Application of flavone compound in preparation of product for preventing and treating skin light injury

Abstract

The invention discloses an application of a flavone compound in preparation of a product for preventing and treating skin light injury. The flavone compound is a compound as shown in a formula 1. The flavone compound can effectively relieve skin light injury caused by UVB (medium-wave ultraviolet B), and can be applied to preparation of cell necrosis inhibitors and drugs, health food, cosmetics and hygienic products for preventing or treating or improving skin light injury caused by UVB exposure.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to application of a flavonoid compound in preparing a product for preventing and treating UVB-induced skin photodamage. Background Art

[0002] Skin photodamage is a general term for the various harmful effects of prolonged exposure of the skin to the sun's ultraviolet radiation. When the skin is exposed to the sun for a long time, it is easily affected by ultraviolet radiation, causing acute and chronic skin lesions, such as external aging (photoaging), skin inflammation, etc., and may lead to skin cancer. About 80% of facial skin aging is caused by ultraviolet exposure.

[0003] Excessive exposure to ultraviolet radiation, especially UVB components, can cause erythema, edema, hyperplasia, pigmentation, photoaging, immunosuppression and skin cancer. UVB rays reach the superficial dermis of the skin, causing skin inflammatory cell infiltration, epidermal / dermal hyperplasia, and skin collagen loss. After the skin is continuously irradiated by UVB, it is also easy to cause melanin deposition, causing the skin color to darken. On the other hand, the repeated stimulation of melanin-forming cells by ultraviolet radiation may cause the latter's genetic material mutation or abnormal death, causing skin melanoma or vitiligo.

[0004] Existing studies have shown that UVB radiation can cause cell death and inflammatory responses, including programmed cell death and inflammasome activation. UVB irradiation can cause organelle damage and activation of necrosis signals, ultimately leading to phosphorylation of the effector protein MLKL, which in turn destroys cell membrane integrity, causing cell death and releasing damage-related molecules. At the same time, UVB can cause intracellular oxidative stress and mitochondrial damage, ultimately leading to the production of intracellular reactive oxygen species and mitochondrial superoxide, accompanied by a decrease in mitochondrial membrane potential.

[0005] MLKL protein phosphorylation during cell programmed necrosis can be detected by Western blotting (WB); the integrity of the cell membrane can be detected by lactate dehydrogenase (LDH) release assay.

[0006] Skin photodamage can accelerate skin tissue aging, chronic inflammation, and even induce skin cancer. In recent decades, air pollution and damage to the ozone layer have made skin diseases caused by ultraviolet rays gradually become a huge challenge for the public health system. In the United States, more than 3.5 million new cases of non-melanoma skin cancer are diagnosed each year. In my country, although the incidence of skin cancer is relatively low, it is on the rise. According to a report from the Chinese Center for Disease Control and Prevention, the incidence of non-melanoma skin cancer ranks sixth in China, second only to breast cancer.

[0007] For the prevention and treatment of skin photodamage, one is to block the impact of ultraviolet radiation on cells, and the other is to inhibit the occurrence of post-sun inflammation. However, traditional sunscreen or anti-inflammatory drugs have many defects. For example, traditional physical and chemical sunscreens have adverse reactions such as clogging pores, affecting sweat gland function, inducing allergic reactions, and poor treatment effects on post-sun inflammation; traditional anti-inflammatory drugs such as steroids or non-steroidal anti-inflammatory drugs have certain toxicity, easily cause hormone disorders, abdominal pain, indigestion, etc., and cannot intervene in programmed cell death, and have poor targeting.

[0008] Flavonoids are widely found in vegetables, fruits, and tea in our daily diet. They are also the active ingredients of some traditional medicinal materials. They have been proven to have good antioxidant, anti-inflammatory, antibacterial, and anti-tumor activities, and are relatively safe.

[0009] The flavonoid compounds (C 20 H 18 O6) Application against UVB photodamage. Summary of the invention

[0010] The purpose of the present invention is to overcome the defects of the prior art and provide an application of a flavonoid compound in the preparation of a product for preventing and treating skin photodamage. The compound can be used as a protective agent to prevent and treat skin inflammation caused by UVB radiation and alleviate skin damage; the compound also shows inhibitory activity on UVB-induced cell necrosis and can effectively alleviate the structural damage of skin tissue after UVB irradiation. It can be used as an effective ingredient for inhibiting UVB-induced skin photodamage to prevent and treat UVB-related skin sunburn, or as an additive in functional cosmetics, health foods and sanitary products.

[0011] The technical solution to achieve the above object is: use of a flavonoid compound in the preparation of a product for preventing and treating skin photodamage, wherein the flavonoid compound is a compound C of formula (1) 20 H 18 O6, molecular formula is:

[0012]

[0013] The use of the above-mentioned flavonoid compound, wherein the flavonoid compound protects the skin from light damage caused by UVB by inhibiting cell necrosis. The skin light damage inducing condition is UVB radiation, which manifests as skin redness, dehydration, skin structure changes, and increased levels of cell necrosis in skin tissue.

[0014] The above-mentioned application of flavonoid compounds, wherein in UVB-induced skin photodamage, cell necrosis is triggered by MLKL protein phosphorylation. The cells include keratinocytes and macrophages, and the photodamage refers to cell death, cell structure destruction, cell oxidative stress, etc., which occur in the above-mentioned cells after UVB irradiation.

[0015] Preferably, the protection against UVB light damage refers to improving the cell mortality rate after UVB irradiation, inhibiting oxidative stress after UVB irradiation, and protecting cell integrity.

[0016] Preferably, the reducing cell death and protecting cell integrity refers to at least one of inhibiting the release of cell LDH after UVB irradiation and inhibiting the phosphorylation of MLKL protein.

[0017] The above-mentioned application of flavonoid compounds, wherein the effective concentration of the flavonoid compounds for inhibiting UVB-induced skin photodamage is 0.125% to 0.50% w / v. When the concentration of the flavonoid compounds in each product is 0.125% to 0.5% w / v, it can significantly inhibit the skin damage on the back of mice after UVB irradiation, which is manifested by significantly reversing the skin inflammatory cell infiltration caused by UVB, significantly reversing the hyperplasia of the epidermis / dermis, significantly reversing the loss of skin collagen, and significantly inhibiting the skin cell necrosis caused by UVB.

[0018] The use of the above-mentioned flavonoid compound, wherein the flavonoid compound effectively alleviates cellular oxidative stress and inhibits the production of intracellular reactive oxygen and mitochondrial superoxide induced by UVB irradiation.

[0019] The use of the above-mentioned flavonoid compound, wherein the flavonoid compound reduces the damage of cell mitochondria after UVB irradiation and inhibits the decrease of mitochondrial membrane potential.

[0020] In the above-mentioned application of flavonoid compounds, the skin photodamage caused by UVB includes at least one of sunburn, skin redness induced by solar radiation, skin aging induced by solar radiation, skin cancer induced by solar radiation and skin pigmentation induced by solar radiation.

[0021] The above-mentioned application of flavonoid compounds, wherein the product includes at least one of medicines, health foods, cosmetics and sanitary products.

[0022] The above-mentioned application of flavonoid compounds, wherein the dosage form of the medicine and health food is at least one of injection, film, ointment, cream, paste, gel, coating, plaster, patch, aerosol, spray, extract, tablet, dispersible tablet, lozenge, orodisintegrating tablet, sustained-release tablet, capsule, soft capsule, pill and granule.

[0023] The above-mentioned application of flavonoid compounds, wherein the type of the cosmetics includes at least one of sunscreen, sunscreen milk, sunscreen gel, facial cleanser, toner, floral water, sunscreen spray, sunscreen lotion, facial cream, facial mask and facial gel.

[0024] In the above-mentioned application of flavonoid compounds, the type of sanitary products includes at least one of wet wipes, sanitary wipes, sanitary napkins, diapers, band-aids and medical gauze.

[0025] According to the results of animal experiments, the flavonoid compound can be applied to animal models to play a protective role in UVB-induced animal skin redness, structural changes, skin dehydration, skin collagen loss, and skin inflammation, thereby alleviating the damage of UVB radiation to the skin.

[0026] According to cell experiments, the flavonoid compound can inhibit UVB-induced oxidative stress, mitochondrial damage, programmed cell necrosis and inflammasome activation in keratinocytes (such as HaCaT cells) and human / mouse macrophages (such as THP-1 and J774A.1 cells), and reduce tissue damage and inflammatory response.

[0027] The flavonoid compound of the present invention is used in the preparation of a product for preventing and treating skin photodamage. The compound has the function of protecting the skin from UVB damage. The compound can reverse skin redness and swelling, skin collagen loss, skin inflammatory cell infiltration, skin water loss and skin cell necrosis caused by UVB radiation at a concentration of 0.125% to 0.5% (w / v); at the same time, the compound has good antioxidant effect and can effectively alleviate the oxidative stress and mitochondrial damage of keratinocytes and macrophages caused by UVB, indicating that the compound has the effect of antioxidant and inhibiting photodamage. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a diagram showing the damage of the mouse back skin obtained in Example 1 of the present application after continuous UVB irradiation (in the compound 1 pretreatment group, the degree of damage to the mouse back skin was significantly reduced);

[0029] Figure 2 This is a statistical chart of the redness and swelling results of the mouse back skin obtained in Example 1 of the present application (in the compound 1 pretreatment group, the redness and swelling of the mouse skin was significantly reduced, indicating that the compound can effectively alleviate the skin redness and swelling caused by UVB);

[0030] Figure 3 This is a microscopic image of the H&E pathological staining section of mouse skin tissue in Example 2 of the present application;

[0031] Figure 4 This is a statistical graph of the measurement results of the epidermal thickness of mouse skin tissue in Example 2 of the present application;

[0032] Figure 5 This is a statistical graph of the measurement results of the dermis thickness of mouse skin tissue in Example 2 of the present application;

[0033] Figure 6 This is a microscopic image of a Masson pathologically stained section of mouse skin tissue in Example 3 of the present application;

[0034] Figure 7 This is a statistical chart of the collagen fiber content in mouse skin tissue in Example 3 of the present application;

[0035] Figure 8 This is a statistical diagram of the measurement results of mouse skin moisture content in Example 4;

[0036] Fig. 9 This is the result of immunoblotting of MLKL protein phosphorylation in mouse skin tissue after treatment with compound 1 in Example 5;

[0037] Fig.10 This is a microscopic image of the cell morphology of keratinocytes HaCaT after treatment with compound 1 in Example 7;

[0038] Fig.11 This is the result of immunoblotting of MLKL protein phosphorylation in keratinocytes HaCaT after treatment with compound 1 in Example 7;

[0039] Fig.12 This is a fluorescence micrograph of the reactive oxygen probe DCFH-DA in keratinocytes HaCaT after treatment with compound 1 in Example 8;

[0040] Fig.13 This is a statistical graph of the fluorescence intensity of the reactive oxygen probe DCFH-DA in keratinocytes HaCaT after treatment with compound 1 in Example 8;

[0041] Fig.14 These are the results of the LDH release test of macrophages J774A.1 and THP-1 after treatment with compound 1 in Example 9 (A is the result of the LDH release test of J774A.1, and B is the result of the LDH release test of THP-1);

[0042] Fig.15 Fluorescence microscopic imaging of the active oxygen probe DCFH-DA in J774A.1 cells after treatment with compound 1 in Example 13 and its fluorescence intensity statistical graph (A is the fluorescence microscopic imaging graph, and B is the fluorescence intensity statistical graph);

[0043] Fig.16 This is the fluorescence microscopic imaging of the mitochondrial superoxide probe MSR in J774A.1 cells after treatment with compound 1 in Example 14;

[0044] Fig.17It is the fluorescence microscopic imaging of the mitochondrial membrane potential probe JC-1 in J774A.1 cells after treatment with compound 1 in Example 15 and the fluorescence intensity statistics of JC-1 aggregates / monomers (A is the fluorescence microscopic imaging diagram, and B is the fluorescence intensity statistics diagram). DETAILED DESCRIPTION

[0045] In order to enable those skilled in the art to better understand the technical solution of the present invention, the specific implementation methods thereof are described in detail below in conjunction with the accompanying drawings:

[0046] Application of a flavonoid compound in the preparation of a product for preventing and treating skin photodamage, wherein the flavonoid compound is a compound C of formula (1) 20 H 18 O6, molecular formula is:

[0047]

[0048] In the following examples, compound 1 is used to refer to the flavonoid compound.

[0049] Example 1: Experiment on Compound 1 Alleviating UVB-Induced Skin Damage in Mice

[0050] (1) Experimental methods:

[0051] Preparation of topical hydrogel containing compounds at specified concentrations: weigh 1 g of Carbomer 940, 5 g of glycerol, and 0.2 g of polysorbate 90 (Tween 80), mix well in 30 ml of distilled water, add 10 ml of 40 mg / ml sodium hydroxide solution and 1% propylparaben ethanol solution, mix well, add water to 70 g, leave at room temperature for 1 to 2 hours to allow Carbomer to fully absorb water, continue to add distilled water to 100 g and stir well to obtain a transparent and uniform hydrogel matrix, use centrifugation (500×g, 10 minutes) to remove bubbles for use. Dissolve compound 1 in anhydrous ethanol, and then mix with the hydrogel matrix at the specified final concentration to obtain a gel containing compound 1, which is stored in the dark.

[0052] Animal grouping and depilatory treatment: ICR mice were randomly divided into different dose modeling groups in groups of 5, and the UVB modeling dose was 300mJ / cm2. For the study of the protective effect of compound 1 on UVB light damage to mouse skin, mice were randomly divided into 5 groups in groups of 5: Sham group, modeling group (UVB+hydrogel matrix), low-dose treatment group (UVB+0.125% compound 1), medium-dose treatment group (UVB+0.25% compound 1), and high-dose treatment group (UVB+0.5% compound 1). For all grouped mice, the hair in a fixed area on the back of the mice was shaved short using a small animal shaving machine one day in advance, and then the hair was further removed using a depilatory containing potassium thioglycolate. After about 2 to 3 minutes, the depilatory cream was wiped clean with a sterile cotton ball dipped in saline to avoid residue. In order to reduce the impact of hair growth during the experiment, the mice were depilated once every 2 days.

[0053] Drug administration and modeling: The mice were anesthetized with isoflurane, and the UVB lamp was preheated for 5 to 10 minutes to stabilize its power. The sham group was not treated, and the modeling group mice were smeared with a hydrogel matrix without compound 1 on the exposed part of the back skin. The compound 1 treatment group was divided into three groups with different doses and smeared with a hydrogel containing different concentrations of compound 1 to form a uniform gel film on the exposed skin of the back of the mice. After the gel was absorbed, the mice were irradiated with the same dose of UVB for 5 days. During this period, the weight, skin appearance, skin water content, etc. of the mice were continuously recorded, and the degree of redness and swelling of the irradiated part of the skin of the mice was statistically analyzed.

[0054] (2) Experimental results are as follows Figure 1 , Figure 2 As shown, it can be seen that as time goes by, the skin of mice irradiated by UVB gradually shows signs of damage on the 5th day, while the redness, swelling and lesions of the mouse skin in the group pretreated with hydrogel containing 0.125% to 0.5% (w / v) are significantly improved.

[0055] Example 2: Compound 1 reduces UVB-induced inflammatory cell infiltration and structural changes in mouse skin

[0056] (1) Experimental methods:

[0057] The animal grouping, modeling, and drug administration methods of this example are the same as those of Example 1.

[0058] After the mice were killed on the 5th day, a 10 mm diameter skin tissue sampler was used to sample the skin on the back of the mice, and the samples were placed in a tissue fixative (10% formalin) to denature the protein to maintain the cell morphology and structure. Alcohol was used from low to high concentrations to gradually remove the water in the tissue block. The tissue block was then placed in xylene, a transparent agent that is soluble in both alcohol and paraffin, for transparent treatment. The alcohol in the tissue block was replaced with xylene, and the transparent tissue block was placed in the melted paraffin liquid and placed in a wax melting box for insulation. After the paraffin is completely immersed in the tissue block, embed it: first prepare a container (such as a folded small paper box), pour in the melted paraffin, quickly clamp the tissue block that has been soaked in paraffin and put it in it, and after cooling and solidification, use a slicer to cut it into 5-8 μm thick slices, put it in heated water to iron it flat, and then stick it on a slide and dry it in a 45°C constant temperature box. The samples were stained with H&E stain, then dehydrated with ethanol and cleared with xylene, and sealed with neutral gum and coverslip for observation under a microscope.

[0059] (2) Experimental results:

[0060] The experimental results can be found in Figure 3 to Figure 5 The mouse skin tissue samples were stained with H&E and imaged using a microscope. It was found that compound 1 could dose-dependently inhibit the abnormal proliferation of the epidermis and dermis after UVB irradiation and reduce inflammatory cell infiltration.

[0061] Example 3: Compound 1 reduces UVB-induced collagen loss in mouse skin

[0062] (1) Experimental methods:

[0063] The animal grouping, modeling, and drug administration methods of this example are the same as those of Example 1.

[0064] After the mice were killed on the 5th day, a 10 mm diameter skin tissue sampler was used to sample the skin on the back of the mice, and the samples were placed in a tissue fixative (10% formalin) to denature the protein to maintain the cell morphology and structure. Alcohol was used from low to high concentrations to gradually remove the water in the tissue block. The tissue block was then placed in xylene, a transparent agent that is soluble in both alcohol and paraffin, for transparent treatment. The alcohol in the tissue block was replaced with xylene, and the transparent tissue block was placed in the melted paraffin liquid and placed in a wax melting box for insulation. After the paraffin is completely immersed in the tissue block, embed it: first prepare a container (such as a folded small paper box), pour in the melted paraffin, quickly clamp the tissue block that has been soaked in paraffin and put it in it, and after cooling and solidification, use a slicer to cut it into 5-8 μm thick slices, put it in heated water to iron it flat, and then stick it on a slide and dry it in a 45°C constant temperature box. Masson stain was used for staining, followed by ethanol dehydration and xylene clearing, and then neutral gum and coverslip sealing were used for observation under a microscope. ImageJ software was used to analyze the images and quantitatively analyze the collagen staining.

[0065] (2) Experimental results:

[0066] Collagen fibers are an important component of dermal connective tissue and are responsible for maintaining skin elasticity. The aging process of the skin is accompanied by a decrease in the content of collagen fibers. Masson staining distinguishes collagen fibers and muscle fibers in tissues by the difference in the permeability of collagen fibers and muscle fibers to different anionic dyes. Generally speaking, collagen fibers appear blue (collagen fibers have greater permeability and are stained with aniline blue, which has larger molecules), and muscle fibers appear red (muscle fibers have less permeability and are stained with fuchsin and ponceau red, which have smaller molecules).

[0067] The results of the above experiments are as follows Figure 6 and Figure 7 As shown, it can be seen that compound 1 can dose-dependently reverse the decrease in collagen fiber content in mouse skin tissue caused by UVB irradiation, indicating that compound 1 can protect skin elasticity from UVB radiation damage, suggesting that it has anti-aging effects on the skin.

[0068] Example 4: Compound 1 reduces UVB-induced skin water loss in mice

[0069] (1) Experimental methods:

[0070] The animal grouping, modeling, and drug administration methods of this example are the same as those of Example 1.

[0071] On the 5th day, the mice were killed and the skin of the back of the mice was sampled using a 10 mm diameter skin tissue sampler, and the wet weight of each sample was weighed using an analytical balance. Each sample was then placed in a drying oven for ventilation and drying to a constant weight, and its dry weight was weighed using an analytical balance to calculate the skin water content (%) = (wet weight - dry weight) / wet weight × 100%. The data of the drug administration group were compared with those of the model group.

[0072] (2) Experimental results:

[0073] Skin moisture content is an important indicator for measuring skin health and judging skin aging. The occurrence of skin diseases is often accompanied by a decrease in skin moisture content.

[0074] The experimental results are as follows Figure 8 As shown, after UVB irradiation, the water content of mouse skin decreased by about 6% compared with the normal group, while compound 1 alleviated the skin water loss caused by UVB at a concentration of 0.125% to 0.5%, indicating that compound 1 has the effect of moisturizing the skin.

[0075] Example 5: Compound 1 inhibits UVB-induced cell necrosis in mouse skin tissue

[0076] (1) Experimental methods:

[0077] The animal grouping, modeling, and drug administration methods of this example are the same as those of Example 1.

[0078] Mouse skin tissue protein extraction: After the mice were killed on the 5th day, the skin tissues of mice in each group were sampled, and the skin tissue samples of each group were weighed with equal weight, cut into pieces of 1-2 mm in size, and transferred to a porcelain mortar and ground into fine powder under liquid nitrogen freezing. The powder was transferred to a pre-cooled 1.5 ml EP tube, and RIPA lysis buffer containing phosphatase inhibitors and protease inhibitors was added at a ratio of 10 mg / 100 μL. The tube was incubated on ice for 30 minutes and ultrasonically disrupted (6% power, 2s on / 2s off, 15 cycles, operated under ice-water mixture cooling), followed by centrifugation using a high-speed refrigerated centrifuge (12,000 g, 10 minutes, 4°C), the supernatant was collected, and 6×SDS loading buffer was added, the tube was treated in a boiling water bath for 5-8 minutes, and stored in a -20°C or -80°C freezer for use.

[0079] Protein detection: The samples prepared in the previous step were subjected to immunoblotting analysis to detect the levels of MLKL protein and its phosphorylated form p-MLKL. The antibodies used were anti-p-MLKL and anti-MLKL, and the internal reference protein was β-actin.

[0080] (2) Experimental results:

[0081] The characteristic of cell necrosis is that the phosphorylated MLKL protein is localized in the cell membrane and forms a pore-like structure, which causes increased cell membrane permeability, ultimately resulting in cell structural damage and the release of damage-associated molecular patterns, activating the innate immune mechanism.

[0082] The experimental results of this embodiment are as follows Fig. 9 As shown, UVB radiation causes phosphorylation of MLKL protein in mouse skin tissue, while the compound 1-treated group does not show p-MLKL bands or the bands are significantly lighter than those in the model group, indicating that the compound can effectively inhibit the phosphorylation of MLKL protein, thereby inhibiting UVB-induced cell necrosis.

[0083] Example 6: Compound 1 inhibits UVB-induced keratinocyte necrosis

[0084] (1) Experimental methods:

[0085] Human keratinocytes HaCaT were cultured at 4×10 5 The cells were inoculated at a density of 100 μl / well in a 12-well plate and cultured overnight. The next day, the medium was replaced with serum-free Ultra-fectin medium containing compound 1. After incubation for 30 minutes, the medium was discarded and replaced with 200 μl 37°C warm PBS per well. The cells were irradiated with UVB lamps at a dose of 100 mJ / cm 2 After irradiation, PBS was discarded and the cells were incubated with serum-free Ultra-fectin medium containing compound 1 for 6 hours.

[0086] The treatments for each group in the above experiment were:

[0087] Treatment 1 was the normal group without UVB irradiation;

[0088] Treatment 2 was the model group, in which 0.1% (v / v) DMSO was added, also serving as the vehicle control;

[0089] In treatment 3, the added substance was compound 1 at a concentration of 5 μM;

[0090] In treatment 4, the added substance was compound 1 at a concentration of 10 μM;

[0091] In treatment 5, the added substance was compound 1 at a concentration of 20 μM;

[0092] Treatments 3 to 5 were the experimental groups.

[0093] The morphological changes of cells in each treatment group were imaged under bright field using an inverted fluorescence microscope.

[0094] Total cell proteins were extracted using RIPA lysis buffer containing phosphatase inhibitors and protease inhibitors, and then treated with 6×SDS loading buffer. The extracted proteins were analyzed by immunoblotting, and β-actin was used as the internal reference protein.

[0095] The experimental results of this example are shown in Fig.10 , Fig.11 , Fig.10 It can be seen that after the model group was irradiated with UVB, the keratinocytes showed necrosis-like morphological changes; after pretreatment with compound 1, the morphological changes of the cells were alleviated and returned to normal.

[0096] Fig.11 It can be seen that the phosphorylated MLKL protein band level in the model group increased significantly, while the phosphorylated MLKL protein band level in the compound 1 group decreased significantly, indicating that compound 1 can effectively inhibit the necrosis of HaCaT cells induced by UVB radiation.

[0097] Example 7: Compound 1 reduces UVB-induced reactive oxygen species production in keratinocytes

[0098] Human keratinocytes HaCaT were cultured at 4×10 5 The cells were inoculated at a density of 100 μl / well in a 12-well plate and cultured overnight. The next day, the medium was replaced with serum-free Ultra-fectin medium containing compound 1. After incubation for 30 minutes, the medium was discarded and replaced with 200 μl 37°C warm PBS per well. The cells were irradiated with UVB lamps at a dose of 100 mJ / cm 2 After irradiation, PBS was discarded and the cells were incubated with serum-free Ultra-Fectin medium containing compound 1 for 6 hours.

[0099] The treatments for each group in the above experiment were:

[0100] Treatment 1 was the normal group without UVB irradiation;

[0101] Treatment 2 was the model group, in which 0.1% (v / v) DMSO was added, also serving as the vehicle control;

[0102] In treatment 3, the added substance was compound 1 at a concentration of 10 μM;

[0103] Use 500 μL / well of serum-free Ultra-fectin medium containing 20 μM DCFH-DA and incubate the cells at 37°C in the dark for 20 minutes to load the probe. Gently wash the cells three times with warm serum-free medium to remove unbound DCFH-DA. For detection of DCF signals: use a fluorescence microscope to set the excitation / emission wavelength to 495nm / 529nm to image DCF fluorescence-positive cells; use a SpectraMax multi-function microplate reader, set the reading mode to "Fluorescence", and the reading method to "Wellscan" to read the DCF fluorescence intensity at multiple sites in the well plate under 495nm excitation light.

[0104] (2) Experimental results:

[0105] DCFH-DA (2',7'-Dichlorodihydrofluorescein diacetate) is a membrane-permeable reactive oxygen species fluorescent probe that can penetrate the cell membrane into living cells and is ultimately oxidized by ROS to DCF (2'-7' dichlorofluorescein). The fluorescence intensity of DCF can be used to indicate the level of reactive oxygen species in cells.

[0106] The experimental results of this embodiment are as follows Fig.12 and Fig.13 As shown, after UVB irradiation of keratinocytes, the cells in the model group showed obvious DCF fluorescence, while the DCF fluorescence of the compound 1 pretreatment group decreased significantly, indicating that compound 1 can effectively reduce the production of reactive oxygen species (ROS) in HaCaT cells induced by UVB irradiation.

[0107] Example 8: Compound 1 inhibits UVB radiation-induced macrophage LDH release

[0108] (1) Experimental methods:

[0109] Mouse macrophage J774A.1 or human macrophage THP-1 were cultured at 2×10 5 The cells were inoculated at a density of 100 μL / well in a 24-well plate and cultured overnight (THP-1 cells were also incubated with 100 ng / mL PMA to induce adhesion). The next day, the culture medium was removed and replaced with PBS preheated at 37°C, 100 μL / well, and the cells were irradiated with UVB at a dose of 50 mJ / cm 2 PBS was discarded and replaced with serum-free Ultra-fectin medium containing compound 1 and incubated for 6 hours.

[0110] The treatments for each group in the above experiment were:

[0111] Treatment 1 was the normal group without UVB irradiation;

[0112] Treatment 2 was the model group, in which 0.1% (v / v) DMSO was added, also serving as the vehicle control;

[0113] In treatment 3, the added substance was compound 1 at a concentration of 5 μM;

[0114] In treatment 4, the added substance was compound 1 at a concentration of 10 μM;

[0115] In treatment 5, the added substance was compound 1 at a concentration of 20 μM;

[0116] Treatments 3 to 5 were the experimental groups.

[0117] 100 μL of cell culture supernatant was collected and placed in a 96-well plate. Non-Radioactive Cytotoxicity Assay, Promega; USA), add the detection reagent, place at room temperature for 15 minutes, place the 96-well plate in an ELISA reader and read the OD 490 After the values ​​were calculated, the LDH release rate (%) was calculated according to the following formula.

[0118] LDH release rate = (OD 490 -OD in normal group 490 ) / (OD of the model group 490 -OD in normal group 490 )×100%

[0119] (2) Experimental results:

[0120] LDH (lactate dehydrogenase) is an enzyme in the glycolysis process and is widely present in cells. Under normal circumstances, the cell membrane of living cells is intact and has low permeability, so LDH is not released outside the cell; however, cell pyroptosis or necrosis causes the membrane to be damaged, and LDH is released outside the cell. This process is also accompanied by the release of intracellular substances and DAMPs (damage-associated molecular patterns), which in turn causes inflammation.

[0121] The experimental results of this embodiment are as follows Fig.14 . Fig.15 Compound 1 can inhibit the release of LDH from both macrophages in a dose-dependent manner, indicating that compound 1 can alleviate the damage of macrophages caused by UVB radiation and protect cell integrity.

[0122] Example 9: Compound 1 inhibits UVB-induced reactive oxygen species production in macrophages

[0123] (1) Experimental methods:

[0124] Mouse macrophages J774A.1 were cultured at 1×10 6The cells were seeded at a density of 100 μL / well in a 6-well plate and cultured overnight. The next day, the culture medium was removed and replaced with PBS preheated at 37°C, 100 μL / well, and the cells were irradiated with UVB at a dose of 50 mJ / cm 2 PBS was discarded and replaced with serum-free Ultra-fectin medium containing compound 1 and incubated for 6 hours.

[0125] The treatments for each group in the above experiment were:

[0126] Treatment 1 was the normal group without UVB irradiation;

[0127] Treatment 2 was the model group, in which 0.1% (v / v) DMSO was added, also serving as the vehicle control;

[0128] In treatment 3, the added substance was compound 1 at a concentration of 5 μM;

[0129] In treatment 4, the added substance was compound 1 at a concentration of 10 μM;

[0130] In treatment 5, the added substance was compound 1 at a concentration of 20 μM;

[0131] Treatments 3 to 5 were the experimental groups.

[0132] DCFH-DA was used to detect the level of ROS in cells. The specific method is shown in Example 8.

[0133] (2) Experimental results:

[0134] Abnormal accumulation of ROS in cells can cause cellular oxidative stress and is an important inducer of inflammasome activation, cell apoptosis and necrosis.

[0135] The experimental results of this embodiment are as follows Fig.15 As shown in the figure, after UVB irradiation, the DCFHA-DA fluorescence level in mouse macrophages in the model group increased significantly, while compound 1 treatment decreased it in a dose-dependent manner. This indicates that compound 1 can effectively inhibit the production of cellular ROS caused by UVB.

[0136] Example 10: Compound 1 inhibits UVB-induced mitochondrial superoxide production

[0137] (1) Experimental methods:

[0138] Mouse macrophages J774A.1 were cultured at 1×10 6 The cells were seeded at a density of 100 μL / well in a 6-well plate and cultured overnight. The next day, the culture medium was removed and replaced with PBS preheated at 37°C, 100 μL / well, and the cells were irradiated with UVB at a dose of 50 mJ / cm 2 PBS was discarded and replaced with serum-free Ultra-fectin medium containing compound 1 and incubated for 6 hours.

[0139] The treatments for each group in the above experiment were:

[0140] Treatment 1 was the normal group without UVB irradiation;

[0141] Treatment 2 was the model group, in which 0.1% (v / v) DMSO was added, also serving as the vehicle control;

[0142] In treatment 3, the added substance was compound 1 at a concentration of 5 μM;

[0143] In treatment 4, the added substance was compound 1 at a concentration of 10 μM;

[0144] In treatment 5, the added substance was compound 1 at a concentration of 20 μM;

[0145] Treatments 3 to 5 were the experimental groups.

[0146] The cell supernatant was discarded, and 500 μL / well of HBSS buffer containing 5 μM MSR was incubated with the cells at 37°C in the dark for 20 min to load the probe. After incubation, Mito-SOX fluorescence was imaged under a fluorescence microscope at an excitation wavelength / emission wavelength of 396 nm / 610 nm.

[0147] (2) Experimental results:

[0148] When tissues or cells are damaged, mitochondrial superoxide accumulates and damages mitochondrial function. It is also an important source of intracellular reactive oxygen species and an important factor in cellular oxidative damage, and is closely related to aging and the occurrence of various diseases. MSR (Mito-SOX Red) is a membrane-permeable mitochondrial superoxide fluorescent probe that can enter living cells and selectively target mitochondria. It is oxidized by mitochondrial superoxide rather than other reactive oxygen species (ROS). Its oxidation product has red fluorescence, and the maximum excitation wavelength / emission wavelength is 396nm / 610nm.

[0149] The experimental results are as follows Fig.16 As shown in the figure, after UVB irradiation, the fluorescence of Mito-SOX Red in mouse macrophages in the model group was significantly enhanced, while that in the compound 1 treatment group was dose-dependently weakened. This indicates that compound 1 can effectively inhibit the level of mitochondrial superoxide induced by UVB radiation.

[0150] Example 11: Compound 1 alleviates UVB-induced mitochondrial membrane potential loss

[0151] (1) Experimental methods:

[0152] Mouse macrophages J774A.1 were cultured at 1×10 6The cells were seeded at a density of 100 μL / well in a 6-well plate and cultured overnight. The next day, the culture medium was removed and replaced with PBS preheated at 37°C, 100 μL / well, and the cells were irradiated with UVB at a dose of 50 mJ / cm 2 PBS was discarded and replaced with serum-free Ultra-fectin medium containing compound 1 and incubated for 6 hours.

[0153] The treatments for each group in the above experiment were:

[0154] Treatment 1 was the normal group without UVB irradiation;

[0155] Treatment 2 was the model group, in which 0.1% (v / v) DMSO was added, also serving as the vehicle control;

[0156] In treatment 3, the added substance was compound 1 at a concentration of 5 μM;

[0157] In treatment 4, the added substance was compound 1 at a concentration of 10 μM;

[0158] In treatment 5, the added substance was compound 1 at a concentration of 20 μM;

[0159] Treatments 3 to 5 were the experimental groups.

[0160] The changes in mitochondrial membrane potential were detected using the JC-1 fluorescent probe. The supernatant was removed, the cells were washed twice with 1×PBS, 5 μg / mL of JC-1 dye (diluted with PBS) was added and incubated in a 37°C incubator for 15 min, and JC-1 was imaged using a fluorescent inverted microscope. The maximum excitation / emission wavelength of JC-1 aggregates is 550 / 600 nm, showing orange fluorescence; the maximum excitation / emission wavelength of JC-1 monomers is 485 / 535 nm, showing green fluorescence. All experiments were repeated three times. The data obtained from the experiments were statistically analyzed using Excel and GraphPad Prism 7.0 software. One-way ANOVA was used to compare the significance of differences between different groups.

[0161] (2) Experimental results:

[0162] Mitochondrial membrane potential (MMP or Δψm) is an important part of maintaining the mitochondrial electron transport chain. Compared with healthy mitochondria, damaged mitochondria generally have lower membrane potential. Therefore, mitochondrial membrane potential becomes an important indicator for determining whether mitochondria are damaged.

[0163] JC-1 is a membrane-permeable cationic fluorescent probe that can enter living cells and accumulate in mitochondria to form JC-1 aggregates. Damaged mitochondria are often unable to maintain membrane potential and thus cannot form JC-1 aggregates, and the fluorescence test for JC-1 aggregates is negative.

[0164] The experimental results of this embodiment are as follows Fig.17 As shown in the figure, after UVB irradiation, the mitochondrial membrane potential of the model group decreased significantly, while compound 1 treatment alleviated the decrease in mitochondrial membrane potential. This shows that compound 1 can effectively reduce mitochondrial damage caused by UVB radiation.

[0165] In summary, the flavonoid compound of the present invention is used in the preparation of products for preventing and treating skin photodamage. The flavonoid compound can effectively alleviate skin photodamage caused by ultraviolet B (UVB), and can be used in the preparation of cell necrosis inhibitors and in the preparation of drugs, health foods, cosmetics, and sanitary products for preventing, treating, or improving skin photodamage after UVB exposure.

[0166] Those skilled in the art should recognize that the above embodiments are only used to illustrate the present invention, and are not intended to limit the present invention. As long as they are within the spirit of the present invention, any changes or modifications to the above embodiments will fall within the scope of the claims of the present invention.

Claims

1. A use of a flavonoid compound in the preparation of a product for preventing and treating UVB-induced skin photodamage, characterized in that: The flavonoid compound is a compound C of formula (1): 20 H 18 O6, molecular formula:

2. The use of the flavonoid compound according to claim 1, characterized in that: The flavonoids protect the skin from photodamage caused by UVB by inhibiting cell necrosis.

3. The use of the flavonoid compound according to claim 2, characterized in that: In UVB-induced skin photodamage, cell necrosis is triggered by MLKL protein phosphorylation.

4. The use of the flavonoid compound according to claim 1, characterized in that: The effective concentration of the flavonoid compound for inhibiting UVB-induced skin light damage is 0.125% to 0.50% w / v.

5. The use of the flavonoid compound according to claim 1, characterized in that: The flavonoid compound effectively alleviates cellular oxidative stress and inhibits the generation of intracellular reactive oxygen and mitochondrial superoxide induced by UVB irradiation.

6. The use of the flavonoid compound according to claim 1, characterized in that: The flavonoid compound reduces the damage of cell mitochondria after UVB irradiation and inhibits the decrease of mitochondrial membrane potential.

7. The use of the flavonoid compound according to claim 1, characterized in that: The UVB-induced skin photodamage includes at least one of sunburn, skin redness and swelling induced by solar radiation, skin aging induced by solar radiation, skin cancer induced by solar radiation, and skin pigmentation induced by solar radiation.

8. The use of the flavonoid compound according to claim 1, characterized in that: The product includes at least one of medicines, health foods, cosmetics and sanitary products.

9. The use of the flavonoid compound according to claim 8, characterized in that: The dosage form of the medicine and health food is at least one of injection, film, ointment, cream, paste, gel, coating, plaster, patch, aerosol, spray, extract, tablet, dispersible tablet, lozenge, orodisintegrating tablet, sustained-release tablet, capsule, soft capsule, pill and granule.

10. The use of the flavonoid compound according to claim 8, characterized in that: The types of the cosmetics include at least one of sunscreen, sunscreen milk, sunscreen gel, facial cleanser, toner, floral water, sunscreen spray, sunscreen lotion, facial cream, facial mask and facial gel.

11. The use of the flavonoid compound according to claim 8, characterized in that: The types of the sanitary products include at least one of wet wipes, sanitary wipes, sanitary napkins, diapers, band-aids and medical gauze.

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