Application of mulberry root bark extract

Abstract

The invention discloses application of a mulberry root bark extract, and particularly relates to application of the mulberry root bark extract in preparation of a product for improving skin cortisol unbalance caused by pressure stress. The mulberry root bark extract provided by the invention can improve skin problems caused by excessive transformation of cortisol due to pressure stress, and improve the expression quantity of skin collagen, skin barrier related protein and skin basement membrane related protein, so that skin barrier damage, skin sensitivity, skin aging, skin aging and the like caused by emotional pressure are effectively relieved. Wrinkles are generated; the skin elasticity is reduced and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cosmetics, and particularly relates to a use of mulberry root bark extract in preparing a product for improving skin cortisol imbalance caused by stress. BACKGROUND

[0002] Mulberry root bark is a traditional Chinese medicinal material with various biological activities. Cortisol, also known as hydrocortisone, is a glucocorticoid secreted by the adrenal cortex after activation of the hypothalamic-pituitary-adrenal axis (HPA axis). It is often referred to as the "stress hormone". When the human body is stimulated by stress, cortisol can be activated locally in the skin. Short-term or long-term abnormal release of cortisol can cause problems such as impaired epidermal barrier function, increased skin sensitivity, increased fragility, and accelerated skin aging.

[0003] There is currently no report on treating skin problems caused by cortisol imbalance due to stress using mulberry root bark extract. SUMMARY

[0004] The present application provides a use of mulberry root bark extract in preparing a product for improving skin cortisol imbalance caused by stress. The mulberry root bark extract can inhibit the excessive conversion of cortisone to cortisol in the skin, thereby preventing or improving skin problems caused thereby.

[0005] In a first aspect, the present application provides a use of mulberry root bark extract in preparing a product for improving skin cortisol imbalance caused by stress.

[0006] In any embodiment of the present application, improving skin cortisol imbalance caused by stress includes preventing or alleviating skin problems related to cortisol imbalance caused by stress.

[0007] In any embodiment of the present application, the skin problems include at least one of skin aging caused by long-term stress, impaired skin barrier, weakened skin basement membrane, skin sensitivity, skin discoloration, decreased skin moisturizing ability, decreased oil control ability, acne, skin inflammation, appearance of tired skin, skin ulcers, skin petechiae, increased capillary fragility, and difficult healing of skin wounds.

[0008] In any embodiment of the present application, the cortisol imbalance includes at least one of increased cortisol content in the skin and enhanced cortisol activity in the skin.

[0009] In any embodiment of the present application, the mulberry root bark extract can inhibit the conversion of cortisone to cortisol.

[0010] In any embodiment of the present application, the preparation method of the mulberry root bark extract includes at least one of solvent extraction, supercritical extraction, and ultrasonic extraction.

[0011] In any embodiment of the present application, the mulberry root extract includes at least one of a mulberry root glycerol extract, a mulberry root water extract, a mulberry root alcohol extract, and a mulberry root ester extract. In any embodiment of the present application, the product includes at least one of a pharmaceutical product and a cosmetic product.

[0012] In any embodiment of the present application, the dosage form of the product includes at least one of a cream, a liquid, a gel, a spray, an aerosol, a patch, and a lyophilized agent.

[0013] In any embodiment of the present application, the concentration of the mulberry root extract in the product is greater than or equal to 0.001% (w / v).

[0014] In any embodiment of the present application, the product further includes an excipient including at least one of a fragrance, a dye, an emulsifier, a stabilizer, a lubricant, a solvent, a humectant, an emollient, a moisturizer, a film-forming agent, a UV absorber, an essential oil, a vitamin, a trace metal, an anti-irritant, an antimicrobial agent, an antioxidant, a chelating agent, a preservative, a pH adjuster, a skin conditioner, a thickening agent, and a silicone compound.

[0015] The present application provides a use of a mulberry root extract in preparing a product for improving skin cortisol imbalance caused by stress. The mulberry root extract of the present application can inhibit the conversion of cortisone to cortisol in the skin under stress, and relieve skin problems such as skin barrier function damage, skin basement membrane weakening, and skin collagen loss caused by emotional stress. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required to be used in the embodiments of the present application will be briefly introduced as follows. Those skilled in the art can also obtain other drawings according to these drawings without creating any creative labor.

[0017] Figure 1 A schematic diagram of the results of the influence of different concentrations of mulberry root extract on the inhibition of cortisol production in Example 1 of the present application (wherein **P<0.0001 in the figure indicates that the difference is extremely extremely extremely significant compared with the cortisone group; and ###p<0.0001 indicates that the difference is extremely extremely extremely significant compared with Experimental Group 1 (cortisone + 0.001% mulberry root)). Figure 2 A schematic diagram of the results of the influence of different concentrations of mulberry root extract on the expression of collagen type I COL-I mRNA and collagen type III COL-III mRNA in Example 2 of the present application (wherein, Figure 2 A is a schematic diagram of the results of the influence of different concentrations of mulberry root extract on the expression of collagen type I COL-I mRNA; Figure 2B is a schematic diagram of the results of the influence of mulberry root-bark extract of different concentrations on collagen COL-III mRNA expression, ##p<0.01 indicates that the difference is extremely significant compared with the normal group; *p<0.05 indicates that the difference is significant compared with the corticosterone group, and ***p<0.001 indicates that the difference is extremely significant compared with the corticosterone group.

[0018] Figure 3 A schematic diagram of the results of the influence of mulberry root-bark extract of different concentrations on keratin, filaggrin and LOR mRNA expression in Example 3 of the present application (Figure A is a schematic diagram of the results of the influence of mulberry root-bark extract of different concentrations on keratin 1 mRNA expression in Example 3 of the present application; Figure B is a schematic diagram of the results of the influence of mulberry root-bark extract of different concentrations on keratin 10 mRNA expression in Example 3 of the present application; Figure C is a schematic diagram of the results of the influence of mulberry root-bark extract of different concentrations on filaggrin FLG mRNA expression in Example 3 of the present application; and Figure D is a schematic diagram of the results of the influence of mulberry root-bark extract of different concentrations on LOR mRNA expression in Example 3 of the present application; ##p<0.01 indicates that the difference is extremely significant compared with the normal group; *p<0.05 indicates that the difference is significant compared with the corticosterone group, and **p<0.01 indicates that the difference is extremely significant compared with the corticosterone group.

[0019] Figure 4 A schematic diagram of the results of the influence of mulberry root-bark extract of different concentrations on skin basement membrane-related gene mRNA expression in Example 4 of the present application (Figure A is a schematic diagram of the results of the influence of mulberry root-bark extract of different concentrations on collagen COL-IV mRNA expression in Example 4 of the present application; Figure B is a schematic diagram of the results of the influence of mulberry root-bark extract of different concentrations on collagen COL-VII mRNA expression in Example 4 of the present application; and Figure C is a schematic diagram of the results of the influence of mulberry root-bark extract of different concentrations on collagen COL-XVII mRNA expression in Example 4 of the present application; ##p<0.01 indicates that the difference is extremely significant compared with the normal group; *p<0.05 indicates that the difference is significant compared with the corticosterone group, **p<0.01 indicates that the difference is extremely significant compared with the corticosterone group, and ***p<0.001 indicates that the difference is extremely significant compared with the corticosterone group. DETAILED DESCRIPTION

[0020] In order to make the application purposes, technical solutions and beneficial technical effects of the present application clearer, the present application will be further described in detail below in combination with examples. It should be understood that the implementation examples described in the present specification are only for the purpose of explaining the present application, and are not intended to limit the present application.

[0021] For simplicity, only some numerical ranges are explicitly recited herein. However, any lower limit can be combined with any upper limit to form a range not explicitly recited; and any lower limit can be combined with any other lower limit to form a range not explicitly recited, and likewise any upper limit can be combined with any other upper limit to form a range not explicitly recited. Further, although each point or individual number within a range is encompassed, unless the context clearly dictates otherwise, this is not necessarily the intention. Thus, each point or individual number can serve as its own lower or upper limit to, in combination with, any other point or individual number, or in combination with other lower or upper limits, to form a range not explicitly recited.

[0022] It has to be noted that, as used herein, the terms "includes", "containing", "having" or "comprising" and conjugations thereof are used in their open-ended sense and do not exclude other elements not explicitly listed. In other words, these terms are intended to be equivalent to the term "comprising". It is further noted that, as used herein, the terms "consisting of" and "consisting essentially of" and conjugations thereof are used in their narrowest sense to mean that the listed elements are the only ones that are present in the composition, process, method, article, or apparatus. In other words, these terms are intended to be equivalent to the term "consisting of".

[0023] Unless otherwise indicated, the values for the parameters mentioned in the application can be measured using any method known in the art (for example, they can be tested according to the methods given in the examples of the application). Unless otherwise indicated, the test temperature for the parameters mentioned in the application is 25°C and the test pressure is standard atmospheric pressure.

[0024] The foregoing summary of the application is not intended to describe every disclosed embodiment or implementation of the application. The example embodiments are described more fully below. At various places in the application, guidance is provided by a series of examples, which can be used in various combinations. In each instance, the recited examples are intended to be representative only and should not be interpreted as exhaustive.

[0025] Morus alba L. and has anti-inflammatory, antioxidant, antibacterial and other biological activities. It is often used in traditional Chinese medicine for clearing heat, reducing lung, relieving asthma and coughing. Cortisol, also known as hydrocortisone, is a glucocorticoid secreted by the adrenal cortex after activation of the hypothalamic-pituitary-adrenal axis (HPA axis). It is often referred to as the "stress hormone". When the human body is stimulated by psychological or physiological stress, the local skin can activate cortisol through the steroidogenic enzyme system. Short-term or long-term abnormal release of cortisol can damage the lipid metabolism of the stratum corneum, reduce the intercellular adhesion of keratinocytes, and inhibit the expression of key barrier proteins such as filaggrin and loricrin, thereby leading to impaired epidermal barrier function, increased trans-epidermal water loss, increased skin sensitivity and increased skin fragility. In addition, it can also inhibit collagen synthesis and promote matrix metalloproteinase expression, thereby accelerating the aging process of the skin.

[0026] Currently, there is no report on the use of Morus alba L. extract to treat skin problems caused by cortisol imbalance. Therefore, it is urgent to develop a new use of Morus alba L. extract to overcome the shortcomings of the prior art.

[0027] The first aspect of the embodiments of the present application provides a use of a Morus alba L. extract in the preparation of a product for improving skin cortisol imbalance caused by stress.

[0028] The Morus alba L. extract of the present application can effectively improve various skin problems caused by excessive conversion of cortisol. Its mechanism is to inhibit the excessive generation of cortisol, significantly up-regulate the expression of skin barrier-related proteins (such as filaggrin, loricrin, keratin, etc.) and skin basement membrane-related proteins (such as collagen type IV, collagen type VII, collagen type X VII, etc.), thereby enhancing the skin barrier function and maintaining the structural integrity of the basement membrane. At the same time, the extract can also promote the regeneration and synthesis of collagen type I and collagen type III in the dermis, effectively alleviate the skin barrier damage, sensitive state aggravation, wrinkle formation and skin elasticity decline caused by emotional stress, and show excellent comprehensive skin care effect.

[0029] In some embodiments, improving skin cortisol imbalance caused by stress includes preventing or alleviating skin problems related to cortisol imbalance caused by stress.

[0030] In some embodiments, the skin problems include at least one of skin aging caused by long-term stress, impaired skin barrier, weakened skin basement membrane, skin sensitivity, skin discoloration, decreased skin moisturizing ability, decreased oil control ability, acne, skin inflammation, appearance of tired skin, skin ulcers, skin petechiae, increased capillary fragility, and difficult healing of skin wounds.

[0031] In some embodiments, the cortisol imbalance comprises at least one of an elevated cortisol level in the skin and an enhanced cortisol activity.

[0032] Preferably, the cortisol imbalance comprises at least one of an elevated activity of 11 β-HSD1 in the skin and an increased level of 11 β-HSD1 in the skin.

[0033] As an example, the stress refers to a factor capable of activating the HPA axis to release the secretion of glucocorticoids from the adrenal gland, and the stress can be at least one of psychological, emotional, mental and physical stress.

[0034] In some embodiments, the prevention and / or alleviation of the skin problem caused by the cortisol imbalance comprises upregulating the mRNA expression level of at least one gene of collagen type I, collagen type III, keratin 1, keratin 10, filaggrin, loricrin, collagen type IV, collagen type VII and collagen type XlV.

[0035] In some embodiments, the prevention or alleviation of the skin problem caused by the cortisol imbalance comprises increasing the level of at least one of collagen type I, collagen type III, keratin 1, keratin 10, filaggrin, loricrin, collagen type IV, collagen type VII and collagen type XlV.

[0036] In some embodiments, the prevention or alleviation of the skin problem caused by the cortisol imbalance comprises enhancing the activity of at least one of collagen type I, collagen type III, keratin 1, keratin 10, filaggrin, loricrin, collagen type IV, collagen type VII and collagen type XlV.

[0037] By upregulating the mRNA expression, protein level and activity of barrier and structural key components such as collagen type I, collagen type III, keratin 1, keratin 10, filaggrin, loricrin, collagen type IV, collagen type VII and collagen type XlV, the skin barrier function is systematically enhanced from the molecular level to the tissue level, the basement membrane integrity is maintained, and the dermal collagen regeneration and stability are promoted, thereby synergistically alleviating the problems of skin barrier damage, sensitive state, wrinkle formation and elasticity decline caused by the cortisol imbalance induced by emotional stress.

[0038] In some embodiments, the mulberry root bark extract is capable of inhibiting the conversion of cortisone to cortisol.

[0039] In some embodiments, the mulberry root bark extract, as an 11β-HSD1 inhibitor, can reduce the production of cortisol, e.g., the production of cortisol in the skin, by inhibiting the activity of 11β-HSD1 in the skin, thereby preventing and / or alleviating skin problems caused by cortisol imbalance. For example, when cortisol is elevated, it can cause skin water-oil imbalance and skin barrier function damage caused by increased oil secretion from the skin surface, decreased natural moisturizing factors such as hyaluronic acid, decreased expression of epidermal structural proteins (e.g., keratin K1, keratin K10, loricrin, filaggrin, etc.), and the like; further damage to the skin barrier and skin aging caused by decreased true epidermal junction structural proteins (e.g., tight junction proteins, laminin) and degradation of dermal extracellular matrix (e.g., hyaluronic acid, collagen); and skin problems such as wrinkles, decreased skin strength, and the like caused by flattening of the basement membrane.

[0040] In some embodiments, the method of preparing the mulberry root bark extract comprises at least one of solvent extraction, supercritical extraction, and ultrasonic extraction.

[0041] Optionally, the mulberry root bark extract comprises at least one of a mulberry root bark glycerol extract, a mulberry root bark water extract, a mulberry root bark alcohol extract, and a mulberry root bark ester extract.

[0042] In some embodiments, the product comprises at least one of a pharmaceutical product and a cosmetic product.

[0043] In some embodiments, the dosage form of the product comprises at least one of a cream, a liquid, a gel, a spray, an aerosol, a patch, and a lyophilized agent.

[0044] In some embodiments, the concentration of the mulberry root bark extract in the product is greater than or equal to 0.001% (w / v).

[0045] As an example, the concentration of the mulberry root bark extract in the product can be 0.001% (w / v), 0.002% (w / v), 0.003% (w / v), 0.004% (w / v), 0.005% (w / v), 0.006% (w / v), 0.007% (w / v), 0.008% (w / v), 0.009% (w / v), 0.01% (w / v), 0.012% (w / v), 0.014% (w / v), 0.016% (w / v), 0.018% (w / v), 0.02% (w / v), 0.022% (w / v), 0.024% (w / v), 0.026% (w / v), 0.028% (w / v), 0.03% (w / v), 0.035% (w / v), 0.04% (w / v), 0.045% (w / v), or 0.05% (w / v).

[0046] As examples, the product can be formulated into a lotion (for gentle moisturization of the skin), a serum (to supplement the nutrients required by the skin), a cream (for deep nourishment), a massage cream (for massage to promote absorption), an essence (with high concentration of active ingredients), an eye cream (for eye care), a cleansing cream (for gentle cleansing), a cleansing foam (for rich foam cleansing), a cleansing water (for refreshing cleansing), a mask (for concentrated care), a spray (for convenient moisturization), or a powder (for convenient portability and use), etc.

[0047] This diverse selection of dosage forms allows the cosmetic composition of the present application to better adapt to different skin types and usage scenarios. For example, for oily skin, a light lotion or spray may be more preferred; while for dry skin, a nourishing cream or massage cream may be more popular. At the same time, different dosage forms also facilitate the packaging and use of the product, such as the portability of powder for travel, and the use of spray for large-area skin moisturization.

[0048] Optionally, the product further comprises an auxiliary material, which includes at least one of a fragrance, a dye, an emulsifier, a stabilizer, a lubricant, a solvent, a humectant, a softener, a wetting agent, a film-forming agent, a UV absorber, an essential oil, a vitamin, a trace metal, an anti-irritant, an antimicrobial agent, an antioxidant, a chelating agent, a preservative, a pH adjuster, a skin conditioner, a thickening agent, and a silicone compound.

[0049] Optionally, the mulberry root bark extract can be extracted from one or more parts of the flowers, leaves, fruits, or stems of mulberry root bark, which means that active extracts can be obtained from different parts of mulberry root bark, and different parts may contain different concentrations and types of active ingredients.

[0050] In this application, keratin 1 refers to Keratin 1.

[0051] In this application, keratin 10 refers to Keratin 10.

[0052] Keratin 1 (Keratin 1) and Keratin 10 (Keratin 10) are the main intermediate filament proteins in keratinocytes, which form a complex network of cytoskeletal fibers in the cytoplasm, providing mechanical support to keratinocytes, maintaining the morphological and structural integrity of the cells, enabling the stratum corneum to withstand external mechanical pressure and friction, and preventing skin damage. During the differentiation process of keratinocytes, the expression levels of keratin 1 and keratin 10 change, and they interact with other proteins to regulate the differentiation process of cells, promote the formation and maturation of the stratum corneum, and thus enhance the barrier function of the skin.

[0053] In this application, filaggrin refers to Filaggrin.

[0054] Filaggrin (FLG) is an important molecule that connects keratin fibers in the stratum corneum of human skin. With the assistance of FLG monomers, keratin and fibers are regularly aggregated to form a solid physical barrier in the outermost layer of the epidermis, which can prevent the loss of epidermal moisture and the invasion of external allergens, further consolidating the physical barrier function of the skin. At the same time, FLG can be degraded in the stratum corneum of the epidermis to form small amino acid molecules, which have a moisturizing function and are called "natural moisturizing factors". They play an important role in moisturizing and barrier integrity.

[0055] In this application, Loricrin refers to Loricin.

[0056] Loricrin (LOR) is one of the main components of the cornified envelope. It cross-links with other proteins such as involucrin, filaggrin, and transglutaminase to form a highly cross-linked insoluble protein network, reinforcing the structure of the cornified envelope, making it more robust and stable, thereby enhancing the mechanical strength and compression resistance of keratinocytes. In addition, Loricrin can interact with proteins such as involucrin to tightly connect keratin fibers with other cell structures of keratinocytes, forming an integrated cytoskeletal network, making the structure of the stratum corneum more compact and ordered, and improving the integrity of the skin barrier.

[0057] In this application, Collagen Type I refers to Collagen Type I, abbreviated as I-Col or COL-I in English. It is the most important fiber-forming collagen in the extracellular matrix of dermal cells and is the core protein that maintains the mechanical strength and structural integrity of the skin. Type I collagen is synthesized in fibroblasts and secreted into the extracellular matrix, where it is modified by enzymes and self-assembled into thick fiber bundles, forming a solid scaffold structure for the skin. Under normal circumstances, the balance between the continuous synthesis and orderly degradation of type I collagen is the key to maintaining skin elasticity and tensile strength. Excessive action of cortisol can significantly inhibit the collagen synthesis ability of fibroblasts, while promoting the expression of matrix metalloproteinases and accelerating the degradation of type I collagen, leading to a loose and broken collagen network structure in the dermis, resulting in skin collapse, wrinkle formation, and a significant decrease in elasticity.

[0058] In the present application, collagen type III refers to Collagen Type III, abbreviated as III-Col or COL-III in English, which is an important fibrous reticular collagen in the extracellular matrix of the dermis, often co-distributed with collagen type I, and together forms the structural basis of skin flexibility and elasticity. After being synthesized and secreted in fibroblasts, collagen type III participates in the formation of a fine fiber network, and plays a key role in maintaining the integrity and elasticity of the skin soft tissue structure. Under normal circumstances, the stable expression and orderly assembly of collagen type III are important conditions for ensuring the delicacy, suppleness and wound repair capacity of the skin. Excessive action of cortisol can inhibit the function of fibroblasts, interfere with the synthesis and secretion of collagen type III, and destroy the normal assembly of its fiber network, leading to dermal reticular structure disorder and reduced elasticity, and further aggravating skin fragility, sensitivity and texture abnormalities.

[0059] In the present application, collagen type IV refers to Collagen Type IV, abbreviated as IV-Col or COL-IV in English, which is the main component of the basement membrane reticular structure and is directly involved in the composition of the extracellular matrix after being synthesized in cells. Under normal circumstances, the expression and update of collagen type IV gene help maintain the stability and function of the basement membrane. The action of cortisol may interfere with this update process, making the basement membrane unable to repair and rebuild in time, further aggravating the weakening of the basement membrane, and reducing its support and protection effect on cells.

[0060] In the present application, collagen type VII refers to Collagen Type VII, abbreviated as VII-Col or COL-VII in English, which is mainly generated by basal keratinocytes and dermal fibroblasts. It forms anchoring fibrils at the dermal-epidermal junction, firmly attaching the epidermal basement membrane to the dermal extracellular matrix. The stability of these two layers is the key to the tightness and elasticity of the skin. When the expression of collagen type VII gene decreases, the number and quality of anchoring fibers will be affected, leading to loose connection between the epidermis and the dermis, and decreased stability of the basement membrane, which is prone to skin lesions such as blisters.

[0061] In the present application, collagen type XVII refers to Collagen Type XVII, abbreviated as XVII-Col or COL-XVII in English, which is the core component of the basement membrane hemidesmosome, mainly located in the hair follicle stem cells of the hair follicle bulb and the epidermal stem cells at the junction of the epidermis and the dermis. Its role is to tightly connect the epidermis and the dermis through the basement membrane zone to promote epidermal replacement and stabilize the basement membrane. The protein encoded by collagen type XVII plays a key role in maintaining the proliferation and differentiation of epidermal stem cells and the stability of the basement membrane. The decrease of collagen type XVII gene expression will lead to the impairment of epidermal stem cell function, affecting the normal update and repair of the epidermis, and further making the basement membrane lose normal cell support, become fragile and unstable.

[0062] In the present application, cortisone refers to a naturally occurring glucocorticoid produced by the zona fasciculata of the adrenal gland.

[0063] Examples The present application is described in more detail by the following examples, which are only illustrative and not limitative since various modifications and variations of the application disclosed will be apparent to those skilled in the art. Unless otherwise stated, all parts, percentages and ratios reported in the following examples are based on mass, and all reagents used in the examples are commercially available or synthesized according to conventional methods and used as received without further purification, and the instruments used in the examples are commercially available.

[0064] Example 1 Inhibition of the conversion of corticosterone to cortisone by mulberry root bark extract 1. Experimental materials and reagents The main materials and reagents used in the experiment include: mulberry root bark extract, DMEM culture medium (Hyclone), corticosterone (Sigma), dimethyl sulfoxide (DMSO, Sigma), immortalized human keratinocytes (HaCaT cells, Chinese Academy of Sciences Cell Bank), human cortisol (Cortisol) ELISA detection kit (Shanghai Enzyme-linked Biotechnology Co., Ltd.).

[0065] 2. Preparation of experimental sample solutions (1) Preparation of mulberry root bark extract powder: Dry mulberry root bark was crushed and passed through a 20-mesh sieve. The crushed mulberry root bark was extracted twice by heating reflux extraction with 10 times the amount of 70% ethanol solution, each for 2 hours. The extract solutions were combined and concentrated under reduced pressure to a paste. The paste was suspended in water and then loaded onto a pretreated AB-8 macroporous adsorption resin column. The column was eluted with 3 column volumes of pure water, 20% ethanol solution, 50% ethanol solution and 80% ethanol solution, respectively, in gradient. The 50% ethanol elution fraction rich in target components was collected, concentrated under reduced pressure, and then freeze-dried to obtain mulberry root bark extract powder.

[0066] (2) Experimental sample solution 1: An appropriate amount of corticosterone and mulberry root bark extract powder was accurately weighed and dissolved in DMSO, followed by serial dilution with DMEM medium to prepare a mixed solution containing 3 μM corticosterone and 0.001% (w / v) mulberry root bark extract.

[0067] (3) Experimental sample solution 2: The preparation method is the same as that of experimental sample solution 1, and a mixed solution containing 3 μM corticosterone and 0.0001% (w / v) mulberry root bark extract is finally obtained.

[0068] (4) Corticosterone solution (used in the model group): An appropriate amount of corticosterone was accurately weighed, dissolved in DMSO, and then diluted with DMEM medium to prepare a corticosterone solution with a concentration of 3 μM.

[0069] (5) Control group solution: DMSO was diluted with DMEM medium to a final concentration of 0.2% (v / v), consistent with all experimental groups.

[0070] 3. Cell culture and experimental grouping HaCaT cells were seeded in a 24-well cell culture plate at a density of 8 x 10 4 cells / well, 500 μL of complete DMEM medium was added to each well, and the plate was incubated in a 37°C, 5% CO2 incubator for 24 hours. When the cell confluence reached 70%-80%, the experimental grouping was performed: Untreated group (normal group): The original culture medium was aspirated and replaced with an equal volume of control group solution.

[0071] Model group (corticosterone group): The original culture medium was aspirated and replaced with an equal volume of 3 μM corticosterone solution.

[0072] Experimental group 1 (corticosterone + 0.001% mulberry root bark): The original culture medium was aspirated and replaced with an equal volume of experimental sample solution 1 containing 3 μM corticosterone + 0.001% (w / v) mulberry root bark extract.

[0073] Experimental group 2 (corticosterone + 0.0001% mulberry root bark): The original culture medium was aspirated and replaced with an equal volume of experimental sample solution 2 containing 3 μM corticosterone + 0.0001% (w / v) mulberry root bark extract.

[0074] Each group of cells was continued to be cultured for 24 hours.

[0075] 4. Detection of corticosterone content After the culture was completed, the cell culture supernatant from each well was collected and centrifuged (3000 rpm, 10 min) to obtain the clear supernatant. The operation was strictly performed according to the instructions of the human corticosterone ELISA detection kit, and the corticosterone content in the cell culture supernatant from each well was detected by an enzyme-labeled instrument. Taking the corticosterone content of the untreated group as the reference, the change in corticosterone content of the model group and the experimental groups was calculated, and taking the corticosterone content of the model group as the reference, the corticosterone conversion rate of the experimental groups was calculated.

[0076] The calculation formula for the corticosterone conversion rate is: Corticosterone conversion rate = [(corticosterone content of experimental group - corticosterone content of untreated group) / (corticosterone content of model group - corticosterone content of untreated group)] x 100%.

[0077] GraphPad prism6 software was used for plotting, and the significance of cortisol conversion rate between groups was tested. *p<0.05 indicates a statistically significant difference.

[0078] Results as shown in Figure 1 Compared with the model group (containing only 3 μM corticosterone), the cortisol conversion rate of HaCaT cells treated with 0.0001% (w / v) and 0.001% (w / v) mulberry root bark extract in the experimental group was significantly decreased, indicating that mulberry root bark extract can significantly inhibit the conversion of corticosterone to cortisol in HaCaT cells, and inhibit the generation of cortisol in a concentration-dependent manner. The effect of 0.001% (w / v) mulberry root bark extract on inhibiting the generation of cortisol was significantly better than that of 0.0001% (w / v) mulberry root bark extract.

[0079] Example 2 Mulberry root bark extract promotes collagen expression 1. Experimental materials and reagents The main materials and reagents used in this experiment include: mulberry root bark extract (same as example 1), corticosterone (Sigma), dimethyl sulfoxide (DMSO, Sigma), human skin fibroblasts (HDF-α cells, ATCC, USA), fibroblast basal medium (FBM medium, ScienCell), total RNA extraction kit (TaKaRa, Japan), reverse transcription kit (Thermo Fisher, USA), SYBR Green qPCR premix reagent (Thermo Fisher, USA). The specific primers of COL-I, COL-III and the internal reference gene GAPDH were synthesized by Genechem (Shanghai) Co., Ltd.

[0080] 2. Preparation of experimental sample solution (1) Experimental sample solution 3: Accurately weigh the appropriate amount of corticosterone and mulberry root bark extract powder, dissolve in DMSO, then serially dilute with FBM basal medium, finally prepare a mixed solution containing 3 μM corticosterone and 0.001% (w / v) mulberry root bark extract.

[0081] (2) Experimental sample solution 4: The preparation method is the same as experimental sample solution 3, and a mixed solution containing 3 μM corticosterone and 0.0001% (w / v) mulberry root bark extract is finally obtained.

[0082] (3) Corticosterone solution (model group): Accurately weigh the appropriate amount of corticosterone, dissolve in DMSO, then dilute with FBM basal medium, prepare a corticosterone solution with a concentration of 3 μM.

[0083] (4) Control solution: FBM basal medium was used to dilute DMSO, with a final concentration of 0.2% (v / v), consistent with all experimental groups.

[0084] 3. Cell culture and experimental grouping HDF-a cells were seeded in 6-well cell culture plates at a density of 2 x 10 4 cells / well, with 2 mL of complete FBM medium added to each well, and incubated in a 37°C, 5% CO2 incubator for 24 hours. When the cell confluence reached 70-80%, the cells were subjected to experimental grouping: Untreated group (normal group): The original culture medium was aspirated and replaced with an equal volume of control solution.

[0085] Model group (corticosterone group): The original culture medium was aspirated and replaced with an equal volume of 3 μM corticosterone solution.

[0086] Experimental group 3 (corticosterone + 0.001% mulberry root bark): The original culture medium was aspirated and replaced with an equal volume of experimental sample solution 3 (containing 3 μM corticosterone + 0.001% (w / v) mulberry root bark extract).

[0087] Experimental group 4 (corticosterone + 0.0001% mulberry root bark): The original culture medium was aspirated and replaced with an equal volume of experimental sample solution 4 (containing 3 μM corticosterone + 0.0001% (w / v) mulberry root bark extract).

[0088] Each group of cells was continued to culture for 24 hours.

[0089] 4. Real-time fluorescent quantitative PCR (qRT-PCR) detection The culture medium in each well was aspirated and washed twice with PBS, and RNA was extracted from each well according to the instructions of the RNA extraction kit. Total RNA was reverse transcribed into cDNA using a reverse transcription kit, and the target mRNA expression level was detected on a real-time quantitative PCR instrument using a SYBR Green kit. GAPDH was used as an internal control, and the relative expression of the target gene mRNA was calculated according to 2-ΔΔCt. GraphPad prism6 software was used for plotting and testing the significance of COL-I and COL-III mRNA expression levels in each group of cells.

[0090] The results are shown in Figure 2 A and Figure 2As shown in FIG. B, the mRNA expression levels of COL-I and COL-III in HDF-a cells after treatment with corticosterone alone decreased significantly; compared with cells treated with corticosterone solution alone, the mRNA expression of collagen I in fibroblasts was significantly promoted by mulberry root bark extract at concentrations of 0.0001% (w / v) and 0.001% (w / v); compared with cells treated with corticosterone solution alone, the mRNA expression of collagen III in fibroblasts was significantly promoted by mulberry root bark extract at a concentration of 0.001% (w / v), which played an important role in delaying skin aging under stress.

[0091] Example 3: Mulberry Root Bark Extract Against Stress-Induced Skin Barrier Damage 1. Experimental materials and reagents The main materials and reagents used in this experiment include: mulberry root bark extract (same as Example 1), corticosterone (Sigma), dimethyl sulfoxide (DMSO, Sigma), human immortalized keratinocytes (HaCaT cells), DMEM medium (Hyclone), total RNA extraction kit (Japan TaKaRa), reverse transcription kit (Thermo Fisher, USA), SYBR Green qPCR premix reagent (Thermo Fisher, USA). The specific primers of keratin 1 (KRT1), keratin 10 (KRT10), filaggrin (FLG), loricrin (LOR) and the internal reference gene GAPDH were synthesized by Sheng Wu Bioengineering (Shanghai) Co., Ltd.

[0092] 2. Preparation of experimental sample solutions (1) Experimental sample solution 5: The appropriate amount of corticosterone and mulberry root bark extract powder was accurately weighed, dissolved in DMSO, and then serially diluted with DMEM medium to prepare a mixed solution containing 3 μM corticosterone and 0.001% (w / v) mulberry root bark extract.

[0093] (2) Experimental sample solution 6: The preparation method is the same as experimental sample solution 5, and a mixed solution containing 3 μM corticosterone and 0.0001% (w / v) mulberry root bark extract is finally obtained.

[0094] (3) Corticosterone solution (model group): The appropriate amount of corticosterone was accurately weighed, dissolved in DMSO, and then diluted with DMEM medium to prepare a corticosterone solution with a concentration of 3 μM.

[0095] (4) Control solution: DMEM medium was used to dilute DMSO, with a final concentration of 0.2% (v / v), consistent with all experimental groups.

[0096] 3. Cell culture and experimental grouping HaCaT cells were seeded in 6-well cell culture plates at a density of 4 x 10 5 cells / well, with 2 mL of complete DMEM medium added to each well, and incubated in a 37°C, 5% CO2 incubator for 24 hours. When the cell confluence reached 70-80%, the experimental grouping was performed: Untreated group (normal group): The original culture medium was aspirated and replaced with an equal volume of control solution.

[0097] Model group (corticosterone group): The original culture medium was aspirated and replaced with an equal volume of 3 μM corticosterone solution.

[0098] Experimental group 5 (corticosterone + 0.001% mulberry root bark): The original culture medium was aspirated and replaced with an equal volume of experimental sample solution 5 (containing 3 μM corticosterone + 0.001% (w / v) mulberry root bark extract).

[0099] Experimental group 6 (corticosterone + 0.0001% mulberry root bark): The original culture medium was aspirated and replaced with an equal volume of experimental sample solution 6 (containing 3 μM corticosterone + 0.0001% (w / v) mulberry root bark extract).

[0100] Each group of cells was continued to be cultured for 24 hours.

[0101] 4. Real-time fluorescent quantitative PCR (qRT-PCR) detection The culture medium in each well was aspirated and washed twice with PBS, and RNA was extracted from each well according to the instructions of the RNA extraction kit. Total RNA was reverse transcribed into cDNA using a reverse transcription kit, and the target mRNA expression level was detected on a real-time quantitative PCR instrument using a SYBR Green kit. GAPDH was used as an internal control, and the relative expression of the target gene mRNA was calculated according to 2-ΔΔCt. GraphPad prism6 software was used for plotting and testing the significance of Keratin1, Keratin10, FLG, and LOR mRNA expression levels in each group of cells.

[0102] The results are shown in Table 1. Figure 3As shown in FIG. 3A, 3B, 3C, 3D, the mRNA expression levels of Keratin1, Keratin10, FLG, and LOR in the model group (corticosterone group) of HaCaT cells were significantly decreased. Compared with the cells treated with corticosterone solution alone, the mulberry root bark extract at a concentration of 0.001% (w / v) could significantly promote the expression of Keratin1, Keratin10, FLG, and LOR mRNA in keratinocytes, and played an important role in skin repair under stress.

[0103] Example 4: Effect of mulberry root bark extract on resisting stress-induced weakening of skin basement membrane 1. Experimental materials and reagents The main materials and reagents used in this experiment include: mulberry root bark extract (same as Example 1), corticosterone (Sigma), dimethyl sulfoxide (DMSO, Sigma), human immortalized keratinocytes (HaCaT cells), DMEM medium (Hyclone), total RNA extraction kit (Japan TaKaRa), reverse transcription kit (Thermo Fisher, USA), SYBR Green qPCR premix reagent (Thermo Fisher, USA). The specific primers of collagen type IV, collagen type VII, collagen type XVII and the internal reference gene GAPDH were synthesized by Shenguo Bioengineering (Shanghai) Co., Ltd.

[0104] 2. Preparation of experimental sample solutions (1) Experimental sample solution 7: The appropriate amount of corticosterone and mulberry root bark extract powder was accurately weighed and dissolved in DMSO, and then serially diluted with DMEM medium to prepare a mixed solution containing 3 μM corticosterone and 0.001% (w / v) mulberry root bark extract.

[0105] (2) Experimental sample solution 8: The preparation method is the same as that of experimental sample solution 7, and a mixed solution containing 3 μM corticosterone and 0.0001% (w / v) mulberry root bark extract is finally obtained.

[0106] (3) Corticosterone solution (model group): The appropriate amount of corticosterone was accurately weighed, dissolved in DMSO, and then diluted with DMEM medium to prepare a corticosterone solution with a concentration of 3 μM.

[0107] (4) Control solution: DMSO was diluted with DMEM medium to a final concentration of 0.2% (v / v), consistent with all experimental groups.

[0108] 3. Cell culture and experimental grouping HaCaT cells were seeded in 6-well plates at a density of 4×105 The cells were seeded in 6-well cell culture plates at a density of 1.5 x 105 cells / well in 2 mL of complete DMEM medium and incubated at 37 °C in a 5% CO2 incubator for 24 hours. When the cells reached 70-80% confluence, they were divided into groups for experimental treatment: Untreated group (normal group): The original culture medium was removed and replaced with an equal volume of control solution. Model group (corticosterone group): The original culture medium was removed and replaced with an equal volume of 3 μM corticosterone solution.

[0109] Experimental group 7 (corticosterone + 0.001% mulberry root bark): The original culture medium was removed and replaced with an equal volume of experimental sample solution 7 containing 3 μM corticosterone + 0.001% (w / v) mulberry root bark extract.

[0110] Experimental group 8 (corticosterone + 0.0001% mulberry root bark): The original culture medium was removed and replaced with an equal volume of experimental sample solution 8 containing 3 μM corticosterone + 0.0001% (w / v) mulberry root bark extract. The cells in each group were continued to be cultured for 24 hours.

[0111] 4. Real-time fluorescent quantitative PCR (qRT-PCR) detection The culture medium in each well was removed and washed twice with PBS. RNA was extracted from each well according to the instructions of the RNA extraction kit. Total RNA was reverse transcribed into cDNA using a reverse transcription kit, and the target mRNA expression level was detected on a real-time quantitative PCR instrument using a SYBR Green kit. GAPDH was used as an internal reference, and the relative expression of the target gene mRNA was calculated according to 2-ΔΔCt. GraphPad prism6 software was used for plotting and testing the significance of COL-4, COL-7, and COL-17 mRNA expression levels in cells among the groups.

[0112] The results are shown in Figures Figure 4 As shown in Figures A, 4B, and 4C, the expression levels of COL-4, COL-7, and COL-17 mRNA in HaCaT cells in the model group (corticosterone group) were significantly decreased. Compared with cells treated only with corticosterone solution, 0.001% (w / v) mulberry root bark extract significantly promoted the expression of COL-4, COL-7, and COL-17 mRNA, indicating that 0.001% (w / v) mulberry root bark extract can significantly counteract the weakening of the basement membrane caused by stress and effectively antagonize the inhibition of the expression of basement membrane components in human keratinocytes induced by corticosterone, significantly up-regulating the mRNA expression levels of type IV collagen, type VII collagen, and type XVII collagen.

[0113] In addition, the mulberry root-bark extract prepared in Examples 1 to 4 (final extract concentration 0.001% (w / v)) was used to treat cells (e.g., HaCaT cells) that were not induced by corticosterone to simulate stress, and the results showed that the mulberry root-bark extract prepared in Example 1 did not significantly improve the expression of skin collagen-related genes (Collagen Type I and Collagen Type III), skin barrier-related genes (Keratin 1, Keratin 10, Filaggrin, and Loricrin), and skin basement membrane weakening-related genes (Collagen-IV, Collagen-VII, and Collagen-XVII) in cells (e.g., HaCaT cells) that were not induced by corticosterone. Thus, it was further confirmed that the mulberry root-bark extract selectively and significantly improved skin barrier function damage, skin basement membrane weakening, and skin collagen loss caused by the imbalance of cortisol under stress. The mulberry root-bark extract can selectively and effectively alleviate skin barrier damage, skin sensitivity, wrinkle formation, and skin elasticity decline caused by emotional stress.

[0114] The above description is merely a specific implementation of the present application, and those skilled in the art can clearly understand that, for the convenience and brevity of description, the specific working process described above can refer to the corresponding process in the foregoing method embodiments, which will not be described again here. It should be understood that the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of various equivalent modifications or replacements within the technical scope disclosed in the present application, and these modifications or replacements should be covered in the protection scope of the present application.

Claims

1. Use of a mulberry root bark extract in the preparation of a product for improving skin cortisol imbalance caused by stress.

2. Use according to claim 1, characterized in that, The improvement of the skin cortisol imbalance caused by stress includes prevention or alleviation of skin problems associated with cortisol imbalance caused by stress.

3. Use according to claim 2, characterized in that, The skin problems include at least one of skin aging caused by long-term stress, skin barrier damage, skin basement membrane weakening, skin sensitivity, skin discoloration, skin moisture retention capacity reduction, oil control capacity reduction, acne, skin inflammation, tired skin appearance, skin ulcer, skin petechiae, increased capillary fragility, and difficult healing of skin wounds.

4. Use according to claim 1, characterized in that, The cortisol imbalance includes at least one of increased cortisol content in the skin and enhanced cortisol activity in the skin.

5. Use according to claim 1, characterized in that, The mulberry root bark extract can inhibit the conversion of cortisone to cortisol.

6. Use according to claim 1, characterized in that, The preparation method of the mulberry root bark extract includes at least one of solvent extraction, supercritical extraction, and ultrasonic extraction. The mulberry root bark extract includes at least one of mulberry root bark glycerol extract, mulberry root bark water extract, mulberry root bark alcohol extract, and mulberry root bark ester extract.

7. Use according to claim 1, characterized in that, The product includes at least one of a pharmaceutical product and a cosmetic product.

8. Use according to claim 1, characterized in that, The dosage form of the product includes at least one of a cream, a liquid, a gel, a spray, an aerosol, a film, and a lyophilizate.

9. Use according to claim 1, characterized in that, The concentration of the mulberry root bark extract in the product is greater than or equal to 0.001% (w / v).

10. Use according to claim 1, characterized in that, The product further includes at least one of a flavoring agent, a dye, an emulsifying agent, a stabilizer, a lubricant, a solvent, a humectant, a softener, a wetting agent, a film-forming agent, a UV absorber, an essential oil, a vitamin, a trace metal, an anti-irritant, an antimicrobial agent, an antioxidant, a chelating agent, a preservative, a pH adjuster, a skin conditioner, a thickening agent, and a silicone compound.

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