Application of aescin in preparation of products for inducing autophagy of skin cells

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of aescin in preparation of a product for inducing autophagy of skin cells. According to the application disclosed by the invention, research finds that aescin can induce autophagy of skin keratinocytes for the first time, and the expression level of autophagy-related proteins LC3-I and LC3-II in the skin keratinocytes can be remarkably improved; in addition, aescin and ursolic acid have a synergistic effect in the aspect of inducing skin keratinocyte autophagy, and are safe and non-irritant to skin. Therefore, the aescin and the composition of the aescin and the ursolic acid can be used for preparing products for inducing autophagy of skin cells so as to resist aging, remove freckles, whiten skin, control oil and strengthen a skin cuticle barrier, and have the advantages of remarkable curative effect and high safety.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the application of aescin in preparing a product for inducing skin cell autophagy. Background Art

[0002] Normal skin cells will grow and decompose repeatedly. When people are young, the speed of cell growth and repair is greater than the speed of aging. However, with the increase of age, the increase of mental stress, the accumulation of bad living habits, and the damage caused by ultraviolet rays, chemicals, etc., the speed of cell repair gradually slows down the speed of cell destruction, thus forming skin aging. One of the common characteristics of aging cells is the accumulation of abnormal proteins in the cytoplasmic matrix, which needs to rely on cell autophagy to clean up. Autophagy is a cell self-digestion process that removes "cellular waste" (including abnormally modified proteins or protein aggregates, and damaged organelles) through lysosomes to maintain normal cell homeostasis. When the skin is stimulated or damaged by the outside world, autophagy can help remove harmful substances in damaged cells and prevent them from causing damage to surrounding healthy cells. Autophagy can also degrade a considerable part of the melanosomes containing melanin, thereby reducing the melanin content in melanocytes, and then producing the effects of lightening spots, whitening and removing yellowing, and improving the uniformity of skin color. In addition, autophagy also plays an important role in the process of sebaceous gland fat production. Studies have shown that autophagy-activating peptides can inhibit the production of fat by sebaceous cells and reduce the amount of fat on the skin surface. Therefore, by stimulating skin cell autophagy, pathological or non-pathological skin problems associated with excessive sebum production can be improved.

[0003] For the skin, in addition to removing waste, another important role of cell autophagy is to restore cell vitality. The nutrients and proteins obtained by the hydrolytic enzymes of the lysosome can be used by damaged cells and normal cells, promoting the repair and regeneration of damaged skin cells and strengthening the natural vitality of cells. Therefore, cell autophagy plays an important role in the maintenance, strengthening and repair of the skin barrier. Moreover, it is particularly important that this mechanism is completely different from other products used to protect the skin barrier. At present, most skin products that protect the skin barrier generally reduce the damage to the skin barrier by reducing irritation, eliminating allergenic or inflammatory factors, and resisting external invasion. At the same time, they use the metabolism of the skin itself to achieve the effect of maintaining or repairing the skin barrier. The product itself does not produce the repair effect of activating the skin, nor can it further strengthen the skin barrier. However, the lysosome is the proteolytic system most affected by aging, so the activity of cell autophagy decreases with age. Therefore, it is necessary to seek more drugs that induce skin autophagy to provide more options for the purposes of delaying skin aging, removing spots and whitening, reducing oil secretion, and strengthening the skin barrier. Summary of the invention

[0004] In view of the above technical problems, the present invention provides an application of aescin in preparing a product for inducing skin cell autophagy. The present invention has found through research that aescin can induce skin keratinocyte autophagy and can be used to prepare products for inducing skin cell autophagy, such as anti-aging products, freckle removal products, whitening products, oil control products, skin barrier strengthening products, etc.

[0005] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions: A first aspect of the present invention provides use of aescin in preparing a product for inducing skin cell autophagy, wherein the active ingredient of the product comprises aescin.

[0006] Aescin is a natural saponin compound. Existing studies have shown that aescin has multiple pharmacological activities, such as downregulating serum TNF-α levels, inhibiting IL-8 expression, and anti-oxidation. However, the above effects are not related to inducing skin cell autophagy. So far, no studies have proven that aescin can induce skin cell autophagy.

[0007] The present invention has discovered for the first time through research that aescin has the effect of inducing autophagy, and can significantly increase the expression levels of autophagy-related proteins LC3-I and LC3-II in skin keratinocytes. Therefore, aescin can be used to prepare products that induce skin cell autophagy, such as anti-aging products, freckle removal products, whitening products, oil control products, and skin barrier strengthening products.

[0008] Preferably, the skin cells are skin keratinocytes.

[0009] Preferably, the product is an anti-aging product by inducing skin cell autophagy.

[0010] Preferably, the product is a product that removes freckles by inducing autophagy of skin cells.

[0011] Preferably, the product is a product that whitens skin by inducing autophagy in skin cells.

[0012] Preferably, the product is a product that controls oil by inducing autophagy of skin cells.

[0013] Preferably, the product is a product that strengthens the skin stratum corneum barrier by inducing skin cell autophagy.

[0014] Further preferably, the product is a product for repairing and / or soothing sensitive skin.

[0015] More preferably, the product is a product for increasing skin moisture content, reducing skin transepidermal water loss, and soothing and removing redness.

[0016] Preferably, the product is a skin external preparation.

[0017] Exemplarily, the topical skin preparations include powders, lotions, tinctures, oils, emulsions, ointments, plasters, pastes, paints, gels, aerosols, and the like.

[0018] More preferably, the skin external preparation is an ointment or a gel. Ointments and gels are convenient to use and are easy to maintain on the skin surface.

[0019] Exemplarily, the raw materials of the above-mentioned ointment can be the following raw materials in mass fractions: 73.5-89.5 parts of water, 4.5-5.5 parts of glycerol, 1.8-2.2 parts of emulsifier, 9-11 parts of white oil, 0.9-1.1 parts of sodium polyacrylate, 0.18-0.22 parts of aescin, and 0.18-0.22 parts of methylparaben. Specifically, the following raw materials may be included: 81.6 parts of water, 5 parts of glycerol, 2 parts of emulsifier, 10 parts of white oil, 1 part of sodium polyacrylate, 0.2 parts of aescin, and 0.2 parts of methylparaben. Among them, the emulsifier can be a mixture of glyceryl stearate and stearate in a mass ratio of 40:60, such as a mixture of Emulgade 165 CN glyceryl stearate and PEG-100 stearate of BASF (China) Co., Ltd. in a mass ratio of 40:60.

[0020] The above ointment can be prepared by the following preparation method: (1) Disperse sodium polyacrylate, methylparaben and glycerin into water, stir and heat to 80-90°C to completely melt; (2) Mix the white oil and emulsifier, stir and heat to 80-90°C to completely melt them; (3) Add the mixture from step (2) to the mixture from step (1), homogenize and emulsify to ensure uniform distribution, and cool to 40-50°C; (4) dissolving aescin and adding it to the mixture (3), stirring to ensure uniform distribution; (5) Pour the mixture obtained in step (4) into a mold or container and cool it to room temperature to obtain a finished ointment.

[0021] Exemplarily, the raw materials of the gel include, by weight: 84-102 parts of water, 0.9-1.1 parts of thickener, 4.5-5.5 parts of glycerol, 0.15-0.25 parts of methylparaben, 0.18-0.22 parts of aescin, 0.4-0.6 parts of betaine, and 0.018-0.022 parts of high molecular weight hyaluronic acid. Specifically, the following raw materials may be included: 93 parts of water, 1 part of thickener, 5 parts of glycerol, 0.2 parts of methylparaben, 0.2 parts of aescin, 0.6 parts of betaine, and 0.02 parts of high molecular weight hyaluronic acid. The thickener may be sodium polyacryloyldimethyl taurate (SILK).

[0022] The above gel can be prepared by the following preparation method: (1) Slowly add thickener and high molecular weight hyaluronic acid into glycerin, stirring continuously until evenly dispersed; (2) adding the mixture from step (1), methylparaben and betaine into water, stirring and heating to 80-90°C to completely melt the mixture, and cooling the mixture to 40-50°C; (3) Add aescin to the gel prepared in step (2) and continue stirring to ensure uniform distribution; (4) Cool the mixture in step (3) to room temperature and then pour it into a suitable container to obtain the finished gel.

[0023] Preferably, the active ingredient of the product further includes ursolic acid.

[0024] The present invention has found through research that ursolic acid also has the effect of inducing skin keratinocyte autophagy, and ursolic acid has a synergistic effect when used in combination with aescin, and the effect of inducing skin keratinocyte autophagy at the same dose is significantly better than the use of either alone.

[0025] Preferably, the mass ratio of the aescin to the ursolic acid is 0.8-1.2:1.

[0026] Further preferably, the mass ratio of the aescin to the ursolic acid is 1:1.

[0027] The second aspect of the present invention provides a composition for external use on the skin, comprising aescin and ursolic acid, wherein the mass ratio of the aescin to the ursolic acid is 0.8-1.2:1.

[0028] Preferably, the mass ratio of the aescin to the ursolic acid is 1:1.

[0029] The third aspect of the present invention provides a skin external product, which is a skin external product for inducing skin cell autophagy, and its active ingredient includes the above-mentioned skin external composition.

[0030] Preferably, the skin cells are skin keratinocytes.

[0031] Preferably, the skin external product is an anti-aging product by inducing skin cell autophagy.

[0032] Preferably, the skin external product is a product for removing spots by inducing autophagy of skin cells.

[0033] Preferably, the skin external product is a product that whitens skin by inducing autophagy in skin cells.

[0034] Preferably, the skin external product is a product that controls oil by inducing autophagy of skin cells.

[0035] Preferably, the skin external product is a product that strengthens the skin stratum corneum barrier by inducing skin cell autophagy.

[0036] Further preferably, the skin external product is a product that repairs and / or soothes sensitive skin by inducing skin cell autophagy.

[0037] More preferably, the skin external product is a product for increasing skin moisture content, reducing skin transepidermal water loss, and soothing and removing redness.

[0038] Exemplarily, the dosage forms of the skin topical product include powders, lotions, tinctures, oils, emulsions, ointments, plasters, pastes, film coatings, gels, aerosols, and the like.

[0039] Preferably, the skin external product is a gel or an ointment. The corresponding product can be prepared by adding aescin and other active ingredients to common excipients of gel or ointment according to conventional methods.

[0040] Exemplarily, the raw materials of the above-mentioned ointment can be the following raw materials in mass fractions: 73.5-89.5 parts of water, 4.5-5.5 parts of glycerol, 1.8-2.2 parts of emulsifier, 9-11 parts of white oil, 0.9-1.1 parts of sodium polyacrylate, 0.16-0.24 parts of aescin, 0.2 parts of ursolic acid, and 0.18-0.22 parts of methylparaben. Specifically, the following raw materials may be included: 81.6 parts of water, 5 parts of glycerol, 2 parts of emulsifier, 10 parts of white oil, 1 part of sodium polyacrylate, 0.2 parts of aescin, 0.2 parts of ursolic acid, and 0.2 parts of methylparaben. Among them, the emulsifier can be a mixture of glyceryl stearate and stearate in a mass ratio of 40:60, such as a mixture of Emulgade 165 CN glyceryl stearate and PEG-100 stearate of BASF (China) Co., Ltd. in a mass ratio of 40:60.

[0041] The above ointment can be prepared by the following preparation method: (1) Disperse sodium polyacrylate, methylparaben and glycerin into water, stir and heat to 80-90°C to completely melt; (2) Mix the white oil and emulsifier, stir and heat to 80-90°C to completely melt them; (3) Add the mixture from step (2) to the mixture from step (1), homogenize and emulsify to ensure uniform distribution, and cool to 40-50°C; (4) dissolving aescin and ursolic acid and adding them to the mixture (3), stirring to ensure uniform distribution; (5) Pour the mixture obtained in step (4) into a mold or container and cool it to room temperature to obtain a finished ointment.

[0042] Exemplarily, the raw materials of the gel include, by weight: 84-102 parts of water, 0.9-1.1 parts of thickener, 4.5-5.5 parts of glycerol, 0.15-0.25 parts of methylparaben, 0.16-0.24 parts of aescin, 0.2 parts of ursolic acid, 0.4-0.55 parts of betaine, and 0.018-0.022 parts of high molecular weight hyaluronic acid. Specifically, the following raw materials may be included: 93 parts of water, 1 part of thickener, 5 parts of glycerol, 0.2 parts of methylparaben, 0.2 parts of aescin, 0.2 parts of ursolic acid, 0.4 parts of betaine, and 0.02 parts of high molecular weight hyaluronic acid. The thickener may be sodium polyacryloyldimethyl taurate (SILK).

[0043] The above gel can be prepared by the following preparation method: (1) Slowly add thickener and high molecular weight hyaluronic acid into glycerin, stirring continuously until evenly dispersed; (2) adding the mixture from step (1), methylparaben and betaine into water, stirring and heating to 80-90°C to completely melt the mixture, and cooling the mixture to 40-50°C; (3) Add aescin and ursolic acid to the gel prepared in step (2) and continue stirring to ensure uniform distribution; (4) Cool the mixture in step (3) to room temperature and then pour it into a suitable container to obtain the finished gel.

[0044] The beneficial effects of the present invention are: (1) The present invention is the first to discover the outstanding efficacy of aescin in inducing skin cell autophagy. This discovery is an innovative breakthrough in promoting skin repair, beauty, anti-aging, and improving skin barrier in aescin, filling the technical gap in the application of aescin in inducing skin cell autophagy; (2) The present invention has discovered for the first time through research that aescin can significantly increase the expression levels of autophagy-related proteins LC3-I and LC3-II in skin keratinocytes, and for skin keratinocytes pretreated with IL-4 and IL-13, aescin can also increase transmembrane resistivity, reduce paracellular flux, and exert autophagy, indicating that the mechanism of aescin's action on skin keratinocytes is completely different from the mechanism of action of other drugs that can only eliminate inflammatory factors but cannot stimulate active cell repair by inducing autophagy; (3) The present invention also found that aescin and ursolic acid have a synergistic effect in inducing skin keratinocyte autophagy. And experimentally verified that for non-inflamed but sensitive skin, the combined use of the two can significantly increase skin moisture content, reduce skin transepidermal water loss, and soothe and remove redness, indicating that the combined use of the two can effectively strengthen the skin barrier; (4) Compared with chemical drugs that induce skin keratinocyte autophagy, the natural compounds aescin and ursolic acid in the present invention have higher safety and are suitable for long-term use; the present invention has proved through experiments that the skin topical preparation made with aescin and ursolic acid as active ingredients is non-irritating to the skin; (5) Aescin and its combination with ursolic acid can be made into a variety of external dosage forms, including but not limited to common forms such as creams and gels, which are convenient for daily and clinical use. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The expression level of GFP-LC3 and the formation of autophagosomes after 6 hours of aescin treatment of GFP-LC3 HeLa cells in Example 9 of the present invention; Figure 2 The protein expression levels of LC3-1 and LC3-II after HaCaT cells were treated with aescin alone, chloroquine (CQ) alone, and aescin and chloroquine in combination in Example 10 of the present invention; Figure 3 is the protein expression level of LC3-1 and LC3-II after treating HaCaT cells with different concentrations of aescin for 6 hours in Example 10 of the present invention; Figure 4 is the protein expression level of LC3-1 and LC3-II after HaCaT cells were treated with 20 μM aescin for different time periods in Example 10 of the present invention; Figure 5 is the effect of aescin on cell transmembrane resistivity and paracellular flux in Example 10 of the present invention; Figure 6 The expression level of GFP-LC3 and the formation of autophagosomes after GFP-LC3 HeLa cells were treated with ursolic acid for 6 hours in Example 11 of the present invention; Figure 7 The protein expression levels of LC3-1 and LC3-II after the HaCaT cells were treated with ursolic acid alone, bafilomycin A1 alone, and ursolic acid and bafilomycin A1 in combination in Example 11 of the present invention; Figure 8 is the protein expression level of LC3-1 and LC3-II after treating HaCaT cells with different concentrations of ursolic acid for 6 hours in Example 11 of the present invention; Fig. 9 The expression level of GFP-LC3 and the formation of autophagosomes after 6 hours of treatment of GFP-LC3 HeLa cells with aescin and ursolic acid in Example 11 of the present invention; Fig.10The protein expression levels of LC3-1 and LC3-II after HaCaT cells were treated with aescin alone, ursolic acid alone, and aescin and ursolic acid in combination in Example 11 of the present invention; Fig.11 The gel containing aescin and ursolic acid in Example 12 of the present invention changes the water content of the stratum corneum of the facial skin of the test subjects. Compared with the baseline value (D0), *** P <0.001; Fig.12 The following is the change of transepidermal water loss rate of the facial skin of the test subjects by the gel containing aescin and ursolic acid in Example 12 of the present invention. Compared with the baseline value (D0), *** P <0.001; Fig.13 The gel containing aescin and ursolic acid in Example 12 of the present invention changes the hemoglobin content in the facial skin of the subjects. Compared with the baseline value (D0), *** P <0.001; Fig.14 The changes in the redness values ​​of the facial skin of the subjects treated with the gel containing aescin and ursolic acid in Example 12 of the present invention are shown in FIG. 1 . Compared with the baseline value (D0), *** P <0.001; Fig.15 The diagrams show the effect of the gel containing aescin and ursolic acid in Example 12 of the present invention on the red areas on the faces of some subjects. The upper diagram shows the improvement of the redness on the facial skin of subject 13 (female, 31 years old), and the lower diagram shows the improvement of the redness on the acne area of ​​subject 27 (female, 27 years old). DETAILED DESCRIPTION

[0046] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work fall within the scope of protection of the present invention.

[0047] Autophagy can delay skin aging, remove spots and whiten the skin, reduce oil secretion, and strengthen the skin barrier. Moreover, after autophagy is activated, the nutrients and proteins decomposed by the hydrolases of the lysosomes can also be used by damaged cells and normal cells, thereby promoting the repair and regeneration of damaged skin cells and strengthening the natural vitality of cells. This mechanism is completely different from the mechanism of action of products that can only reduce the damage to the skin barrier by external factors but do not activate the skin's own repair effect and cannot strengthen the skin barrier. They need to utilize the skin's own metabolism to maintain or repair the skin barrier.

[0048] However, lysosomes are greatly affected by aging, which causes the activity of cell autophagy to decrease with age. Therefore, it is necessary to seek more drugs that induce skin autophagy to provide more options for delaying skin aging, removing spots and whitening, reducing oil secretion, and strengthening the skin barrier.

[0049] The present invention discovered for the first time through research that aescin has the effect of inducing autophagy and can significantly increase the expression levels of autophagy-related proteins LC3-I and LC3-II in skin keratinocytes.

[0050] Based on the above research, an embodiment of the present invention provides the use of aescin in preparing a product for inducing skin cell autophagy.

[0051] The embodiment of the present invention further provides a composition for external use on the skin, comprising aescin and ursolic acid, wherein the mass ratio of the aescin to the ursolic acid is 0.8-1.2:1.

[0052] An embodiment of the present invention further provides a skin external application product, which is a skin external application product for inducing skin cell autophagy, and its active ingredient includes the above-mentioned skin external application composition.

[0053] The scheme of the present invention is described below through specific embodiments.

[0054] The emulsifier used in the following examples is a mixture of Emulgade 165 CN glyceryl stearate and PEG-100 stearate in a mass ratio of 40:60 produced by BASF (China) Co., Ltd.

[0055] Unless otherwise specified, the reagents, drugs or instruments used in the following examples were obtained from commercial sources.

[0056] Example 1 This embodiment provides an ointment for inducing skin cell autophagy, which can be used for anti-aging, freckle removal, whitening and strengthening the stratum corneum barrier of the skin.

[0057] The raw materials of the ointment are: 81.6 parts of water, 5 parts of glycerin, 2 parts of emulsifier, 10 parts of white oil, 1 part of sodium polyacrylate, 0.2 parts of aescin and 0.2 parts of methylparaben.

[0058] The preparation method comprises the following steps: (1) Disperse sodium polyacrylate, methylparaben and glycerin in 80.6 parts of water, stir and heat to 80-90°C to completely melt.

[0059] (2) Mix the white oil and emulsifier, stir and heat to 80-90℃ until they are completely melted.

[0060] (3) Add the mixture from step (2) to the mixture from step (1), homogenize and emulsify to ensure uniform distribution, and cool to 40-50°C.

[0061] (4) Dissolve aescin in 1 part water and add to mixture (3), stirring to ensure even distribution.

[0062] (5) Pour the mixture obtained in step (4) into a mold or container and cool it to room temperature to obtain a finished ointment.

[0063] Example 2 This embodiment provides an ointment for inducing skin cell autophagy, which can be used for anti-aging, freckle removal, whitening and strengthening the stratum corneum barrier of the skin.

[0064] The raw materials of the ointment are: 81.6 parts of water, 5 parts of glycerin, 2 parts of emulsifier, 10 parts of white oil, 1 part of sodium polyacrylate, 0.2 parts of aescin, 0.2 parts of ursolic acid, and 0.2 parts of methylparaben.

[0065] The preparation method comprises the following steps: (1) Disperse sodium polyacrylate, methylparaben and glycerin in 80.6 parts of water, stir and heat to 80-90°C to completely melt.

[0066] (2) Mix the white oil and emulsifier, stir and heat to 80-90℃ until they are completely melted.

[0067] (3) Add the mixture from step (2) to the mixture from step (1), homogenize and emulsify to ensure uniform distribution, and cool to 40-50°C.

[0068] (4) Dissolve aescin and ursolic acid in 1 part water and add to mixture (3), stirring to ensure even distribution.

[0069] (5) Pour the mixture obtained in step (4) into a mold or container and cool it to room temperature to obtain a finished ointment.

[0070] Example 3 This embodiment provides an ointment for inducing skin cell autophagy, which can be used for anti-aging, freckle removal, whitening and strengthening the skin stratum corneum barrier. The raw materials of the ointment are: 73.5 parts of water, 4.5 parts of glycerol, 1.8 parts of emulsifier, 9 parts of white oil, 0.9 parts of sodium polyacrylate, 0.16 parts of aescin, 0.2 parts of ursolic acid, and 0.18 parts of methylparaben. The preparation method is the same as that of Example 2.

[0071] Example 4 This embodiment provides an ointment for inducing skin cell autophagy, which can be used for anti-aging, freckle removal, whitening and strengthening the skin stratum corneum barrier. The raw materials of the ointment are: 89.5 parts of water, 5.5 parts of glycerol, 2.2 parts of emulsifier, 11 parts of white oil, 1.1 parts of sodium polyacrylate, 0.24 parts of aescin, 0.2 parts of ursolic acid, and 0.22 parts of methylparaben. The preparation method is the same as that of Example 2.

[0072] Example 5 This embodiment provides a gel that induces skin cell autophagy, and the gel can be used for anti-aging, freckle removal, whitening and strengthening the stratum corneum barrier of the skin.

[0073] The raw materials of the gel are: 93 parts of water, 1 part of thickener SILK, 5 parts of glycerin, 0.2 parts of methylparaben, 0.2 parts of aescin, 0.6 parts of betaine, and 0.02 parts of high molecular weight hyaluronic acid.

[0074] The preparation method comprises the following steps: (1) Slowly add thickener SILK and high molecular weight hyaluronic acid into glycerin and stir continuously until evenly dispersed.

[0075] (2) Add the mixture from step (1), methylparaben and betaine into water, stir and heat to 80-90°C to completely melt, and cool to 40-50°C.

[0076] (3) Add aescin to the gel prepared in step (2) and continue stirring to ensure even distribution.

[0077] (4) Cool the mixture in step (3) to room temperature and then pour it into a suitable container to obtain the finished gel.

[0078] Example 6 This embodiment provides a gel that induces skin cell autophagy, and the gel can be used for anti-aging, freckle removal, whitening and strengthening the stratum corneum barrier of the skin.

[0079] The raw materials of the gel are: 93 parts of water, 1 part of thickener SILK, 5 parts of glycerin, 0.2 parts of methylparaben, 0.2 parts of aescin, 0.2 parts of ursolic acid, 0.4 parts of betaine, and 0.02 parts of high molecular weight hyaluronic acid.

[0080] The preparation method comprises the following steps: (1) Slowly add thickener SILK and high molecular weight hyaluronic acid into glycerin and stir continuously until evenly dispersed.

[0081] (2) Add the mixture from step (1), methylparaben and betaine into water, stir and heat to 80-90°C to completely melt, and cool to 40-50°C.

[0082] (3) Add aescin and ursolic acid to the gel prepared in step (2) and continue stirring to ensure uniform distribution.

[0083] (4) Cool the mixture in step (3) to room temperature and then pour it into a suitable container to obtain the finished gel.

[0084] Example 7 This embodiment provides a gel that induces skin cell autophagy, and the gel can be used for anti-aging, freckle removal, whitening and strengthening the stratum corneum barrier of the skin.

[0085] The raw materials of the gel are: 84 parts of water, 0.9 parts of thickener SILK, 4.5 parts of glycerol, 0.15 parts of methylparaben, 0.16 parts of aescin, 0.2 parts of ursolic acid, 0.45 parts of betaine, and 0.018 parts of high molecular weight hyaluronic acid. The preparation method is the same as that of Example 6.

[0086] Example 8 This embodiment provides a gel that induces skin cell autophagy, and the gel can be used for anti-aging, freckle removal, whitening and strengthening the stratum corneum barrier of the skin.

[0087] The raw materials of the gel are: 102 parts of water, 1.1 parts of thickener SILK, 5.5 parts of glycerin, 0.25 parts of methylparaben, 0.24 parts of aescin, 0.2 parts of ursolic acid, 0.55 parts of betaine, and 0.022 parts of high molecular weight hyaluronic acid. The preparation method is the same as that of Example 6.

[0088] Example 9 This example provides the effect of aescin in inducing autophagy in Hela cells.

[0089] 1. Methods Hela cells expressing the autophagosome marker GFP-LC3 were seeded in 6-well plates containing DMEM medium (containing 10% fetal bovine serum) at a rate of 2 × 10 cells / well. 5 Cells were incubated at 37°C, 5% CO2 overnight.

[0090] Aescin solution (solvent: DMSO, concentration: 20 mM) was added to the wells seeded with GFP-LC3 Hela cells to make the final concentration of aescin in the culture medium to be 20 μM, and the cells were treated for 6 hours.

[0091] The cells were collected, lysed and proteins were extracted to detect the expression level of GFP-LC3, and the formation of autophagosomes was observed by immunofluorescence microscopy.

[0092] 2. Results like Figure 1 As shown in Figure 2, aescin can significantly increase the expression level of autophagosome marker GFP-LC3 in GFP-LC3 Hela cells (** P <0.01), inducing the formation of a large number of autophagosomes, indicating that aescin can induce autophagy in GFP-LC3 Hela cells.

[0093] Example 10 This example provides the effect of aescin in inducing autophagy in skin keratinocytes.

[0094] 1. Methods 1.1 HaCaT cells, a skin keratinocyte cell line, were seeded in 6-well plates containing DMEM medium (containing 10% fetal bovine serum) at a rate of 2 × 10 cells per well. 5 Cells were incubated at 37°C, 5% CO2 overnight.

[0095] Aescin solution (solvent is DMSO, concentration is 20 mM) was added to the wells seeded with HaCaT cells to make the final concentration of aescin in the culture medium to 20 μM, and the cells were treated for 6 hours; or chloroquine (CQ) solution was added to make the final concentration of chloroquine in the culture medium to 15 μM, and the cells were treated for 4 hours; or aescin solution was first added to make the final concentration of aescin in the culture medium to 20 μM, and the cells were treated for 2 hours, and then chloroquine solution was added to make the final concentration of chloroquine in the culture medium to 15 μM, and the cells were treated together for 4 hours.

[0096] Different volumes of aescin solution (solvent: DMSO, concentration: 20 mM) were added to the wells seeded with HaCaT cells to make the final concentration of aescin in the culture medium 5-50 µM for 6 hours.

[0097] Aescin solution (solvent is DMSO, concentration is 20 mM) was added to the wells seeded with HaCaT cells, so that the final concentration of aescin in the culture medium was 20 μM, and HaCaT cells were treated for different time periods.

[0098] The cells were collected, lysed and proteins were extracted, and SDS-PAGE electrophoresis and membrane transfer were performed. The expression levels of autophagy-related proteins (LC3-I and LC3-II) were detected by Western blot, and the formation of autophagosomes was observed by immunofluorescence microscopy.

[0099] 1.2 HaCaT cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C and 5% CO2. After pretreatment with IL-4 (final concentration of 100 ng / ml) and IL-13 (final concentration of 100 ng / ml) for 12 hours, aescin (final concentration of 20 μM) was added for 48 hours, and the cell transmembrane resistivity and paracellular flux were detected using an epithelial transmembrane cell resistance meter and an ELISA reader. DMSO treatment was the negative control group, and IL-4 and IL-13 treatment was the positive control group.

[0100] 2. Results like Figure 2~Figure 4 As shown, aescin can significantly increase the expression levels of autophagy-related proteins LC3-I and LC3-II in HaCaT cells, indicating that aescin can significantly increase the autophagy level of skin keratinocytes.

[0101] Studies have shown that the pathophysiological effects of Th2 cytokines interleukin-4 (IL-4) and interleukin-13 (IL-13) include destruction of the skin barrier and inhibition of autophagy, but there are no reports that aescin can inhibit IL-4 and IL-13. Therefore, theoretically, when aescin is used in HaCaT cells pretreated with IL-4 and IL-13, it will be difficult to repair HaCaT cells through autophagy due to the inhibitory effects of IL-4 and IL-13 on autophagy. Figure 5 As shown, for HaCaT cells pretreated with IL-4 and IL-13, aescin could significantly increase the cell transmembrane resistivity and reduce the paracellular flux (* P <0.05), indicating that aescin inhibits IL-4 and IL-13, and can still play an autophagic role in skin keratinocytes with elevated IL-4 and IL-13, thereby restoring the skin barrier damage caused by IL-4 and IL-13, which further indicates that the mechanism of aescin's action on skin keratinocytes is completely different from the mechanism of action of other drugs that can only eliminate inflammatory factors but cannot stimulate cell active repair by inducing autophagy.

[0102] Embodiment 11 This example provides the effect of aescin combined with ursolic acid in inducing cell autophagy.

[0103] 1. Methods 1.1 Effect of ursolic acid on autophagy in Hela cells Hela cells expressing the autophagosome marker GFP-LC3 were seeded in 6-well plates containing DMEM medium (containing 10% fetal bovine serum) at a rate of 2 × 10 cells / well. 5 Cells were incubated at 37°C, 5% CO2 overnight.

[0104] Ursolic acid solution (solvent: DMSO) was added to the wells seeded with GFP-LC3 Hela cells to make the final concentration of ursolic acid in the culture medium 40 μM, and the cells were treated for 6 hours.

[0105] The cells were collected, lysed and proteins were extracted to detect the expression level of GFP-LC3, and the formation of autophagosomes was observed by immunofluorescence microscopy.

[0106] 1.2 Effect of ursolic acid on inducing autophagy in skin keratinocytes HaCaT cells, a skin keratinocyte cell line, were seeded in a 6-well plate containing DMEM medium (containing 10% fetal bovine serum) at a rate of 2 × 10 cells / well. 5 Cells were incubated at 37°C, 5% CO2 overnight.

[0107] Add ursolic acid solution (the solvent is DMSO) to the wells seeded with HaCaT cells to make the final concentration of ursolic acid in the culture medium to 40 μM, and treat for 6 hours; or add bafilomycin A1 (Baf A1) solution to make the final concentration of bafilomycin A1 in the culture medium to 100 nM, and treat for 2 hours; or first add ursolic acid solution to make the final concentration of ursolic acid in the culture medium to 40 μM, treat for 4 hours, then add bafilomycin A1 solution to make the final concentration of bafilomycin A1 in the culture medium to 100 nM, and treat together for 2 hours.

[0108] Different volumes of ursolic acid solution were added to the wells seeded with HaCaT cells to make the final concentration of ursolic acid in the culture medium 10-100 μM and treated for 6 hours.

[0109] 1.3 Effect of aescin combined with ursolic acid on autophagy induction in Hela cells Hela cells expressing the autophagosome marker GFP-LC3 were seeded in 6-well plates containing DMEM medium (containing 10% fetal bovine serum) at a rate of 2 × 10 cells / well. 5 Cells were incubated at 37°C, 5% CO2 overnight.

[0110] Aescin solution (the solvent is DMSO) was added to the wells seeded with GFP-LC3 Hela cells to make the final concentration of aescin in the culture medium to 10 μM, and the cells were treated for 6 hours; or ursolic acid solution (the solvent is DMSO) was added to make the final concentration of ursolic acid in the culture medium to 10 μM, and the cells were treated for 6 hours; or aescin solution and ursolic acid solution were added to make the final concentrations of aescin and ursolic acid in the culture medium to 5 μM, and the cells were treated for 6 hours.

[0111] The cells were collected, lysed and proteins were extracted to detect the expression level of GFP-LC3, and the formation of autophagosomes was observed by immunofluorescence microscopy.

[0112] 1.4 Effect of aescin combined with ursolic acid on inducing autophagy in skin keratinocytes HaCaT cells, a skin keratinocyte cell line, were seeded in a 6-well plate containing DMEM medium (containing 10% fetal bovine serum) at a rate of 2 × 10 cells / well. 5 Cells were incubated at 37°C, 5% CO2 overnight.

[0113] Aescin solution (the solvent is DMSO) was added to the wells seeded with HaCaT cells to make the final concentration of aescin in the culture medium to be 10 μM, and the cells were treated for 6 hours; or ursolic acid solution (the solvent is DMSO) was added to make the final concentration of ursolic acid in the culture medium to be 10 μM, and the cells were treated for 6 hours; or aescin solution and ursolic acid solution were added to make the final concentrations of aescin and ursolic acid in the culture medium to be 5 μM each, and the cells were treated for 6 hours.

[0114] 2. Results like Figure 6 As shown in Figure 2, ursolic acid can significantly increase the expression level of the autophagosome marker GFP-LC3 in GFP-LC3 Hela cells (** P <0.01), inducing the formation of a large number of autophagosomes, indicating that ursolic acid can induce autophagy in GFP-LC3 Hela cells.

[0115] like Figure 7 , Figure 8 As shown, ursolic acid can significantly increase the expression levels of autophagy-related proteins LC3-I and LC3-II in HaCaT cells, indicating that ursolic acid can significantly increase the autophagy level of skin keratinocytes.

[0116] like Fig. 9 As shown in the figure, the combination of aescin and ursolic acid can significantly increase the expression level of the autophagosome marker GFP-LC3 in GFP-LC3Hela cells at a lower concentration and induce the formation of a large number of autophagosomes. The GFP-LC3 expression level and the amount of autophagosome formation are significantly higher than those of aescin or ursolic acid alone at the same dose (** P <0.01), indicating that the combination of aescin and ursolic acid has a synergistic effect in inducing autophagy in GFP-LC3 Hela cells.

[0117] like Fig.10 As shown in the results, the combined use of aescin and ursolic acid can significantly increase the expression levels of autophagy-related proteins LC3-I and LC3-II in HaCaT cells at a lower concentration, and is significantly better than the use of aescin or ursolic acid alone at the same dose, indicating that the combined use of aescin and ursolic acid also has a significant synergistic effect on improving the autophagy level of skin keratinocytes.

[0118] Example 12 This example provides the effect of a topical product containing aescin and ursolic acid on inducing autophagy of skin cells.

[0119] 1. Methods 1.1 Skin safety assessment Thirty participants were selected for each group according to the subject inclusion criteria, including 15 males and 15 females, with an average age of 32.45 years. The test substance (the gel that induces skin cell autophagy in Example 6) was placed in the spot tester, with an amount of about 0.020g-0.025g semi-solid. The control well was a blank control (no substance was placed), and the spot tester with the test substance was applied to the back or forearm of the subject with a low-allergenic tape, and gently pressed with the palm of the hand to evenly apply it to the skin for 24 hours. After removing the test substance spot tester (until the indentation disappears), observe the skin reaction for 0.5, 24, and 48 hours, and record the results according to the skin reaction grading standard in the "Technical Specifications for Safety of Cosmetics" (2015 edition).

[0120] 1.2 Soothing and repairing efficacy test 33 healthy men and women aged 18-40 who met the test requirements were selected, with an average age of 30.36 years old. Their facial skin was sensitive, with a lactic acid stimulation test score greater than 5 points, redness, tightness, tingling and other problems, but no obvious inflammatory symptoms. The clinical efficacy of soothing and repairing was evaluated through human testing for 28 days. The specific test method is to use the test substance twice a day, once in the morning and once in the evening, cleanse the face normally, and after using toner, take an appropriate amount of the test substance and apply it evenly on the facial skin. Use it continuously for 28 days, and measure the water content of the skin stratum corneum, the transepidermal water loss rate of the skin, the skin hemoglobin content and facial image acquisition before use and on the 14th and 28th days respectively.

[0121] 2. Results 2.1 Skin safety test results Table 1 Skin safety test results

[0122] As shown in the results of Table 1, the test product did not cause irritation to the skin, which shows that the topical product containing aescin and ursolic acid in the present invention is safe to the skin.

[0123] 2.2 Water content of the stratum corneum The test results of the moisture content of the stratum corneum of the skin of 33 subjects after using the test product are shown in Table 2 and Fig.11 .

[0124] Table 2 Difference analysis of water content in facial skin stratum corneum

[0125] The test results showed that compared with the baseline value (D0), the water content of the stratum corneum increased by 18.74% and 42.51% at D14 and D28 respectively. P The values ​​were all less than 0.001, which was statistically significant. It can be seen that after using the topical product containing aescin and ursolic acid of the present invention, the moisture content of the subject's skin increased significantly, that is, the topical product had a certain moisturizing effect.

[0126] 2.3 Transepidermal water loss (TEWL) The results of the transepidermal water loss rate of the 33 subjects in this trial after using the test product are shown in Table 3 and Fig.12 .

[0127] Table 3 Analysis of differences in transepidermal water loss rate of facial skin

[0128] The test results showed that the transepidermal water loss rate decreased by 10.22% and 15.73% respectively at D14 and D28 compared with the baseline value (D0). The paired sample t-test analysis results showed that the D14 and D28 were significantly P The values ​​were all less than 0.001, and the differences were statistically significant. This indicates that after using the topical product containing aescin and ursolic acid in the present invention, the water loss of the facial skin of the subjects was significantly reduced, indicating that the topical product has a repairing effect on the skin.

[0129] 2.4 Skin hemoglobin content The results of the test on the hemoglobin content in the facial skin of 33 subjects after using the test product are shown in Table 4 and Fig.13 .

[0130] Table 4 Analysis of differences in hemoglobin content in facial skin

[0131] The test results showed that compared with the baseline value (D0), the skin hemoglobin content decreased by 7.58% and 15.95% at D14 and D28 respectively. P The values ​​were all less than 0.001, which was statistically significant. It can be seen that after using the topical product containing aescin and ursolic acid in the present invention, the hemoglobin content in the facial skin of the subjects decreased, indicating that the topical product has a soothing and redness-removing effect on the skin.

[0132] 2.5 Facial Image Acquisition The test results of facial skin redness values ​​of 33 subjects after using the test product are shown in Table 5 and Fig.14 .

[0133] Table 5 Analysis of differences in facial skin redness values

[0134] The test results showed that compared with the baseline value (D0), the skin redness value decreased by 11.41% and 19.05% at D14 and D28 respectively. P The values ​​were all less than 0.001, which was statistically significant. It can be seen that after using the topical product containing aescin and ursolic acid in the present invention, the red area on the face of the test subject was reduced, indicating that the topical product has the effect of removing redness from the skin.

[0135] Sensitive skin has a weakened skin barrier function, and the skin is prone to inflammation such as redness, tightness, itching and stinging. The effects were compared by collecting facial red area images taken by the subjects using the test samples D14 and D28 VISIA 7. Fig.15 As shown, after the subjects used the topical product containing aescin and ursolic acid of the present invention, the area of ​​the red zone was significantly reduced or lightened.

[0136] The results of the present embodiment show that the topical product containing the combination of aescin and ursolic acid significantly improves multiple indicators of sensitive skin, including increasing the water content of the stratum corneum, reducing the transepidermal water loss rate, and reducing the hemoglobin content and red zone value. These data all show the effectiveness of the combination of aescin and ursolic acid in strengthening the skin barrier and relieving skin discomfort symptoms (such as redness, tightness and tingling). Therefore, it can be concluded that the composition has a good effect of strengthening the skin barrier, can soothe and repair sensitive skin, and is suitable for daily care of sensitive skin.

[0137] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. The use of aescin in preparing a product for inducing skin cell autophagy, characterized in that: The active ingredients of the product include aescin.

2. The use according to claim 1, characterized in that: The skin cells are skin keratinocytes; and / or The product is an anti-aging product by inducing skin cell autophagy; and / or The product is a product that removes freckles by inducing skin cell autophagy; and / or The product is a product that whitens skin by inducing skin cell autophagy; and / or The product is a product that controls oiliness by inducing skin cell autophagy; and / or The product is a product that strengthens the skin stratum corneum barrier by inducing skin cell autophagy; and / or The product is used to increase the moisture content of the skin, reduce the transepidermal water loss rate of the skin, soothe and remove redness; and / or The product is a skin external preparation.

3. The use according to claim 1 or 2, characterized in that: The active ingredient of the product also includes ursolic acid.

4. The use according to claim 3, characterized in that: The mass ratio of the aescin to the ursolic acid is 0.8-1.2:

1.

5. The use according to claim 4, characterized in that: The mass ratio of the aescin to the ursolic acid is 1:

1.

6. A composition for external use on skin, characterized in that: The invention comprises aescin and ursolic acid, wherein the mass ratio of the aescin to the ursolic acid is 0.8-1.2:

1.

7. The composition for external use on skin according to claim 6, characterized in that The mass ratio of the aescin to the ursolic acid is 1:

1.

8. A skin external product, characterized in that: The skin external product is an external product for inducing skin cell autophagy, and its active ingredient includes the skin external composition according to claim 6 or 7.

9. The skin external product according to claim 8, characterized in that: The skin cells are skin keratinocytes; and / or The skin external product is an anti-aging product by inducing skin cell autophagy; and / or The skin external product is a product that removes spots by inducing skin cell autophagy; and / or The skin external product is a product that whitens skin by inducing skin cell autophagy; and / or The skin external product is a product that controls oiliness by inducing skin cell autophagy; and / or The skin external product is a product that strengthens the skin stratum corneum barrier by inducing skin cell autophagy.

10. The skin external product according to claim 8 or 9, characterized in that: The skin external product is a product used to increase the moisture content of the skin, reduce the transepidermal water loss rate of the skin, and soothe and remove redness.

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