Method for effectively improving dark skin color by using SERPINA1 inhibitor or antagonist

Abstract

The invention relates to a method for effectively improving dark skin color by using an SERPINA1 inhibitor or an antagonist, and particularly provides application of an inhibitor or an antagonist of a SerpinA1 gene or a coding protein of the SerpinA1 gene to preparation of a composition or a preparation, and the composition or the preparation is used for improving the dark skin color. It is found for the first time that the inhibitor or antagonist of the SerpinA1 gene or the encoded protein of the SerpinA1 gene can effectively improve the darkness of the skin color.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine and in particular to a method for effectively improving dull skin color using a SERPINA1 inhibitor or antagonist. Background Art

[0002] Compared with other skin problems, Chinese women's demands for whitening and skin color improvement are at the forefront among the global population. When it comes to changes in skin color, the general perception is that it is related to exogenous stimuli, such as ultraviolet radiation stimulating melanocytes to secrete more melanin, and the oxygen free radicals produced by ultraviolet radiation stimulate skin inflammation. Inflammatory factors further promote melanocytes to secrete melanin; therefore, the development plans of traditional oral or skin care and beauty products are all through the reduction of melanin, reducing the production and transport of melanin. Although these solutions are very classic and effective, market feedback in recent years has found that products developed under similar mechanism backgrounds can no longer meet consumers' demands for improving skin color, nor have they achieved the expected psychological effects. With the advancement of science, a large number of studies have found that changes in the living environment and living behaviors of urban people may be the reason why existing products are not effective in improving skin color, that is, modern urban people are less exposed to ultraviolet rays for a long time, and the problem of dark skin may be caused by other factors, such as long-term use of electronic devices, bad living habits, and long-term psychological high pressure. Mental stress has a particularly significant impact on skin health. A 2004 study based on 25 healthy men found that mental stress can slow down wound healing; in addition, mental stress can also damage the skin barrier function and delay the repair process of the skin barrier.

[0003] In addition, the skin often appears dull during the normal aging process. The core reason is poor skin barrier function, obstructed metabolism (desquamation) of the stratum corneum, and accumulation of old and dead skin cells. The normal metabolism of old and dead skin cells is an important part of the successful differentiation process of the epidermis, ensuring the proper moisture content, skin smoothness and elasticity, and appropriate pH value of the skin surface.

[0004] Currently, the application of oral, cosmetic, and skin care products is at a blank stage for people under stress who have dull skin due to problems with their skin's ability to metabolize keratin.

[0005] Therefore, there is an urgent need in this field to screen compounds on this basis to find compounds that inhibit the expression of the SerpinA1 gene, so as to fundamentally open up new ideas for whitening and improving the skin color of modern urban stressed people. Summary of the invention

[0006] The purpose of the present invention is to provide a compound that inhibits the expression of the SerpinA1 gene. By inhibiting the expression of the SerpinA1 gene, the expression of KLK5 and KLK7 in the KLK family is regulated, and the ability related to epidermal keratin differentiation is regulated to achieve a whitening effect, which opens up new ideas for whitening and improving skin color for modern urban stressed people.

[0007] In a first aspect of the present invention, there is provided a use of an inhibitor or antagonist of the SerpinA1 gene or its encoded protein for preparing a composition or preparation for improving dull skin color.

[0008] In another preferred embodiment, the composition or preparation is used to improve dull skin color in people under stress.

[0009] In another preferred embodiment, the composition or preparation improves dull skin tone by affecting keratin metabolism.

[0010] In another preferred embodiment, the composition or preparation indirectly affects the KLK family by directly regulating SerpinA1, thereby improving dull skin tone.

[0011] In another preferred embodiment, the KLK family includes KLK5 and KLK7.

[0012] In another preferred embodiment, the inhibitor or antagonist is selected from the following group: small molecule compounds, antibodies, polypeptides, nucleic acids, or a combination thereof.

[0013] In another preferred embodiment, the inhibitor or antagonist includes polyphenol flavonoids.

[0014] In another preferred embodiment, the polyphenol flavonoids include quercetin, baicalin, oleuropein, hydroxytyrosol, anthocyanin, phloretin, naringenin, wogonin, fisetin, golden cinnamon isoflavones, and luteolin.

[0015] In another preferred embodiment, the inhibitor or antagonist includes quercetin, baicalin, oleuropein, and hydroxytyrosol.

[0016] In another preferred embodiment, the concentration of the inhibitor or antagonist is 10-1200 μg / mL.

[0017] In another preferred embodiment, when the inhibitor or antagonist is quercetin, the concentration is 20-40 μg / mL.

[0018] In another preferred embodiment, when the inhibitor or antagonist is baicalin, the concentration is 900-1200 μg / mL.

[0019] In another preferred embodiment, when the inhibitor or antagonist is oleuropein, the concentration is 80-160 μg / mL.

[0020] In another preferred embodiment, when the inhibitor or antagonist is hydroxytyrosol, the concentration is 10-40 μg / mL.

[0021] In another preferred embodiment, the composition includes a pharmaceutical composition or a cosmetic composition.

[0022] In another preferred embodiment, the composition contains (a) an inhibitor or antagonist of the SerpinA1 gene or its encoded protein; and (b) a pharmaceutically acceptable carrier or a cosmetically acceptable carrier.

[0023] In another preferred embodiment, the composition is liquid, solid, or semi-solid.

[0024] In another preferred embodiment, the composition is an oral preparation.

[0025] In another preferred embodiment, the dosage form of the composition is selected from the following group: liquid dosage form, semisolid dosage form or solid dosage form, more preferably ointment, cream, emulsion, oil, powder, solution, gel, spray, foam, suspension, lotion or sticks.

[0026] In another preferred embodiment, the composition is a facial cream, lotion, essence, cleanser, essence, eye cream or facial mask essence.

[0027] In another preferred embodiment, in the composition, the component (a) accounts for 0.01-5wt%, preferably 0.1-2wt%, of the total weight of the composition.

[0028] In another preferred embodiment, the composition or preparation can be used alone or in combination for improving dull skin tone.

[0029] In another preferred embodiment, the combined use includes: combined use with other substances that improve dull skin tone (such as vitamin C and its derivatives, phenethyl resorcinol, nonapeptide-1, bisabolol).

[0030] The second aspect of the present invention provides a composition comprising:

[0031] (a1) a first active ingredient for improving dull skin tone, wherein the first active ingredient comprises an inhibitor or antagonist of the SerpinA1 gene or its encoded protein;

[0032] (a2) a second active ingredient for improving dull skin tone, the second active ingredient comprising: other substances for improving dull skin tone; and

[0033] (b) a pharmaceutically acceptable carrier.

[0034] In another preferred embodiment, the inhibitor or antagonist is selected from the following group: small molecule compounds, antibodies, polypeptides, nucleic acids, or a combination thereof.

[0035] In another preferred embodiment, the inhibitor or antagonist includes polyphenol flavonoids.

[0036] In another preferred embodiment, the polyphenol flavonoids include quercetin, baicalin, oleuropein, hydroxytyrosol, anthocyanin, phloretin, naringenin, wogonin, fisetin, golden cinnamon isoflavones, and luteolin.

[0037] In another preferred embodiment, the inhibitor or antagonist includes quercetin, baicalin, oleuropein, and hydroxytyrosol.

[0038] In another preferred embodiment, in the composition, the component (a1) accounts for 0.01-5wt%, preferably 0.1-2wt%, of the total weight of the composition.

[0039] In another preferred embodiment, in the composition, the component (a2) accounts for 0.1-10 wt % of the total weight of the composition.

[0040] In another preferred embodiment, the weight ratio of the first active ingredient to the second active ingredient is 1:100 to 100:1, preferably 1:10 to 10:1.

[0041] In another preferred embodiment, the other substances for improving dull skin color are selected from the following group: vitamin C and its derivatives, phenethyl resorcinol, nonapeptide-1, bisabolol, or a combination thereof.

[0042] In another preferred embodiment, the composition may contain a single compound or a mixture of multiple compounds.

[0043] In another preferred embodiment, the composition is used to prepare a medicine or preparation for improving dull skin color.

[0044] In another preferred embodiment, the composition includes a pharmaceutical composition or a cosmetic composition.

[0045] In another preferred embodiment, the composition is liquid, solid, or semi-solid.

[0046] In another preferred embodiment, the composition is an oral preparation.

[0047] In another preferred embodiment, the dosage form of the composition is selected from the following group: liquid dosage form, semisolid dosage form or solid dosage form, more preferably ointment, cream, emulsion, oil, powder, solution, gel, spray, foam, suspension, lotion or sticks.

[0048] In another preferred embodiment, the composition is a facial cream, lotion, essence, cleanser, essence, eye cream or facial mask essence.

[0049] In another preferred embodiment, the total content of the active ingredient (a1) and the active ingredient (a2) is 0.01-10 wt %, preferably 0.1-10 wt %, of the total weight of the composition.

[0050] The third aspect of the present invention provides a medicine kit, comprising:

[0051] (i) a first container, and an active ingredient (a1) an inhibitor or antagonist of the SerpinA1 gene or its encoded protein, or a drug containing the active ingredient (a) in the first container; and

[0052] (ii) a second container, and the active ingredient (a2) or other substances for improving dull skin color, or a medicine containing the active ingredient (a2) in the second container.

[0053] In another preferred embodiment, the inhibitor or antagonist is selected from the following group: small molecule compounds, antibodies, polypeptides, nucleic acids, or a combination thereof.

[0054] In another preferred embodiment, the inhibitor or antagonist includes polyphenol flavonoids.

[0055] In another preferred embodiment, the polyphenol flavonoids include quercetin, baicalin, oleuropein, hydroxytyrosol, anthocyanin, phloretin, naringenin, wogonin, fisetin, golden cinnamon isoflavones, and luteolin.

[0056] In another preferred embodiment, the inhibitor or antagonist includes quercetin, baicalin, oleuropein, and hydroxytyrosol.

[0057] In another preferred embodiment, the first container and the second container are the same or different containers.

[0058] In another preferred embodiment, the drug in the first container is a single-ingredient preparation containing an inhibitor or antagonist of the SerpinA1 gene or its encoded protein.

[0059] In another preferred embodiment, the medicine in the second container is a single-ingredient preparation containing other medicines for improving dull skin color.

[0060] In another preferred embodiment, the dosage form of the drug is an oral dosage form.

[0061] In another preferred embodiment, the dosage form of the drug is a liquid dosage form, a semisolid dosage form or a solid dosage form, more preferably an ointment, a cream, an emulsion, an oil, a powder, a solution, a gel, a spray, a foam, a suspension, a lotion or a stick.

[0062] In another preferred embodiment, the medicine kit further comprises instructions for administering the active ingredient (a1) and the active ingredient (a2) in combination to improve dull skin tone.

[0063] In another preferred embodiment, the dosage forms of the preparation containing the active ingredient (a1) inhibitor or antagonist of SerpinA1 gene or its encoded protein or containing other drugs for improving dull skin color include ointments, creams, emulsions, oils, powders, solutions, gels, sprays, foams, suspensions, lotions or sticks.

[0064] The fourth aspect of the present invention provides a use of the composition described in the second aspect of the present invention or the medicine kit described in the third aspect of the present invention for preparing a medicament for improving dull skin color.

[0065] A fifth aspect of the present invention provides a method for improving dull skin tone, comprising the steps of:

[0066] An inhibitor or antagonist of the SerpinA1 gene or its encoded protein, the pharmaceutical composition according to the second aspect of the present invention, or the drug kit according to the third aspect of the present invention is administered to a subject in need.

[0067] In another preferred embodiment, the administration includes oral administration or external application to the skin.

[0068] In another preferred embodiment, the subject includes a human or a non-human mammal.

[0069] In another preferred embodiment, the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.

[0070] A sixth aspect of the present invention provides a method for screening candidate drugs for improving dull skin color, comprising the steps of:

[0071] (a) in a test group, in a culture system, in the presence of a test substance, culturing cells expressing the SerpinA1 gene or its encoded protein for a period of time T1, and detecting the expression amount E1 and / or activity A1 of the SerpinA1 gene or its encoded protein in the culture system of the test group;

[0072] And in a control group without the test substance and with the same other conditions, detecting the expression amount E2 and / or activity A2 of the SerpinA1 gene or its encoded protein in the culture system of the control group;

[0073] (b1) comparing E1 and E2, if E1 is significantly lower than E2, it indicates that the test substance is a candidate drug for improving dull skin color; and / or

[0074] (b2) Compare A1 and A2. If A1 is significantly lower than A2, it indicates that the test substance is a candidate drug for improving dull skin color.

[0075] In another preferred embodiment, the cells are mammalian cells.

[0076] In another preferred embodiment, the cells are selected from the following group: epidermal cells.

[0077] In another preferred embodiment, the cells include human immortalized epidermal cells (HaCaT cells).

[0078] In another preferred embodiment, the cells are cells cultured in vitro.

[0079] In another preferred embodiment, the “significantly lower than” means E1 / E2≤1 / 2, preferably ≤1 / 3, and more preferably ≤1 / 4.

[0080] In another preferred embodiment, the “significantly lower than” means A1 / A2≤1 / 2, preferably ≤1 / 3, more preferably ≤1 / 4.

[0081] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0082] In another preferred embodiment, the method comprises step (c): administering the candidate drug determined in step (a) to a non-human mammal, thereby determining its effect on the dull skin color of the non-human mammal.

[0083] In another preferred embodiment, the test substance is selected from the following group: small molecule compounds, antibodies, polypeptides, nucleic acids, or a combination thereof.

[0084] In another preferred embodiment, the test substance includes polyphenol flavonoids.

[0085] In another preferred embodiment, the polyphenol flavonoids include quercetin, baicalin, oleuropein, hydroxytyrosol, anthocyanin, phloretin, naringenin, wogonin, fisetin, golden cinnamon isoflavones, and luteolin.

[0086] In another preferred embodiment, the test substances include quercetin, baicalin, oleuropein, and hydroxytyrosol.

[0087] In another preferred embodiment, the concentration of the test substance is 10-1200 μg / mL.

[0088] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 Shown is the harvesting of epidermal layer tissue of the skin.

[0090] Figure 2 Showing the relationship between stress and dull skin.

[0091] Figure 3 The relationship between SERPINA1 and skin dullness is shown.

[0092] Figure 4 The relationship between SERPINA1 and KLK5 and KLK7 is shown.

[0093] Figure 5 Shows the safe concentration screening of raw materials in cell experiments.

[0094] Figure 6 The effects of the compounds on SerpinA1 transcription are shown. Each group was compared with the control group. DETAILED DESCRIPTION

[0095] After extensive and in-depth research, the inventor unexpectedly discovered for the first time that an inhibitor or antagonist of the SerpinA1 gene or its encoded protein can effectively improve dull skin color. On this basis, the inventor completed the present invention.

[0096] Dull skin

[0097] As used herein, the term "dull skin" refers to pigmentation, yellowing, rough skin, lack of radiance, uneven skin tone, and the like on the skin.

[0098] SerpinA1

[0099] SerpinA1 is known as α-1 antitrypsin gene in Chinese and Serpin Family A Member 1 in English. This gene encodes a serine protease inhibitor whose targets include elastase, plasmin, thrombin, trypsin, chymosin and plasminogen activator.

[0100] KLK Family

[0101] The Chinese name of KLK is secretory serine protease family, and its English full name is Kallikrein-related peptides. In the KLK family, the correct expression of KLK5 and KLK7 is the basis for normal differentiation and metabolism of the epidermis.

[0102] Inhibitors or antagonists of SerpinA1 gene or its encoded protein

[0103] As used herein, "an inhibitor or antagonist of the SerpinA1 gene or its encoded protein" refers to a small molecule or macromolecular compound that specifically targets the SerpinA1 gene or its encoded protein and inhibits or antagonizes its expression or activity.

[0104] The inhibitor or antagonist of the SerpinA1 gene or its encoded protein of the present invention inhibits the expression of the SerpinA1 gene, thereby affecting the expression of the KLK5 and KLK7 genes, thereby improving the metabolic function of the stratum corneum of the skin and improving dull skin color.

[0105] In addition, the present invention can be used in combination with other substances selected from the following group (such as other drugs for improving dull skin color). Representative examples include (but are not limited to): selected from the following group: vitamin C and its derivatives, phenethyl resorcinol, nonapeptide-1, bisabolol, or a combination thereof.

[0106] In a preferred embodiment, the inhibitor or antagonist of the SerpinA1 gene or its encoded protein comprises polyphenolic flavonoids.

[0107] In a preferred embodiment, the polyphenol flavonoids include quercetin, baicalin, oleuropein, hydroxytyrosol, anthocyanin, phloretin, naringenin, wogonin, fisetin, golden cinnamon isoflavones, and luteolin.

[0108] In a preferred embodiment, the inhibitors or antagonists of the SerpinA1 gene or its encoded protein include quercetin, baicalin, oleuropein, and hydroxytyrosol.

[0109] Polyphenol flavonoids

[0110] As used herein, the term "polyphenolic flavonoids" refers to a general term for plant components with several phenolic hydroxyl groups in their molecular structure, including flavonoids, tannins, phenolic acids, anthocyanins, etc. Phenols are compounds found in plant foods that have potential health-promoting effects. They usually have antioxidant and certain anti-inflammatory effects.

[0111] In the present invention, it is discovered for the first time that polyphenolic flavonoids including quercetin, baicalin, oleuropein, hydroxytyrosol, anthocyanin, phloretin, naringenin, wogonin, fisetin, cinnamomea isoflavones and luteolin all improve dull skin tone by indirectly affecting the KLK family through direct regulation of SerpinA1.

[0112] Pharmaceutical or cosmetic composition and method of applying the same

[0113] On the other hand, the present invention also provides a pharmaceutical composition or cosmetic composition, which contains (a) a safe and effective amount of the SerpinA1 gene or its encoded protein inhibitor or antagonist of the present invention; and (b) a pharmaceutically or cosmetically acceptable carrier or excipient. The dosage of the SerpinA1 gene or its encoded protein inhibitor or antagonist of the present invention is generally 0.01-5wt%, preferably 0.1-2wt%, based on the total weight of the pharmaceutical composition or cosmetic composition. In addition, the SerpinA1 gene or its encoded protein inhibitor or antagonist of the present invention can be used alone or together with other therapeutic agents (such as formulated in the same pharmaceutical composition).

[0114] The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier used for the administration of a therapeutic agent. The term refers to such pharmaceutical carriers: they do not induce the production of antibodies harmful to the individual receiving the composition, and there is no excessive toxicity after administration. These carriers are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991). Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, adjuvants and combinations thereof.

[0115] Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as wetting agents or emulsifiers, pH buffer substances, etc. may also be present in these carriers.

[0116] Typically, therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution or suspension in liquid vehicles prior to injection can also be prepared.

[0117] Once formulated, the composition of the present invention can be administered by conventional routes, including (but not limited to): intratumoral, intramuscular, intravenous, subcutaneous, intradermal, or topical administration. The subject to be prevented or treated can be an animal; especially a human.

[0118] When the pharmaceutical composition of the present invention is used for actual treatment, various dosage forms of the pharmaceutical composition can be used according to the usage, preferably intravenous preparations or intratumoral injections.

[0119] These pharmaceutical compositions can be formulated by mixing, diluting or dissolving according to conventional methods, and appropriate pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizers are occasionally added, and the formulation process can be carried out in a conventional manner according to the dosage form.

[0120] For example, the preparation of eye drops can be carried out as follows: the L-type amino acid transporter inhibitor or antagonist of the present invention is dissolved in sterile water (surfactant is dissolved in sterile water) together with the basic substance, the osmotic pressure and pH are adjusted to physiological conditions, and suitable pharmaceutical additives such as preservatives, stabilizers, buffers, isotonic agents, antioxidants and viscosity enhancers can be added arbitrarily, and then completely dissolved.

[0121] The pharmaceutical composition of the present invention can also be administered in the form of a sustained release agent. For example, the inhibitor or antagonist of the SerpinA1 gene or its encoded protein of the present invention can be incorporated into a pill or microcapsule with a sustained release polymer as a carrier, and then the pill or microcapsule is surgically implanted into the tissue to be treated. Examples of sustained release polymers include ethylene-vinyl acetate copolymers, polyhydroxymethylacrylate (polyhydrometaacrylate), polyacrylamide, polyvinyl pyrrolidone, methylcellulose, lactic acid polymers, lactic acid-glycolic acid copolymers, etc., preferably biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers.

[0122] When the pharmaceutical composition of the present invention is used for actual treatment, the dosage of the inhibitor or antagonist of the SerpinA1 gene or its encoded protein of the present invention as the active ingredient can be reasonably determined according to the weight, age, sex, and symptom severity of each patient to be treated.

[0123] As used herein, the term "cosmetically acceptable" ingredient refers to a material that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic response), ie, at a reasonable benefit / risk ratio.

[0124] As used herein, the term "effective amount" refers to any amount of a cosmetic agent as described below, which, when used alone or in combination with another cosmetic or therapeutic agent, improves dull skin tone.

[0125] Preferably, the carrier or excipient on the cosmetic is selected from the following group: moisturizers, skin conditioners, thickeners, emollients, emulsifiers, antioxidants, preservatives, anti-ultraviolet agents, film formers, oil-soluble gelling agents, organically modified clay minerals, resins, flavors, salts, pH adjusters, conditioners, chelating agents, cooling agents, anti-inflammatory agents, skin beautifying ingredients, vitamins, amino acids, nucleic acids, inclusion compounds, solvents (such as water), or combinations thereof.

[0126] The thickener includes fatty alcohols or fatty acids such as lauryl alcohol and fatty acids; alkanolamides such as cocamide; ethers such as cetyl alcohol polyoxyethylene (3) ether; esters such as PEG-40 jojoba oil; amine oxides such as wheat germ aminopropyl amine oxide; celluloses such as carboxymethyl cellulose and hydroxyethyl cellulose; polyacrylic acids such as carbomer, glyceryl acrylate / acrylic acid copolymer, polyacrylate cross-linked polymer-6, sodium acrylic acid (ester) copolymer, polyacrylamide; natural gum and its modified products such as sodium alginate and xanthan gum; and new polymer thickeners such as polyvinyl pyrrolidone, ammonium acryloyldimethyl taurate / vp copolymer, hydroxyethyl acrylate / sodium acryloyldimethyl taurate copolymer, etc., or any combination of two or more thereof in any proportion.

[0127] The moisturizer refers to polyols, natural moisturizing factors, amino acids, high molecular biochemical molecule moisturizers, and combinations thereof, specifically glycerol, 1,2-propylene glycol, 1,3-propylene glycol, butylene glycol, pentylene glycol, hexylene glycol, caprylyl glycol, sorbitol, xylitol, polyethylene glycol, PCA-Na, sodium lactate, urea, hydrolyzed protein, soy protein, collagen, chondroitin sulfate, ceramide, betaine, trehalose, acetyl glucosamine, polyglyceryl ether-26, sodium hyaluronate, D-panthenol, polyamino acid polysaccharide condensation, biosaccharide gum-1, collagen, PEG / PPG-17 / 6 copolymer, methyl glucol polyether-10, silk amino acids, one or more of any proportion.

[0128] The emollient refers to various synthetic esters and natural oils such as isononyl isononanoate, pentaerythritol tetra(ethylhexanoate), cetearyl ethylhexanoate, caprylic / capric triglyceride, tri(ethylhexanoic acid) ester, cocoa butter substitute, grape seed oil, shea butter, isooctyl palmitate, jojoba (SIMMONDSIA CHINENSIS) seed oil, PEG-6 caprylic / capric glycerides, macadamia oil, squalane, camellia seed oil, avocado tree (BUTYROSPERMUM PARKII) fruit fat, grape seed oil, emu oil, hydrogenated polyisobutene, hydrogenated ethylhexyl olive oil, hydrogenated olive oil unsaponifiable matter, polydimethylsiloxane, glyceryl stearate, glyceryl caprylate, etc., or two or more thereof in any proportion.

[0129] The skin conditioning agent refers to raw materials with anti-inflammatory, anti-allergic, soothing, antioxidant, oil control and other effects, such as bisabolol, allantoin, witch hazel extract, oligopeptide-1, oligopeptide-6, polyquaternium-73, peony (PAEONIA OFFICINALIS) root extract, sophora flavescens (SOPHORA ANGUSTIFOLIA) root extract, cnidium monnieri (CNIDIUM MONNIERI) fruit extract, licorice (GLYCYRRHIZA URALENSIS) root extract, Phellodendron amurense (PHELLODENDRON AMURENSE) bark extract, golden chamomile (CHRYSANTHELLUM INDICUM) extract, rose (ROSA RUGOSA) flower extract, European linden (TILIAEUROPAEA) flower extract, arnica (ARNICA MONTANA) flower extract, althaea officinalis (ALTHAEA) OFFICINALIS) root extract, pyridoxine, Phellodendron chinense extract, Portulaca oleracea extract, Astragalus extract, Gentian anti-irritant factor, dipotassium glycyrrhizinate, capryloyl glycine, Centella asiatica extract, zinc pyrrolidone carboxylic acid, copper pyrrolidone carboxylic acid, snake venom tripeptide, palmitoyl tripeptide, acetyl dipeptide, acetyl hexapeptide, chamomile essential oil, etc., or two or more in any proportion. The extract or extracting solution can be commercially available products commonly used in the art. For example, peony root extract contains glycosides, benzoic acid, paeoniflorin ketone, paeonol, gallic acid and its derivatives, etc., which have the effects of improving allergic inflammatory reactions, antibacterial, analgesic, anti-inflammatory, and pain and itching caused by physical or chemical factors such as cold and heat, cosmetics, etc., and has obvious soothing effects, and can be prepared using ZL201810201949.6; golden chamomile (CHRYSANTHELLUM INDICUM) extract contains a variety of triterpenoid compounds, which have soothing, anti-allergic, anti-inflammatory, and antioxidant activities.

[0130] The emulsifier is various surfactants, such as polysorbate-80, PEG-60 hydrogenated castor oil, PEG-50 hydrogenated castor oil, PEG-40 hydrogenated castor oil, nonylphenol polyether-14, PEG-100 stearate, cetearyl alcohol, laureth-7, C12-14 isoparaffin, hydrogenated lecithin, beeswax, disodium lauryl sulfosuccinate, sodium methyl cocoyl taurate, sodium lauroyl sarcosinate, sodium cocoyl glycinate, cocamidopropyl betaine, C12-20 alkyl glucoside, C14-22 alcohol, polyol ester, fatty alcohol polyoxyethylene ether, polymer emulsifier, etc., or two or more thereof in any proportion.

[0131] The antioxidant is one or more of vitamin A, vitamin E, cysteine, ascorbic acid, superoxide dismutase, glutathione peroxidase, coenzyme Q10, tocopherol acetate, ergothioneine, etc. in any proportion.

[0132] The pH regulator is one of triethanolamine, potassium hydroxide, sodium hydroxide, citric acid, sodium citrate, potassium citrate, lactic acid, etc., or two or more thereof in any proportion.

[0133] The preservative is one of the parabens such as methylparaben (methylparaben), ethylparaben, propylparaben, butylparaben; isothiazolinones such as methylisothiazolinone and methylchloroisothiazolinone; formaldehyde releasers such as DMDM ​​hydantoin, imidazolidinyl urea, bis(hydroxymethyl)imidazolidinyl urea; and preservative-free system antibacterial agents such as pentylene glycol, hexylene glycol, octanediol, phenylethyl alcohol, phenylpropyl alcohol, phenoxyethanol, ethylhexylglycerin, glyceryl caprate, capric acid, glyceryl caprylate, sorbitan caprylate, glyceryl undecylenate, p-hydroxyacetophenone, levulinic acid, anisic acid, caprylhydroxamic acid, caprylhydroxamic acid, etc., or two or more of them in any proportion.

[0134] Cosmetics of the present invention are suitable for external use on skin. They can be liquid dosage form, semisolid dosage form or solid dosage form, more preferably ointment, cream, emulsion, oil, powder, solution, gel, spray, foam, suspension, lotion or patch (sticks). They can also be the suspension form of microsphere or nano-microsphere or lipid or polymer carrier form or polymer patch form and controlled release hydrogel form. The compositions of these external applications can be anhydrous dosage form, aqueous solution dosage form or emulsion dosage form.

[0135] The pharmaceutical composition or cosmetic composition of the present invention has a significant effect of improving dull skin color.

[0136] The main advantages of the present invention include:

[0137] 1) The present invention first discovered that dull skin color can be effectively improved by inhibiting the expression of SerpinA1 gene.

[0138] 2) The present invention is the first to discover that the SERPINA1 gene can cause dark skin color by affecting the KLK family.

[0139] 3) The present invention screened out for the first time several active substances that have an inhibitory effect on SerpinA1 gene expression, and used them in the development of products for whitening and improving skin color for stressed people.

[0140] 4) The present invention uses a statistical analysis method to find that the SERPINA1 gene, which is highly expressed in stressed people, causes dark skin by affecting the KLK family.

[0141] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0142] Unless otherwise specified, the materials and reagents used in the examples of the present invention are commercially available products.

[0143] Example 1 In stressed people, SERPINA1 gene expression is upregulated, affecting skin keratin metabolism and causing dull skin tone

[0144] ① Sample recruitment and data collection

[0145] We conducted a preliminary mental stress survey by phone, and selected 60 people with high stress and 60 people without stress from 300 volunteers as candidates. The details are as follows: During the first visit, all 300 volunteers carefully read and signed the informed consent form, and then conducted a two-week self-observation, consciously paying attention to the changes in their mental stress. During the second visit, the completed stress questionnaire was returned. We calculated the stress score based on the questionnaire, and selected the top 60 people with the highest stress and the 60 people with the lowest stress to participate in the formal experiment.

[0146] Before the skin test, volunteers need to perform skin control according to the following requirements.

[0147] (I) Within 5 days before the skin test, wash your face with only water, do not use any skin care products, and avoid excessive exposure to sunlight.

[0148] (ii) Within 24 hours before the skin test, keep your skin in its natural state. Do not wash your face, take a bath, or use any skin care products. Avoid swimming, saunas, or any exercise that causes sweating. Avoid contact between your skin and water.

[0149] The data were collected in a constant temperature and humidity laboratory (25°C, 60%). After arriving at the laboratory, the volunteers waited quietly for 30 minutes to allow their skin to fully adapt to the laboratory environment, and then underwent skin dullness testing and epidermis data collection on the back.

[0150] ② Extraction and sequencing of total RNA from the epidermis

[0151] The epidermis was collected by suction blistering using a special instrument (SB-device, Festo AG & Co. KG / Compressor, ekom, DK50 DM, eVENT). The collection site was the lower back (lower back position). Three pieces of epidermal tissue with a diameter of about 3 mm were collected from each volunteer (such as Figure 1 ). First, the skin waits for 30 minutes under a negative pressure of 180 mmHg to adapt to the negative pressure state, and then adjusts to a negative pressure of 240 mmHg and waits for 2-4 hours. After the epidermis bulges, use a sterile syringe to collect the tissue fluid in the blister, then cut the epidermis along the edge and put it into 3 1.5 mL EP tubes, quickly put them into liquid nitrogen for storage, and then transfer them to a -80°C refrigerator with dry ice.

[0152] Total RNA was extracted using the RNeasy Micro Kit (Qiagen, #74004) according to the instructions of the RNeasy Fibrous Tissue Kit (Qiagen, #74704), and RNA was dissolved in RNase free water and stored in a refrigerator at -80°C for later use. Subsequent quality testing was performed using NanoDrop 2000 and Agilent 2100 instruments in conjunction with electrophoresis patterns, and 109 samples with good quality (total RNA content>0.7μg, RIN>7) were selected for library construction and sequencing. The strand-specific transcriptome of human RNA samples was constructed and sequenced using the Illumina Next Seq 500 sequencing platform, with the mode being 75SE. The average Q30 of the sequencing data was greater than 80%. We then filtered out fragments containing adapter sequences, removed fragments with N content exceeding 3% of the sequence length, and removed fragments containing low-quality (<3) bases exceeding 50% of the sequence length, obtaining 60, 105, 135-131, 244, and 224 clean reads for subsequent analysis. The proportion of bases with quality exceeding Q30 in each sample was between 98% and 100%, indicating that the transcriptome sequencing quality was very good.

[0153] result:

[0154] ① The skin darkening values ​​of volunteers in the stress group were higher than those in the non-stress group ( Figure 2 , 5.41 vs. 3.31, P = 7.36 × 10 -6 , skin dullness value 0-9, the higher the value, the darker the skin).

[0155] ② In the RNA data of the stress group, the expression of SERPINA1 gene was significantly different ( Figure 3 a). In addition, we found that the higher the SERPINA1 gene expression, the higher the degree of skin dullness ( Figure 3 b).

[0156] ③For the classic genes KLK5 and KLK7 of skin keratin metabolism, we found that they were negatively correlated with the expression of SERPINA1 ( Figure 4 , a, b). Based on this, SERPINA1 may cause dull skin tone by affecting keratin metabolism.

[0157] Example 2 Detection of 6 compounds' toxicity to cells

[0158] Experimental methods

[0159] (1) Cell culture

[0160] 1) HaCaT cells were obtained from the cell bank of the Chinese Academy of Sciences Typical Culture Collection Committee. After receiving the cells, they were placed in a CO2 incubator (37°C, 5% CO2) for 2 h.

[0161] 2) Take the cells out of the CO2 incubator, spray them evenly with 75% alcohol, discard the original culture medium in the clean bench, wash them once with 4 mL PBS, add 1 mL of trypsin, discard the trypsin after digestion, add fresh complete culture medium (containing 10% FBS, 1% double antibody), blow them down, and transfer them to a new T25 culture flask for continued culture.

[0162] (2) CCK-8 method to screen safe concentration of compounds

[0163] 1) Inoculate a suspension of human immortalized epidermal cells (HaCaT cells) in a 96-well plate (100 μL / well, 4000 cells / well) and culture for 24 h. Set up a blank group and a control group at the same time;

[0164] 2) Replace the medium with complete medium and add different concentrations of compounds to each well, with 6 replicates per group, and continue incubating in the incubator for 24 h;

[0165] 3) Remove the cell culture supernatant, add 100 μL of 10% CCK-8 culture solution to each well (add along the cell plate wall, mix gently, and do not generate bubbles), and incubate in the incubator for 2 hours;

[0166] 4) Measure the absorbance at 450 nm using an enzyme-labeled instrument;

[0167] 5) Calculation formula:

[0168] Cell survival percentage = [(absorbance value of experimental group - blank control) / (absorbance value of control group - blank control)] × 100%;

[0169] 6) Statistical analysis: Experimental data are expressed as mean ± SEM;

[0170] 6. Experimental results are as follows Figure 5shown.

[0171] 7. Conclusion:

[0172] The safe concentrations are: quercetin (0-40μg / mL), baicalin (0-1200μg / mL), oleuropein (0-160μg / mL), and hydroxytyrosol (0-80μg / mL).

[0173] Example 3 Screening for active substances that have an inhibitory effect on SerpinA1 gene expression

[0174] 1. Cell Culture

[0175] HaCaT cells were obtained from the cell bank of the Type Culture Collection Committee of the Chinese Academy of Sciences. After receiving the cells, they were placed in a CO2 incubator (37°C, 5% CO2) for 2 hours.

[0176] The cells were taken out of the CO2 incubator, sprayed evenly with 75% alcohol, discarded the original culture medium in the clean bench, washed once with 4 mL PBS, added with 1 mL trypsin, and after digestion, the trypsin was discarded, and fresh complete culture medium (containing 10% FBS, 1% double antibody) was added and blown down, and then transferred to a new T25 culture flask for continued culture.

[0177] 2. Screening of safe concentration of active substances in cells

[0178] A suspension of human immortalized epidermal cells (HaCaT cells) was inoculated in a 96-well plate (100 μL / well, 4000 cells / well) and cultured for 24 h. A blank group and a control group were set up at the same time. The medium was replaced with complete culture medium, and different concentrations of compounds were added to each well, with 6 replicates per group, and the cells were incubated in an incubator for 24 h. The cell culture supernatant was removed, and 100 μL of 10% CCK-8 culture medium was added to each well, and the cells were incubated in an incubator for 2 hours. The absorbance at 450 nm was measured using an ELISA reader.

[0179] 3. Detection of the inhibitory effect of active substances on SerpinA1 expression (QPCR method)

[0180] There are 4 compounds in total, namely: quercetin (20-80μg / mL), baicalin (900-1200μg / mL), oleuropein (80-160μg / mL), hydroxytyrosol (10-40μg / mL)

[0181] (1) Inoculate HaCaT cell suspension in a 6-well plate and culture for 24 h; replace the medium with complete culture medium, and set up a control group, quercetin group, baicalin group, oleuropein group, and hydroxytyrosol group, and treat with each compound for 24 h;

[0182] (2) QPCR

[0183] After washing the cells with PBS, Trizol was added to lyse the cells and extract total RNA. RNA reverse transcription reaction was performed using Takara reverse transcription kit (Takara, RR037A) (purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.), and then real-time fluorescence quantitative PCR reaction was performed.

[0184] (3) Results analysis

[0185] Graphpad Prism 8.0.1 statistical software was used for data processing.

[0186] The results are as follows Figure 6 As shown, the results showed that quercetin, baicalin, oleuropein and hydroxytyrosol can effectively inhibit the transcription of SerpinA1 gene.

[0187] In addition, the safety concentration screening of the raw materials had been completed before the in vitro efficacy test in this experiment, so Figure 6 The concentration shown is both the effective concentration and the safe concentration.

[0188] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A use of an inhibitor or antagonist of SerpinA1 gene or its encoded protein, characterized in that: Used for preparing a composition or preparation for improving dull skin color.

2. The use according to claim 1, characterized in that The composition or preparation is used for improving dull skin color of people under stress.

3. The use according to claim 1, characterized in that The composition or preparation indirectly affects the KLK family through direct regulation of SerpinA1, thereby improving dull skin color.

4. The use according to claim 1, characterized in that The inhibitors or antagonists include polyphenolic flavonoids.

5. The use according to claim 4, characterized in that The polyphenol flavonoid substances include quercetin, baicalin, oleuropein, hydroxytyrosol, anthocyanin, phloretin, naringenin, wogonin, fisetin, golden cinnamon isoflavones, and luteolin.

6. The use according to claim 1, characterized in that The concentration of the inhibitor or antagonist is 10-1200 μg / mL.

7. A composition, characterized in that include: (a1) a first active ingredient for improving dull skin tone, wherein the first active ingredient comprises an inhibitor or antagonist of the SerpinA1 gene or its encoded protein; (a2) a second active ingredient for improving dull skin color, wherein the second active ingredient comprises: other substances for improving dull skin color; and (b) a pharmaceutically acceptable carrier.

8. A medicine box, characterized in that: include: (i) a first container, and an active ingredient (a1) an inhibitor or antagonist of the SerpinA1 gene or its encoded protein, or a drug containing the active ingredient (a) in the first container; and (ii) a second container, and the active ingredient (a2) or other substances for improving dull skin color, or a medicine containing the active ingredient (a2) in the second container.

9. Use of the composition according to claim 7 or the kit according to claim 8, characterized in that: Used in the preparation of medicines for improving dull skin tone.

10. A method for screening candidate drugs for improving dull skin color, characterized in that: Includes steps: (a) in a test group, in a culture system, in the presence of a test substance, culturing cells expressing the SerpinA1 gene or its encoded protein for a period of time T1, and detecting the expression amount E1 and / or activity A1 of the SerpinA1 gene or its encoded protein in the culture system of the test group; And in a control group without the test substance and with the same other conditions, detecting the expression amount E2 and / or activity A2 of the SerpinA1 gene or its encoded protein in the culture system of the control group; (b1) comparing E1 and E2, if E1 is significantly lower than E2, it indicates that the test substance is a candidate drug for improving dull skin color; and / or (b2) Compare A1 and A2. If A1 is significantly lower than A2, it indicates that the test substance is a candidate drug for improving dull skin color.

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