, and skin barrier function. The hydration score is based on changes in skin moisture content and quantifies the hydration status through numerical values. The higher the score, the better the hydration.

[0057] from Figure 1 It can be seen that compared with the normal control group, the back skin of the mice in the model group showed different degrees of roughness, dryness, lack of gloss, local desquamation, thickening, reduced elasticity, deeper texture and fine wrinkles on the skin. The clinical symptoms were erythema, nodules, scaling, loss of moisture and pigmentation on the back skin of the mice, indicating that UVB caused damage to the back skin of the mice and the corresponding clinical symptoms appeared. Compared with the model group, the positive drug group, the low-dose group of 18β-glycyrrhetinic acid, the medium-dose group of 18β-glycyrrhetinic acid and the high-dose group of 18β-glycyrrhetinic acid could significantly alleviate the clinical symptoms of the back skin of the mice.

[0058] from Figure 2It can be seen that compared with the normal control group, the skin barrier score (Barrier Score) of the model group was reduced (P<0.001), indicating that UVB irradiation destroyed the skin barrier of mice, making the skin more susceptible to invasion and stimulation by external harmful substances. Due to the damage of the skin barrier, it cannot effectively lock moisture, causing the skin to become dry; the transepidermal water loss score (TEWL Score) increased (P<0.001), indicating that the damage to the skin barrier caused the loss of water through skin evaporation; the hydration score (Hydration Score) decreased (P<0.001), which also showed that the skin water content of the mice in the model group decreased and lost its moisturizing ability. Compared with the model group, the skin barrier score and hydration score of the 18β-glycyrrhetinic acid low-dose group, the 18β-glycyrrhetinic acid medium-dose group and the 18β-glycyrrhetinic acid high-dose group were significantly increased, and the transepidermal water loss score was significantly reduced, indicating that after treatment with different doses of 18β-glycyrrhetinic acid, the skin barrier damage was significantly alleviated and the skin moisturizing ability was significantly improved.

[0059] 5. Effect of 18β-glycyrrhetinic acid on pathological morphology of UVB-damaged mouse skin tissue

[0060] After the 9th day of administration, the skin tissue of the mice was taken to prepare skin tissue sections, which were stained with hematoxylin and eosin (HE). The processing process was as follows:

[0061] Skin tissue sections: Take the mouse skin tissue and immerse it in 4% paraformaldehyde fixative for 24 hours, then take out the mouse skin tissue and trim it, then treat it with 75% ethanol for 4 hours, 85% ethanol for 2 hours, 90% ethanol for 2 hours, 95% ethanol for 1 hour, anhydrous ethanol I for 30 minutes, and anhydrous ethanol II for 30 minutes, then soak it in 50% ethanol and 50% xylene, xylene I and xylene II for 10 minutes each to keep it transparent. Finally, melt paraffin I, paraffin II, and paraffin III at 65℃ for 1 hour each. Embed the skin tissue, slice it after cooling, and the thickness of the skin tissue slice is 4μm.

[0062] HE staining: Dewax the skin tissue sections in xylene twice, 5-10 minutes each time, then rehydrate in anhydrous ethanol, 95% ethanol, 85% ethanol and 75% ethanol solutions, 3 minutes each gradient, and then soak in distilled water for 2 minutes. Stain with hematoxylin solution for 3 minutes, wash off the floating color with distilled water, then differentiate with differentiation solution for 10 seconds, return to blue with tap water for 15 minutes, place in eosin solution for 2 minutes, pour off the excess staining solution, then soak in 75% ethanol, 85% ethanol, 95% ethanol and 100% ethanol (I) for 2-3 seconds, soak in 100% ethanol (II) for 1 minute, and finally transparentize with xylene twice, 1 minute each time, and seal with neutral gum.

[0063] After HE staining, the samples were observed under an optical microscope. The staining results were as follows: Figure 3 shown.

[0064] from Figure 3 It can be seen that the skin tissue structure of the normal control group is intact, the epidermis is uniform in thickness, the keratinization layer is normal, the collagen fibers in the dermis are tightly arranged, and there is no obvious edema and inflammatory cell infiltration. Compared with the normal control group, the epidermis in the model group was significantly thickened, hyperkeratinized, and incomplete keratinization was present; the collagen fiber structure in the dermis was loose, edema was obvious, and there was a large number of inflammatory cell infiltrations of neutrophils and lymphocytes, indicating that UVB significantly damaged the skin barrier of mice. Compared with the model group, the epidermis thickness of the low-dose 18β-glycyrrhetinic acid group was smaller, the degree of keratinization was lower, and the collagen fibers in the dermis were more tightly arranged, the degree of edema was reduced, and the degree of inflammatory cell infiltration was reduced. The epidermis thickness of the medium-dose 18β-glycyrrhetinic acid group was further reduced, the keratinization phenomenon was not obvious, the collagen fiber structure in the dermis was relatively tight, the edema was mild, and the inflammatory cell infiltration was significantly reduced. The epidermal thickness of the high-dose 18β-glycyrrhetinic acid group was close to that of the normal control group, with no obvious hyperkeratosis, and the collagen fibers in the dermis were arranged in an orderly manner, without obvious edema, and the infiltration of inflammatory cells was close to that of the normal control group, with basically no inflammatory reaction. The epidermal thickness of the skin layer of mice in each group was compared, and it was found that the epidermal thickness of the positive drug group, the low-dose 18β-glycyrrhetinic acid group, the medium-dose 18β-glycyrrhetinic acid group and the high-dose 18β-glycyrrhetinic acid group was significantly reduced compared with the model group, and the epidermal thickness of the medium-dose 18β-glycyrrhetinic acid group and the high-dose 18β-glycyrrhetinic acid group was lower than that of the positive drug group. The above experimental results show that 18β-glycyrrhetinic acid has a significant effect on repairing the mouse skin barrier, and medium and high doses of 18β-glycyrrhetinic acid also have a good therapeutic effect on UVB-damaged mouse skin compared with vitamin E.

[0065] 6. Effect of 18β-glycyrrhetinic acid on the collagen fiber morphology of UVB-damaged mouse skin tissue

[0066] Masson staining was performed on the skin tissue sections using a Masson trichrome staining kit. The specific processing process is as follows:

[0067] Masson staining: The above mouse skin tissue sections were routinely dewaxed to water, and then Weigert A solution and B solution were mixed evenly in a ratio of 1:1 before use, and stained with the prepared Weigert iron hematoxylin staining solution for 5 minutes. Then differentiated with acidic ethanol differentiation solution for 5 seconds and washed with water. Then use Masson blueing solution to return to blue for 3 to 5 minutes, and wash with distilled water for 1 minute. Then stain with Ponceau fuchsin staining solution for 8 minutes. Prepare weak acid working solution by mixing distilled water and weak acid solution in a ratio of 2:1, wash with the prepared weak acid working solution for 1 minute, wash with phosphomolybdic acid solution for 2 minutes, and then wash with the prepared weak acid working solution for 1 minute; then stain with aniline blue staining solution for 2 minutes. Wash with the prepared weak acid working solution for 1 minute. Dehydrate quickly with 95% ethanol for 2 to 3 seconds, and dehydrate with anhydrous ethanol 3 times, each time for 5 seconds. Finally, use xylene to make it transparent 3 times, each time for 1 to 2 minutes, and seal the slide with neutral gum.

[0068] After Masson staining, the samples were observed under an optical microscope. Figure 4 shown.

[0069] from Figure 4 It can be seen that the collagen fibers in the dermis of the normal control group are evenly distributed, tightly arranged, and evenly stained, showing the typical characteristics of healthy skin. Compared with the normal control group, the collagen fibers in the dermis of the model group were significantly reduced, loosely arranged, unevenly stained, and increased fiber breakage, accompanied by obvious matrix degradation, indicating that the collagen fibers in the model group were severely damaged due to UVB irradiation. Compared with the model group, the collagen fibers in the dermis of the low-dose 18β-glycyrrhetinic acid group increased, were more tightly arranged, the fiber breakage was alleviated, and the degree of matrix degradation was reduced. The distribution of collagen fibers in the dermis of the medium-dose 18β-glycyrrhetinic acid group was more uniform, the arrangement was tighter, the staining was more uniform, and the fiber breakage and matrix degradation were significantly reduced. The structure of collagen fibers in the dermis of the high-dose 18β-glycyrrhetinic acid group was close to that of the normal control group, the fiber distribution almost returned to normal, the arrangement was tight, the staining was uniform, and there was no obvious fiber breakage and matrix degradation. The above results show that 18β-glycyrrhetinic acid has a significant effect on the repair of dermal collagen fiber breakage.

[0070] Effects of 18β-glycyrrhetinic acid on pathological characteristics of UVB-damaged mouse skin

[0071] The skin tissue sections were stained with Sirius red, and the specific processing process was as follows:

[0072] Sirius red staining: Dewax the mouse skin tissue sections routinely, then mix Weigert A solution and B solution in a 1:1 ratio before use, and add the prepared Weigert iron hematoxylin staining solution for 10 minutes, and wash with distilled water for 10 seconds to wash away the excess staining solution. Soak in tap water for 10 minutes, and wash with distilled water 3 times, each time for 5 seconds. Drip the Sirius red staining solution for 15 minutes, rinse with running water to remove the staining solution on the surface of the section, and soak in 75% ethanol, 85% ethanol, 95% ethanol and 100% ethanol (I) for 2-3 seconds, soak in 100% ethanol (II) for 1 minute, and finally transparentize with xylene twice and seal with neutral gum.

[0073] After Sirius red staining, the samples were observed under an optical microscope. The test results were as follows: Figure 5 shown.

[0074] from Figure 5 It can be seen that in the skin of mice in the normal control group, the collagen fibers in the dermis are evenly distributed, tightly arranged and evenly stained, showing the typical characteristics of healthy skin. The collagen fiber structure is intact without obvious breakage or damage, indicating that the skin is in good condition. Compared with the normal control group, the collagen fibers in the model group were significantly reduced, the fibers were loosely arranged, the staining was uneven, and the fiber breakage increased, indicating that the model group had severe collagen fiber damage and structural reconstruction. Compared with the model group, the collagen fiber damage in the low-dose 18β-glycyrrhetinic acid group was slightly alleviated, the fiber breakage phenomenon was reduced, and the fibers were arranged more tightly; the fiber breakage phenomenon of the collagen fibers in the medium-dose 18β-glycyrrhetinic acid group was significantly reduced, the fiber distribution was close to that of the normal control group, the arrangement was tight, and the staining was uniform; the collagen fibers in the high-dose 18β-glycyrrhetinic acid group were tightly arranged, the staining was uniform, there was no obvious fiber breakage phenomenon, and the collagen fiber structure was close to that of the normal control group. Compared with the positive drug group, the Sirius red staining positive area of ​​the 18β-glycyrrhetinic acid low-dose group, the 18β-glycyrrhetinic acid medium-dose group and the 18β-glycyrrhetinic acid high-dose group increased, indicating that the effect of 18β-glycyrrhetinic acid in repairing collagen fibers is significantly better than that of vitamin E in repairing collagen fibers. The above data show that 18β-glycyrrhetinic acid alleviates the phenomenon of UVB-induced collagen fiber rupture in mouse skin, and 18β-glycyrrhetinic acid has a significant effect on repairing collagen fiber damage and protecting the structural integrity of collagen fibers.

[0075] 8. Detection of CAT, SOD and HYP of UVB-damaged mouse skin tissue protein by 18β-glycyrrhetinic acid

[0076] Weigh 30-40 mg of skin tissue, cut the tissue into 1x1 mm or smaller pieces with scissors as samples, and transfer the samples to a centrifuge column for later use. Add 50-80 mg of protein grinding powder to cover the sample, add 100 ul of lysis solution, and then immediately press the sample with a plastic grinding rod to twist and grind for 2-3 minutes, add the remaining 100 ul of lysis solution, and continue grinding for 0.5-1 minute. Cover the lid, centrifuge at 4°C and 12000 rpm for 1 minute, and discard the centrifuge column. Use a pipette to pass through the oil layer to absorb the total skin tissue protein in the supernatant of the tube, and transfer the total skin tissue protein to a new tube for detection of CAT activity, SOD activity and HYP content.

[0077] The catalase (CAT) activity detection kit, superoxide dismutase (SOD) activity detection kit and hydroxyproline (HYP) content detection kit were used respectively, and the experimental operation was carried out according to the kit instructions to determine the CAT activity, SOD activity and HYP content in mouse skin tissue protein. The test results are as follows Figure 6 shown.

[0078] CAT is an important antioxidant enzyme responsible for decomposing hydrogen peroxide in cells, reflecting the skin's ability to resist oxidative stress. SOD can catalyze the conversion of superoxide free radicals into hydrogen peroxide, thereby reducing the damage of free radicals to cells. It is mainly used to evaluate the function of the skin's antioxidant defense system. HYP is an important component of collagen and a sign of the integrity and regeneration of skin tissue structure. The determination of HYP content mainly reflects the synthesis and repair ability of skin collagen.

[0079] from Figure 6 It can be seen that compared with the normal control group, the CAT activity, SOD activity and HYP content of the model group were significantly reduced, indicating that the resistance and antioxidant capacity of UVB-damaged mouse skin to oxidative stress were significantly reduced, and skin collagen was damaged. Compared with the model group, the CAT activity, SOD activity and HYP content of the positive drug group and each dose group of 18β-glycyrrhetinic acid were increased, indicating that each dose group of 18β-glycyrrhetinic acid improved the antioxidant capacity of the skin and improved the collagen synthesis and repair capacity of UVB-damaged mouse skin. In addition, the CAT activity, SOD activity and HYP content of the low-dose 18β-glycyrrhetinic acid group were close to those of the positive drug group, and the CAT activity, SOD activity and HYP content of the medium-dose 18β-glycyrrhetinic acid group and the high-dose 18β-glycyrrhetinic acid group were higher than those of the positive drug group. The above results show that 18β-glycyrrhetinic acid has a good antioxidant and repair effect on UVB-damaged mouse skin tissue protein.

[0080] 2. Experiment on HSF cells repaired by 18β-glycyrrhizic acid in UVB-damaged cells

[0081] 1. Main instruments, materials and reagents

[0082] UV lamp, optical microscope, OLYPUMS image acquisition system; Vitamin E (VE), 18β-Glycyrrhetinic acid (18β-GA), Dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide (MTT);

[0083] Specific antibodies for collagen typeⅢ, MMP2, p53 and p21 were purchased from Proteintech, USA; reactive oxygen species (ROS) were purchased from Bio-Technology Co., Ltd.; cell cycle kit was purchased from Jiangsu Keygen Biotech Co., Ltd.; β-galactosidase staining kit was purchased from Beijing Solebow Technology Co., Ltd.

[0084] 2. Experimental cells: Human skin fibroblasts (HSF) cells were purchased from Purcell Life Science Co., Ltd.

[0085] 3. Modeling and verification of UVB-damaged HSF cell model

[0086] HSF cells in the logarithmic growth phase were taken at 1×10 4 HSF cells were seeded into 96-well plates at a density of 100 cells / well and irradiated with 0, 30, 60, 90, 120, 150, and 300 mJ / cm 2 The HSF cells were irradiated with UVB (wavelength 302 nm) for 60 s, and then the HSF cells were placed in a 5% CO2 incubator for further 24 h.

[0087] MTT was added to the HSF cells cultured in the incubator and then cultured for 4 h. DMSO was then added and the cell activity was detected using an ELISA instrument. The test results were as follows: Figure 7 shown.

[0088] from Figure 7 It can be seen that the cell activity of HSF cells irradiated by UVB decreased to varying degrees. Compared with HSF cells not irradiated by UVB, the cell activity of HSF cells irradiated by UVB at an energy density of 150 mJ / cm 2 The cell viability of HSF cells irradiated with UVB was significantly reduced, so 150 mJ / cm 2 The UVB irradiation dose was used as a method to construct a UVB-damaged HSF cell model.

[0089] Collect 0mJ / cm2 HSF cells irradiated with UVB of 150 mJ / cm 2 The cell cycle of HSF cells irradiated with UVB (referred to as the model group, UVB) was detected by flow cytometry, the expression levels of type III collagen, MMP2, p53 and p21 were detected by Western blotting, and the morphological changes of HSF cells were detected using a β-galactosidase staining kit. Figures 8 to 13 shown.

[0090] The activity of β-galactosidase in senescent cells increases significantly. By detecting the activity of β-galactosidase in cells, it can be determined whether the cells are in a senescent state. The expression of type III collagen, a marker of senescence, decreases significantly with age. MMP2 degrades some of the cleaved collagen fibers, causing them to further degrade into soluble fragments, leading to a decrease in collagen in the dermis and a loose structure. p53 can induce the expression of cyclin-dependent kinase inhibitors (CKIs) such as p21CIP1 / WAF1, thereby inhibiting the activity of cyclin-dependent kinases (CDKs) and causing the cell cycle to arrest in the G1 or G2 phase. The expression level of p21 in senescent cells increases significantly, and by inhibiting the activity of CDKs, the cell cycle is arrested in the G1 phase, leading to cell senescence.

[0091] from Figures 8 to 13 It can be seen that compared with the normal group, the use of 150mJ / cm 2 The proliferation of HSF cells irradiated with medium-wave ultraviolet UVB for 24 hours was inhibited, and the cell morphology changed significantly, with the cell volume increasing and becoming flat, and the refractive index decreasing. Figure 8 , Fig. 9 ) showed that the percentage of positively stained cells in the model group was significantly higher than that in the normal group, indicating that UVB irradiation induced HSF cell senescence. Flow cytometry analysis of the cell cycle results ( Fig.10 , Fig.11 ) showed that compared with the normal group, the proportion of G1 cells in the model group was significantly increased, and the proportion of G2 and S cells was significantly decreased, indicating that HSF cells in the model group were blocked in the G1 phase. Western blotting results ( Fig.12 , Fig.13 ) showed that compared with the normal group, the expression of type III collagen, a marker of aging, in the model group was significantly reduced, while the expression of MMP2, p53 and p21 was significantly increased, indicating that UVB promotes HSF cell aging through multiple pathways. The above results indicate that the UVB-damaged HSF cell model was successfully established.

[0092] 4. Effects of 18β-glycyrrhetinic acid on UVB-damaged HSF cells

[0093] HSF cells in the logarithmic growth phase were taken at 1×10 4 The HSF cells were seeded into 96-well plates at a density of 100 cells / well. The HSF cells were evenly divided into 1 control group (NC), 1 model group (UVB), 1 positive drug group (UVB+VE) and 7 drug-treated groups (UVB+18β-GA). 2 The HSF cells of the model group, the positive drug group and each drug-treated group were irradiated with medium-wave ultraviolet UVB for 60 seconds, and then the HSF cells of each group were placed in a 5% CO2 incubator for further culture for 24 hours.

[0094] 30μmol / L, 60μmol / L, 90μmol / L, 120μmol / L, 150μmol / L, 180μmol / L and 210μmol / L of 18β-glycyrrhetinic acid were added to each of the above-mentioned drug administration groups after culture in the incubator. The treatment method was to first prepare the culture medium containing different concentrations of 18β-glycyrrhetinic acid, and then add the culture medium by changing the cell medium, that is, first suck away the old culture medium, and then add 200μL of culture medium containing different concentrations of 18β-glycyrrhetinic acid to each well; 100μmol / L of vitamin E was added to the positive drug group, and the treatment method was the same as that of the drug administration group; there was no operation in the control group and the model group, and then each group of HSF cells was cultured for 24h, and then MTT was added and cultured for another 4h, DMSO was added, and the cell activity was detected by using an enzyme marker; HSF cells from the control group, model group, positive drug group and drug administration group with the best cell activity were collected, and the reactive oxygen species (ROS) of each group were detected by flow cytometry. The test results are as follows Figures 14 to 16 shown.

[0095] Depend on Fig.14 It can be seen that in the drug-treated group, the cell viability of the UVB-damaged HSF cells treated with 60 μmol / L 18β-glycyrrhetinic acid was the highest. Fig.15 , Fig.16 As shown in the figure, compared with the model group, 60 μmol / L 18β-glycyrrhetinic acid can significantly improve the ROS of UVB-damaged HSF cells, indicating that 18β-glycyrrhetinic acid can effectively reduce the level of ROS in the UVB-damaged HSF cell model, reduce the damage of oxidative stress to cells, and then restore the normal function and vitality of HSF cells. At the same time, the improvement of HSF cell vitality is conducive to promoting the proliferation and survival of HSF cells and promoting the repair of skin tissue. Therefore, compared with the model group, treating HSF cells with different concentrations of 18β-glycyrrhetinic acid can improve the cell vitality of HSF cells after light damage.

[0096] The absorbance values ​​at 490 nm were measured by an ELISA instrument at 0 h, 3 h, 6 h, 9 h, 12 h and 24 h after the addition of 18β-glycyrrhetinic acid in the treatment group of UVB-damaged HSF cells treated with 60 μmol / L 18β-glycyrrhetinic acid. Fig.17 shown.

[0097] Depend on Fig.17 It can be seen that the absorbance value of UVB-damaged HSF cells treated with 60μmol / L 18β-glycyrrhetinic acid increases with the treatment time, among which the absorbance value increases significantly 3 to 12 hours after the addition of 18β-glycyrrhetinic acid. Since the number of cells in the same volume is proportional to the absorbance, the number of HSF cells increases significantly after the addition of 18β-glycyrrhetinic acid, indicating that 18β-glycyrrhetinic acid can promote the proliferation and survival of HSF cells, thereby promoting skin repair. This may be because 18β-glycyrrhetinic acid alleviates the damage caused by UVB irradiation to HSF cells by downregulating the expression of inflammatory factors and oxidative stress indicators.

[0098] The cell cycle of HSF cells damaged by UVB treated with 60 μmol / L 18β-glycyrrhetinic acid in the drug administration group was detected by flow cytometry, and the expression levels of type III collagen (collagen III), MMP2, p53 and p21 were detected by Western blotting. Fig.18 shown.

[0099] Depend on Fig.18 It can be seen that compared with the model group, the expression of aging marker type III collagen in UVB-damaged HSF cells (UVB+18β-GA) treated with 60μmol / L 18β-glycyrrhetinic acid was significantly increased, which can promote cell repair, and the expression of MMP2, p53 and p21 was significantly reduced. This may be because 18β-glycyrrhetinic acid can repair the matrix and DNA damaged by ultraviolet light, inhibit inflammatory response, and improve the stability of photodamaged HSF cells. The above results show the repair effect of 18β-glycyrrhetinic acid on photodamaged HSF cells.

[0100] The above experiments and their results show that 18β-glycyrrhetinic acid can significantly alleviate the clinical symptoms of skin damage such as erythema, nodules, scaling, lack of skin luster and pigmentation in the UVB-damaged mouse skin model; it can significantly reduce the transepidermal water loss score while improving the skin barrier score and hydration score, showing the role of 18β-glycyrrhetinic acid in protecting or repairing the skin barrier and enhancing skin hydration. At the same time, 18β-glycyrrhetinic acid can reduce epidermal thickening, improve keratinization, improve inflammatory cell infiltration, and improve the manifestations of UVB-induced skin collagen fiber damage, including reducing collagen fiber reduction, loose arrangement and fiber breakage, thereby effectively delaying and reducing UVB-induced skin pathology and collagen fiber pathological damage. 18β-glycyrrhetinic acid can also increase the activity of CAT and SOD, as well as the content of HYP, showing the ability of 18β-glycyrrhetinic acid to synthesize and repair skin collagen.

[0101] In the UVB-damaged HSF cell model, 18β-glycyrrhetinic acid can reduce the ROS level of HSF cells, reduce the damage of oxidative stress to cells, and restore the normal function and vitality of HSF cells. 18β-glycyrrhetinic acid can also promote the proliferation and survival of HSF cells. In addition, 18β-glycyrrhetinic acid can increase the expression level of type III collagen and reduce the expression levels of MMP2, p53 and p21.

[0102] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the creative concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. 18 Use of β-glycyrrhetinic acid or an extract containing it in the preparation of drugs or cosmetics for treating or repairing skin photodamage.

2. The use according to claim 1, characterized in that: The medicine or cosmetic is a substance used to protect or repair the skin barrier and enhance skin hydration.

3. The use according to claim 1, characterized in that: The medicine or cosmetic is a substance used to reduce epidermal thickening, improve keratinization, improve inflammatory cell infiltration, and improve the arrangement of collagen fibers in the dermis.

4. The use according to claim 1, characterized in that: The medicine or cosmetic is a substance used to increase the activity of catalase and reduce the level of active oxygen.

5. The use according to claim 1, characterized in that: The medicine or cosmetic is a substance used for promoting the proliferation and survival of fibroblasts.

6. The use according to claim 1, characterized in that: The medicine or cosmetic is a substance for increasing the expression level of type III collagen.

7. The use according to claim 1, characterized in that: The medicine or cosmetic is a substance for reducing the expression levels of MMP2, p53 and p21.

8. The use according to claim 1, characterized in that: The 18β-glycyrrhetinic acid is used in the preparation of a drug or cosmetic for treating or repairing skin photodamage, which has one or more of the following effects: improving skin erythema, induration, scaling, skin gloss, moisture and pigmentation.

9. The use according to any one of claims 1 to 8, characterized in that: The structural formula of the 18β-glycyrrhetinic acid is shown in formula (I):

10. The use according to claim 9, characterized in that: The skin photodamage refers to skin damage caused by ultraviolet rays.

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