A keratinocyte function protective agent and its preparation method and application

Abstract

The present invention discloses a keratinocyte function protective agent and its preparation method and application. The raw materials for preparing the protective agent include raw medicinal materials and Agaricus bisporus; wherein: the raw medicinal materials include the following components in mass percentage: 15%-40% wild chrysanthemum, 15%-40% orientalis leaves, 15%-40% sophora flavescens and 10%-35% liquorice; the keratinocyte function protective agent is obtained by mixing the raw medicinal material extract and the Agaricus bisporus extract, or by compound fermentation of the raw medicinal material and the Agaricus bisporus strain, and then extracting with water. The keratinocyte function protective agent of the present invention is applied to skin external preparations, can repair keratinocyte damage caused by SDS, and effectively promote the expression of cell ELOVL1mRNA.

Description

Technical Field

[0001] The invention belongs to the technical field of natural pharmaceutical chemistry, and in particular relates to a keratinocyte function protective agent and a preparation method and application thereof. Background Art

[0002] The skin barrier is the basis of many important physiological functions of the skin. The epidermis is the outermost layer of the skin barrier and is crucial to the skin barrier function. Keratinocytes are the core component of the epidermis. Keratinocytes continuously proliferate, differentiate, and migrate to form the epidermis. Keratinocytes can be divided into the basal layer, spinous layer, granular layer, and stratum corneum according to their stages of proliferation and differentiation.

[0003] The stratum corneum is the first line of defense of the skin and the physical barrier of the skin. Due to its structural characteristics, it is figuratively likened to a "brick wall structure". The permeability function of the stratum corneum plays a key role in skin moisturizing. The structural composition of the stratum corneum can be summarized into three categories: natural moisturizing factors, intercellular lipids, and stratum corneum cells. Intercellular lipids are mainly dispersed between stratum corneum cells as cells grow and metabolize, and eventually distributed to the skin surface as stratum corneum cells disintegrate.

[0004] The spinous layer of the epidermis will form an organelle: the lamellar body, which carries a variety of lipid precursors. As the spinous layer cells continue to ascend and differentiate, the middle lamellar bodies are formed in the granular layer cells, and gradually move to the periphery of the cells and finally fuse with the cell membrane. After the cells migrate to the stratum corneum, the middle lamellar bodies that are completely fused with the cell membrane will expel lipid precursors and their related enzymes into the intercellular space in the form of exocytosis. On the one hand, the cells continue to differentiate, keratinize, and collapse, and finally form stratum corneum cells; on the other hand, the expelled lipid precursors are catalyzed by enzymes to generate lipid components such as ceramide, fatty acids and cholesterol. The lipid components are constantly squeezed to form a biological liquid crystal membrane structure of a multi-layered layer, so the intercellular lipids of the stratum corneum are formed. The intercellular lipids of the stratum corneum not only have the function of preventing the volatilization of skin moisture, but also can adhere to the stratum corneum cells, and together with the stratum corneum cells, they build a protective wall to reduce the loss of skin moisture and prevent the invasion of exogenous harmful substances.

[0005] The fatty acids in intercellular lipids are mainly long-chain fatty acids, and their expression is regulated by the elongase of very long chain fatty acids (ELOVL). ELOVL1 can use C16:0 saturated fatty acids or C18:1 unsaturated fatty acids as substrates for elongation to generate saturated or unsaturated long-chain fatty acids. Studies have shown that ELOVL1 plays a key role in the formation of myelin in the central nervous system and the skin barrier, and is one of the important factors for keratinocytes to maintain the balance of intercellular lipids.

[0006] Keratinocytes are susceptible to damage by surfactants. Surfactants enter the active epidermis, damage the keratinocyte membrane, reduce the vitality of keratinocytes, cause the release of a large number of inflammatory factors, cause skin inflammation, and even cause disordered expression of skin keratinocyte genes, such as abnormal differentiation and abnormal enzyme expression. Sodium dodecyl sulfate (SDS) is an anionic surfactant that is slightly toxic to the human body. It is used as an emulsifier and detergent with strong degreasing ability. It is often added to detergent products. Direct contact with SDS or SDS residues on clothing are irritating to the skin. Due to its strong cleaning ability and high irritation, long-term use will seriously reduce the skin's defense ability, lead to immature development of epidermal cells, and affect the differentiation and expression of keratinocytes.

[0007] Keratinocytes are also susceptible to oxidative damage due to conditions such as light, heat, and ultraviolet rays, leading to excess free radicals in the skin. Under the attack of free radicals, the DNA chains of skin cells are prone to structural damage, resulting in abnormal expression, causing oxidative damage to keratinocytes, weakening their functions, and causing a decrease in their expression of various barrier-related enzymes. Among them, the decreased expression of ELOVL1 can cause the lipids of the stratum corneum cells to fail to form an ordered structure, which in turn leads to a loose structure of the cell lipids, reducing their adhesion to the stratum corneum cells, and ultimately causing the epidermis to loosen in structure and be unable to resist external stimuli.

[0008] Therefore, it is very important to develop agents that protect keratinocyte function to repair the skin.

[0009] my country's traditional Chinese medicine culture has a long history and abundant raw material resources. With the development of modern biotechnology, the combination of traditional Chinese medicine technology and modern biotechnology has produced a series of excellent drugs. It is necessary to apply the combination of the two to skin topical preparations and develop skin topical preparations that have a protective effect on keratinocytes and their functions. Summary of the invention

[0010] The first object of the present invention is to provide a keratinocyte function protective agent. The second object of the present invention is to provide a method for preparing a keratinocyte function protective agent. The third object of the present invention is to provide the use of the above-mentioned keratinocyte function protective agent in a skin external preparation (e.g., a drug for repairing keratinocyte damage caused by SDS and promoting the expression of cell ELOVL1 mRNA or a drug for promoting the expression of cell ELOVL1 mRNA).

[0011] To achieve the above object, the present invention adopts the following technical solution:

[0012] As a first aspect of the present invention, the present invention provides a keratinocyte function protective agent, and the raw materials for preparing the protective agent include raw medicinal materials and Agaricus bisporus; wherein: the raw medicinal materials include the following components in mass percentage: 15%-40% of wild chrysanthemum, 15%-40% of Platycladus orientalis leaves, 15%-40% of Sophora flavescens and 10%-35% of Licorice;

[0013] For example, the extract of wild chrysanthemum, the extract of Platycladus orientalis, the extract of Sophora flavescens and the extract of Licorice are mixed with the extract of Agaricus bisporus mycelium. Specifically, the method for preparing the keratinocyte function protective agent comprises the following steps:

[0014] (1): Weigh the raw medicinal materials according to the mass percentage, add water 10-20 times the total mass of the raw medicinal materials, extract at 105° C.-115° C. in a high-pressure extraction tank for 1.5-3 h, filter until clear, and take the small molecule filtrate less than 3000 Da to obtain component A;

[0015] (2): taking Agaricus bisporus mycelium in the logarithmic growth period of fermentation, adding water 3-5 times the mass of the Agaricus bisporus mycelium, subjecting the mycelium to cell disruption, centrifuging, removing protein from the supernatant, filtering, taking a small molecule filtrate portion less than 3000 Da, concentrating and freeze-drying, and obtaining component B;

[0016] (3): adding 0.01%-1% of component B by weight of component A to component A, and adding water to make the volume 5-20 times the total weight of the raw medicinal materials to obtain a keratinocyte function protective agent; or, concentrating and freeze-drying the component A, and adding 0.1%-10% of component B by weight of the total weight of the raw medicinal materials, and mixing evenly to obtain a keratinocyte function protective agent;

[0017] Alternatively, wild chrysanthemum, arborvitae leaves, sophora flavescens and liquorice are co-fermented with Agaricus bisporus mycelium to prepare a keratinocyte function protective agent. Specifically, the method for preparing the keratinocyte function protective agent comprises the following steps:

[0018] (a): Weigh the raw medicinal materials according to the mass percentage, add 3-5 times the total mass of the raw medicinal materials with water, and cool to room temperature after sterilization;

[0019] (b): 3-8 ml of liquid Agaricus bisporus spawn is inoculated per 100 g of raw medicinal materials, fermented and cultured for 3-10 days, water is added to make the volume 10-20 times of the total mass of the raw medicinal materials, and the mixture is subjected to slight boiling reflux extraction for 1.5-3 h, then filtered to clarify, and the small molecule filtrate portion less than 3000 Da is taken, concentrated and freeze-dried to obtain a keratinocyte function protective agent; alternatively, the small molecule filtrate portion less than 3000 Da is taken, water is added to make the volume 5-20 times of the total mass of the raw medicinal materials to obtain a keratinocyte function protective agent.

[0020] Preferably, the raw medicinal materials include the following components in percentage by mass: 20%-35% of wild chrysanthemum, 20%-35% of oriental arborvitae leaves, 20%-35% of sophora flavescens and 15%-30% of liquorice.

[0021] According to a specific embodiment of the present invention, the raw medicinal material is composed of the following components in percentage by mass:

[0022] 35% wild chrysanthemum, 25% oriental arborvitae, 25% sophora flavescens and 15% liquorice;

[0023] 20% wild chrysanthemum, 30% oriental arborvitae leaves, 30% sophora flavescens and 20% liquorice;

[0024] 22% wild chrysanthemum, 20% oriental arborvitae leaves, 30% sophora flavescens and 28% liquorice.

[0025] Preferably, the keratinocyte function protective agent also contains an appropriate amount of preservatives. Those skilled in the art can select the appropriate type and content of preservatives according to actual needs.

[0026] In other examples, a suitable amount of preservative may be added to the component A; or a suitable amount of preservative may be added to the small molecule filtrate prepared in step (b). The preservative may be at least one of the ingredients that can be used for preservation in daily cosmetics such as 1,2-hexanediol and p-hydroxyacetophenone.

[0027] Preferably, the preservative is 1,2-hexanediol and / or p-hydroxyacetophenone. The added mass of the 1,2-hexanediol is 0.5-3%, preferably 0.5%; the added mass of the p-hydroxyacetophenone is 0.1-1%, preferably 0.5%.

[0028] As a second aspect of the present invention, the present invention provides a method for preparing the keratinocyte function protective agent described in any one of the above, comprising the following steps:

[0029] (1): Weigh the raw medicinal materials according to the mass percentage, add water 10-20 times the total mass of the raw medicinal materials, extract at 105° C.-115° C. in a high-pressure extraction tank for 1.5-3 h, filter until clear, and take the small molecule filtrate less than 3000 Da to obtain component A;

[0030] (2): taking Agaricus bisporus mycelium in the logarithmic growth period of fermentation, adding water 3-5 times the mass of the Agaricus bisporus mycelium, subjecting the mycelium to cell disruption, centrifuging, removing protein from the supernatant, filtering, taking a small molecule filtrate portion less than 3000 Da, concentrating and freeze-drying, and obtaining component B;

[0031] (3): adding 0.01%-1% of component B by weight of component A to component A, and adding water to make the volume 5-20 times the total weight of the raw medicinal materials to obtain a keratinocyte function protective agent; or, concentrating and freeze-drying the component A, and adding 0.1%-10% of component B by weight of the total weight of the raw medicinal materials, and mixing evenly to obtain a keratinocyte function protective agent;

[0032] According to a specific embodiment of the present invention, in step (1), the extraction time is 2 hours; in steps (1) and (2), the extract is filtered until clarified, filtered through a 2000Da molecular retention membrane, and the small molecule filtrate portion is taken.

[0033] In other embodiments of the present invention, in step (3), 0.5% of 1,2-hexanediol, 0.5% of p-hydroxyacetophenone and 1% of component B are added to component A, respectively, and after mixing, water is added to make the volume 10-15 times the total mass of the raw medicinal materials to obtain a keratinocyte function protective agent.

[0034] In another specific example of the present invention, in step (2), the small molecule filtrate is freeze-dried to obtain component A; in step (3), 0.05% of component B is added to component A, and the mixture is mixed evenly to obtain a keratinocyte function protectant.

[0035] In other embodiments of the present invention, a keratinocyte function protectant is prepared by co-fermenting wild chrysanthemum, arborvitae leaves, sophora flavescens and liquorice with Agaricus bisporus mycelium. Specifically, the method for preparing the keratinocyte function protectant comprises the following steps:

[0036] (a): Weigh the raw medicinal materials according to the above mass percentage, add 3-5 times the total mass of water of the raw medicinal materials, sterilize and cool to room temperature;

[0037] (b): 3-8 ml of liquid Agaricus bisporus spawn is inoculated per 100 g of raw medicinal materials, fermented and cultured for 3-10 days, water is added to make the volume 10-20 times of the total mass of the raw medicinal materials, and the mixture is subjected to slight boiling reflux extraction for 1.5-3 h, then filtered to clarify, and the small molecule filtrate portion less than 3000 Da is taken, concentrated and freeze-dried to obtain a keratinocyte function protective agent; alternatively, the small molecule filtrate portion less than 3000 Da is taken, water is added to make the volume 5-20 times of the total mass of the raw medicinal materials to obtain a keratinocyte function protective agent.

[0038] Preferably, in step (a), the raw medicinal materials include the following components in percentage by mass: 20%-35% of wild chrysanthemum, 20%-35% of Platycladus orientalis leaves, 20%-35% of Sophora flavescens and 15%-30% of Licorice; in step (b), the fermentation culture time is 7-9 days.

[0039] In a specific embodiment of the present invention, in the step (a), water in an amount of about 4 times the total mass of the raw medicinal material is added; in the step (b), the fermentation culture time is 8 days, the water is added to make the volume 16 times the total mass of the raw medicinal material, and the micro-boiling reflux extraction time is 2 hours; the extract is filtered to clarify, filtered through a 2000Da molecular retention membrane, and the small molecule filtrate portion is taken.

[0040] According to some specific examples of the present invention, the method for preparing the keratinocyte function protective agent comprises the following steps:

[0041] (1) Accurately weigh 22% wild chrysanthemum, 20% oriental arborvitae leaves, 30% sophora flavescens, and 28% liquorice by weight, and mix them evenly after coarsely chopping them;

[0042] (2) adding water about 4 times the total mass of the raw medicinal materials, cooling to room temperature after sterilization, inoculating 5 ml or 8 ml of Agaricus bisporus liquid spawn per 100 g of the raw medicinal materials, and fermenting and culturing for 8 days;

[0043] (3) Add water about 16 times the total mass of the raw medicinal materials and reflux for extraction for 2 h;

[0044] (4) After removing the drug residue, the liquid portion is filtered until it is clarified, filtered through a 2000 Da molecular cutoff membrane, and the small molecule filtrate portion is taken, concentrated and freeze-dried to obtain a keratinocyte function protective agent.

[0045] As a third aspect of the present invention, there is provided a keratinocyte function-protecting agent as described in any one of the above items, and use of the keratinocyte function-protecting agent prepared by any one of the preparation methods in a skin external preparation.

[0046] Preferably, there is provided a use of any one of the above-mentioned keratinocyte function protectors in the preparation of a medicament for repairing keratinocyte damage caused by SDS.

[0047] Preferably, there is provided a use of any one of the above-mentioned keratinocyte function-protecting agents in a drug for promoting the expression of cellular ELOVL1 mRNA.

[0048] The keratinocyte function protecting agent of the present invention and its preparation method and application have the following beneficial effects:

[0049] The keratinocyte function protective agent of the present invention is prepared by extracting and preparing natural plant medicinal materials wild chrysanthemum, arborvitae leaf, sophora flavescens and liquorice and Agaricus bisporus using modern biotechnology methods as raw materials. Compared with wild chrysanthemum extract, arborvitae leaf extract, sophora flavescens extract and liquorice extract, and individual components A and B, the keratinocyte function protective agent of the present invention can significantly improve the repair effect of keratinocyte damage caused by SDS, and can effectively promote the expression of cell ELOVL1 mRNA, and is a very effective skin topical preparation.

[0050] The preparation method of the keratinocyte function protective agent of the present invention is obtained by mixing the water extracts of wild chrysanthemum, arborvitae leaves, sophora flavescens and liquorice with the mycelium extract of Agaricus bisporus, or by compound fermentation of wild chrysanthemum, arborvitae leaves, sophora flavescens and liquorice with Agaricus bisporus strains, and then extracting with water. The obtained keratinocyte function protective agent can significantly improve the repair effect of keratinocyte damage caused by SDS, and can effectively promote the expression of ELOVL1 mRNA. The extraction effect of the latter method is better. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The figure is a diagram showing the improvement of the growth rate of SDS-damaged cells by each sample of the embodiment and the comparative example;

[0052] Figure 2 The graph shows the effect of each sample of the embodiment and comparative example on the expression level of ELOVL-1 mRNA in damaged keratinocytes. DETAILED DESCRIPTION

[0053] In order to enable those skilled in the art to more fully understand the technical solution of the present invention, exemplary embodiments of the present invention will be described in more comprehensive and detailed manner below in conjunction with the accompanying drawings.

[0054] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0055] Preparation method of Agaricus bisporus liquid spawn:

[0056] 1. Experimental Reagents

[0057] (1) Experimental strains: Agaricus bisporus 2796 strain was provided by the Experimental Strain Center of Huazhong Agricultural University.

[0058] (2) Culture medium formula:

[0059] Potato dextrose agar (PDA): 200 g potato, 20 g glucose, 20 g agar, 1000 mL water, natural pH, used for slant culture of bacterial strains.

[0060] (3) Primary liquid seed culture medium: 30 g corn starch, 20 g sucrose, 5 g yeast powder, 1 g MgSO4·7H2O, 1.5 g KH2PO4, 0.01 g VB1, 1000 mL water, natural pH.

[0061] (4) Secondary liquid seed culture medium: 31.7 g corn starch, 11.3 g soybean meal, 3.5 g MgSO4·7H2O, 2.5 g KH2PO4, 7.5 g CaCl2, 6.5 g NaCl, and 1000 mL water.

[0062] 2. Cultivation Methods

[0063] (1) Activation of bacterial strains: Cut out 1 cm thick pieces of bacterial strains from the stored slant test tubes. 2 The bacterial blocks of different sizes were inoculated in the middle of the PDA slope and cultured at 25°C for 25 days.

[0064] (2) Primary liquid seed culture: Divide the activated mother seed into approximately 2 cm 2 Inoculate the bacterial blocks of different sizes into the liquid culture medium. Put two bacterial blocks into a 250 mL Erlenmeyer flask, let it stand at 25 °C for 48 h, and then culture it with shaking for 7 days to obtain the first-level liquid seed (shake flask inoculation).

[0065] (3) Secondary liquid seed culture: After the shake flask culture was left to stand at 25°C for 48 hours, it was homogenized with a homogenizer. A 250 mL Erlenmeyer flask was filled with 100 mL of the secondary liquid seed culture medium, and 10% of the primary liquid seeds were inoculated. The culture was shaken on a shaker at 25°C and 150 r / min for 14 days to obtain the Agaricus bisporus liquid culture.

[0066] Example 1

[0067] 1. Preparation of component A:

[0068] (1) Weigh 35 g wild chrysanthemum, 25 g oriental arborvitae leaves, 25 g sophora flavescens and 15 g liquorice, a total of 100 g raw materials;

[0069] (2) Add 1000 g of water to the above raw materials and extract at 105° C. for 2 h in a high-pressure extraction tank;

[0070] (3) Remove the drug residue, filter the liquid portion until it is clear, and then filter it through a 2000Da molecular cutoff membrane to take the small molecule filtrate portion to obtain component A.

[0071] 2. Preparation of component B:

[0072] Take 1 kg of Agaricus bisporus mycelium in the logarithmic growth period of fermentation, add 3 kg of water, crush with a high-pressure homogenizer cell crusher, centrifuge to obtain the supernatant, deproteinize, filter with a 2000 Da molecular retention membrane, collect the small molecule filtrate part, concentrate and freeze-dry to obtain component B.

[0073] 3. Preparation of keratinocyte function protective agent:

[0074] 0.5% of 1,2-hexanediol, 0.5% of p-hydroxyacetophenone, and 0.1% of component B were added to component A, and water was added to make the volume to 1500 g to obtain a keratinocyte function protective agent.

[0075] Example 2

[0076] 1. Preparation of component A:

[0077] (1) Weigh 20 g wild chrysanthemum, 30 g oriental arborvitae leaves, 30 g sophora flavescens and 20 g liquorice, a total of 100 g raw materials;

[0078] (2) Add 1500 g of water to the above raw materials and extract at 110° C. for 2 h in a high-pressure extraction tank;

[0079] (3) Remove the drug residue, filter the liquid part until it is clear, and then filter it through a 2000 Da molecular cutoff membrane. Take the small molecule filtrate part, freeze-dry it, and obtain component A, which weighs 19 g.

[0080] 2. Preparation of component B:

[0081] Take 1 kg of Agaricus bisporus mycelium in the logarithmic growth period of fermentation, add 4 kg of water, crush with a high-pressure homogenizer cell crusher, centrifuge to obtain the supernatant, deproteinize, filter with a 2000 Da molecular retention membrane, collect the small molecule filtrate part, concentrate and freeze-dry to obtain component B.

[0082] 3. Add 1g of component B to component A and mix well to obtain a keratinocyte function protective agent weighing 20g.

[0083] Example 3

[0084] 1. Preparation of component A:

[0085] (1) Weigh 22 g wild chrysanthemum, 20 g oriental arborvitae leaves, 30 g sophora flavescens and 28 g liquorice, a total of 100 g raw materials;

[0086] (2) Add 2000 g of water to the above raw materials and extract at 115° C. for 2 h in a high-pressure extraction tank;

[0087] (3) Remove the drug residue, filter the liquid part until it is clear, filter it through a 2000Da molecular cutoff membrane, take the small molecule filtrate part, and obtain component A, totaling 800g.

[0088] 2. Preparation of component B:

[0089] Take 1kg of Agaricus bisporus mycelium in the logarithmic growth period of fermentation, add 5kg of water, crush with a high-pressure homogenizer cell crusher, centrifuge to obtain the supernatant, deproteinize, filter with a 2000Da molecular cutoff membrane, collect the small molecule filtrate part, concentrate and freeze-dry to obtain component B.

[0090] 3. Add 0.5% of 1,2-hexanediol, 0.5% of p-hydroxyacetophenone and 1% of component B to component A, mix well and add water to make the volume to 1000 g to obtain a keratinocyte function protective agent.

[0091] Example 4

[0092] (1) Weigh 22 g wild chrysanthemum, 20 g oriental arborvitae leaves, 30 g sophora flavescens and 28 g liquorice, a total of 100 g of raw materials, chop them roughly and mix them evenly;

[0093] (2) adding 400 g of water, cooling to room temperature after sterilization, inoculating 5 mL of Agaricus bisporus liquid culture, and fermenting for 8 days;

[0094] (3) Add 1600 g of water and extract under low boiling reflux for 2 h;

[0095] (4) After removing the drug residue, the liquid portion was filtered until clarified, filtered through a 2000 Da molecular cutoff membrane, and the small molecule filtrate portion was concentrated and freeze-dried to obtain a keratinocyte function protective agent weighing 21 g.

[0096] Example 5

[0097] (1) Weigh 22 g wild chrysanthemum, 20 g oriental arborvitae leaves, 30 g sophora flavescens and 28 g liquorice, a total of 100 g of raw materials, chop them roughly and mix them evenly;

[0098] (2) adding 400 g of water, cooling to room temperature after sterilization, inoculating 8 mL of Agaricus bisporus liquid culture, and fermenting for 8 days;

[0099] (3) Add 1600 g of water and extract under low boiling reflux for 2 h;

[0100] (4) After removing the drug residue, the liquid portion was filtered until it was clarified, filtered through a 2000 Da molecular cutoff membrane, and the small molecule filtrate portion was concentrated and freeze-dried to obtain a keratinocyte function protective agent weighing 22 g.

[0101] Comparative Example 1

[0102] (1) Weigh 100 g wild chrysanthemum;

[0103] (2) Add 2000 g of water and extract in a high pressure extraction tank at 115 °C for 2 h;

[0104] (3) After removing the drug residue, the liquid portion was filtered until it was clear, filtered through a 2000 Da molecular cutoff membrane, and the small molecule filtrate portion was taken and freeze-dried to obtain a wild chrysanthemum extract weighing 19 g.

[0105] Comparative Example 2

[0106] (1) Weigh 100 g of Platycladus orientalis leaves;

[0107] (2) Add 2000 g of water and extract in a high pressure extraction tank at 115 °C for 2 h;

[0108] (3) After removing the drug residue, the liquid portion was filtered until it was clear, filtered through a 2000 Da molecular cutoff membrane, and the small molecule filtrate portion was taken and freeze-dried to obtain the Platycladus orientalis leaf extract, weighing 20 g.

[0109] Comparative Example 3

[0110] (1) Weigh 100 g of Sophora flavescens;

[0111] (2) Add 2000 g of water and extract in a high pressure extraction tank at 115 °C for 2 h;

[0112] (3) After removing the drug residue, the liquid portion was filtered until it was clear, filtered through a 2000 Da molecular cutoff membrane, and the small molecule filtrate portion was taken and freeze-dried to obtain the Sophora flavescens extract, weighing 22 g.

[0113] Comparative Example 4

[0114] (1) Weigh 100 g of licorice;

[0115] (2) Add 2000 g of water and extract in a high pressure extraction tank at 115 °C for 2 h;

[0116] (3) After removing the drug residue, the liquid portion was filtered until it was clear, filtered through a 2000 Da molecular cutoff membrane, and the small molecule filtrate portion was taken and freeze-dried to obtain a licorice extract weighing 18 g.

[0117] Effect Example 1 - Verification of the protective effect on keratinocytes

[0118] 1. Experimental reagents and instruments

[0119] T2 cell culture flasks and 96-well plates were purchased from Corning;

[0120] The cell counting kit (CCK-8) was purchased from Dojindo Molecular Technologies;

[0121] Phosphate buffered saline (PBS, 1×) was purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.;

[0122] Fetal bovine serum FBS, DMEM culture medium, trypsin and SDS, etc.

[0123] 2. Sample preparation

[0124] (1) Example and comparative example group: Take 1 / 1000 of the total amount of the product prepared in each of the above examples and comparative examples, and dilute it to 100 g with DMEM medium;

[0125] (2) Group A of Example 3: 1 / 1000 of the total amount of Component A prepared in Example 3 was taken and diluted to 100 g with DMEM medium;

[0126] Example 3 Group B: The B component prepared in Example 3 was diluted to 0.01% using DMEM medium. Specifically, 0.01 g of the B component prepared in Example 3 was diluted to 100 g using DMEM medium.

[0127] The above dilution method can ensure that each group of samples after dilution contains products made from medicinal raw materials of equal quality. Specifically, it is equivalent to that 100g of sample after dilution contains products made from 0.1g of medicinal raw materials.

[0128] The specific preparation method of each group is shown in Table 1.

[0129] Table 1 Sample preparation table of each group of embodiments and comparative examples

[0130]

[0131]

[0132] 3. Cell lines and cell culture

[0133] (1) Cell line: Human immortalized keratinocytes HaCaT, obtained from Shanghai Dermatology Hospital;

[0134] (2) Culture medium: DMEM + 10% FBS;

[0135] (3) Culture conditions: Cultured in a cell culture incubator (5% CO2, 37°C).

[0136] 4. Test on the effect of SDS-induced HaCaT cell injury model

[0137] (1) 10,000 HaCaT cells were seeded in each well of a 96-well plate and grown under the above culture conditions for 24 h, followed by aspiration of the supernatant;

[0138] (2) Add 100 μL of 80 μg / mL SDS to induce HaCaT cell damage, and discard the supernatant after incubation for 24 h;

[0139] (3) 10 μL of each sample prepared in each example and comparative example and diluted with DMEM medium was added, and the mixture was incubated for another 24 h before detection using a CCK-8 kit.

[0140] A PBS group, a blank group, and a model group were set up for control, wherein no cells were inoculated in the PBS wells; the blank group was inoculated with cells and no SDS was used to induce HaCaT cell damage; the model group was inoculated with cells and SDS was used to induce HaCaT cell damage, but no sample was used for treatment; each embodiment and comparative example group was required to add the corresponding sample and incubate again after SDS was used to induce cell damage. At least three replicate wells were set up for each group.

[0141] 5. Test results

[0142] The absorbance was measured and the average value was calculated, and the cell growth rate was calculated relative to the blank group. The results are shown in Table 2 and Figure 1 shown.

[0143] Table 2 Effects on cell growth rate in SDS-induced HaCaT injury model

[0144]

[0145]

[0146] From Table 2 and Figure 1 It can be seen that the cell growth rates in Comparative Example 2 and Comparative Example 3 groups are similar to those in the model group, or even lower than those in the model group; the cell growth rates in Comparative Example 1 group, Comparative Example 4 group, and Example 3A group and Example 3B group are slightly higher than those in the model group; and the cell growth rates in Examples 1-5 are significantly increased relative to the model group.

[0147] This shows that the single Sophora flavescens extract and Platycladus orientalis leaf extract have no repair effect on SDS-induced HaCaT cell damage, the single licorice extract and wild chrysanthemum extract, as well as component A (i.e. a combination of wild chrysanthemum extract, licorice extract, Sophora flavescens extract and Platycladus orientalis leaf extract) and component B (Agaricus bisporus extract) have a slight repair effect on SDS-induced cell damage, but the keratinocyte function protector composed of component A and component B has a significantly improved repair effect on SDS-induced cell damage, and the repair effect is significantly stronger than the superimposed effect of component A and component B.

[0148] In addition, the cell growth rates of Examples 4 and 5 are significantly higher than those of Examples 1-3, indicating that the keratinocyte function protective agent prepared by co-fermenting wild chrysanthemum, licorice, sophora flavescens and Platycladus orientalis with Agaricus bisporus mycelium has a significantly stronger repair effect on damaged cells than the keratinocyte function protective agent prepared by preparing component A and component B separately and then mixing them.

[0149] Effect Example 2 - Effect on the expression of very long chain fatty acid elongase (ELOVL1) mRNA in keratinocytes

[0150] 1. Experimental reagents and instruments

[0151] (1) Reagents:

[0152] DMEM medium, fetal bovine serum, penicillin-streptomycin double antibody, Trypsin-EDTA (0.25%), and phenol red were purchased from Gibco-Thermo Fisher Scientific;

[0153] Phosphate buffered saline (PBS, 1×) was purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.;

[0154] (2) Test kit:

[0155] RNA extraction kit (model: 19231ES50) was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.;

[0156] Reverse transcription kit (model: AG11728) was purchased from Shanghai Lingrui Biotechnology Co., Ltd., and SYBRGreen Pro Taq HS premixed qPCR kit (model: AG11718) was purchased from Shanghai Lingrui Biotechnology Co., Ltd.;

[0157] (3) Instruments: pipette (purchased from eppendorf), CO2 incubator, clean bench, autoclave, high-speed centrifuge, refrigerator, DNA quantifier (model NanoDrop2000C, thermoscientific), Pro FlexTMBase PCR Systerm (model ProFlexPCR, Applied Biosystems), fluorescence quantitative PCR instrument (model QuantStudio 6Flex, purchased from ABI), microplate reader (model Epoch2, purchased from biotek), T25 cell culture flask, 6-well plate (Corning), etc.

[0158] 2. Sample preparation

[0159] The preparation method of each group of samples is consistent with that in Example 1, as shown in Table 1.

[0160] 3. Cell lines and cell culture

[0161] (1) Cell line: Human immortalized keratinocytes HaCaT, obtained from Shanghai Dermatology Hospital;

[0162] (2) Culture medium: DMEM medium + 10% FBS + 1% double antibody;

[0163] (3) Culture conditions: Cultured in a cell culture incubator (5% CO2, 37°C).

[0164] 4. Test on the effect of keratinocytes on the expression of very long chain fatty acid elongase (ELOVL1) mRNA

[0165] (1) 3 × 10 5 HaCaT cells were cultured for 24 h before adding each sample and cultured for another 6 h.

[0166] (2) Add the stimulator (t-BHP) at a final concentration of 200 μM and continue culturing for 2 h;

[0167] (3) Total RNA was extracted using an RNA extraction kit, cDNA was amplified using a reverse transcription kit and a qPCR kit, and then detected using a real-time fluorescence quantitative PCR detection system.

[0168] A blank group and a model group were set up for comparison, wherein, in the blank group, no sample was added after the cells were inoculated, and the cells were not treated with stimulants; in the model group, no sample was added after the cells were inoculated, but the cells were treated with stimulants.

[0169] Detection gene: ELOVL1. Primer list is shown in Table 3.

[0170] Table 3 Primer list

[0171]

[0172] 5. Test results

[0173] Real-time fluorescence quantitative PCR was used to detect the relative expression of each group. The results are shown in the attached figure. Figure 2 shown.

[0174] The relative expression level of ELOVL1 mRNA in the model group was significantly lower than that in the blank group, indicating that t-BHP oxidative damage significantly inhibited the expression of ELOVL1 mRNA in keratinocytes.

[0175] The relative mRNA expression levels of wild chrysanthemum extract (comparative example group 1) and Sophora flavescens extract (comparative example group 3) were lower than those of the model group, indicating that wild chrysanthemum extract and Sophora flavescens extract slightly aggravated the decrease in ELOVL1 mRNA expression caused by t-BHP oxidative damage.

[0176] The relative expression levels of ELOVL1 mRNA in Example 3A group (component A comprising wild chrysanthemum extract, Platycladus orientalis leaf extract, licorice extract and Sophora flavescens extract), Example 3B group (component B comprising Agaricus bisporus extract), Comparative Example 2 group (Platycladus orientalis leaf extract) and Comparative Example 4 group (licorice extract) were higher than those in the model group, indicating that the Platycladus orientalis leaf extract, licorice extract, component A and component B all slightly improved the decreased expression level of ELOVL1 mRNA caused by t-BHP oxidative damage.

[0177] The relative expression of mRNA in Examples 1-5 was significantly higher than that in the model group and each comparative example, and even significantly higher than that in the blank group, indicating that the keratinocyte function protectants in Examples 1-5 can effectively inhibit the decrease in ELOVL1 mRNA expression caused by t-BHP oxidative damage, and can promote the expression of ELOVL1 mRNA. In addition, the expression of ELOVL1 mRNA in Examples 4 and 5 was also higher than that in Examples 1-3, indicating that the keratinocyte function protectant prepared by co-fermenting wild chrysanthemum, licorice, sophora flavescens and Platycladus orientalis with Agaricus bisporus mycelium has a stronger promoting effect on the expression of ELOVL1 mRNA than the promoting effect of preparing component A and component B separately and then mixing them to prepare the keratinocyte function protectant.

[0178] The above description is only an example of the implementation mode of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A keratinocyte function protective agent, characterized in that: The raw materials for preparing the protective agent include raw medicinal materials and Agaricus bisporus; wherein: the mass percentage of each component of the raw medicinal materials is: 15%-40% of wild chrysanthemum, 15%-40% of Platycladus orientalis leaves, 15%-40% of Sophora flavescens and 10%-35% of Licorice; The method for preparing the keratinocyte function protective agent comprises the following steps: (1): Weigh the raw medicinal materials according to the mass percentage, add water 10-20 times the total mass of the raw medicinal materials, extract at 105°C-115°C in a high-pressure extraction tank for 1.5-3h, filter until clear, and take the small molecule filtrate less than 3000Da to obtain component A; (2): Take Agaricus bisporus mycelium in the logarithmic growth period of fermentation, add water 3-5 times the mass of the Agaricus bisporus mycelium, perform cell disruption treatment, centrifuge to obtain the supernatant to remove protein, and then filter, take the small molecule filtrate with a molecular weight less than 3000Da, concentrate and freeze-dry to obtain component B; (3): Adding 0.01%-1% of component B by weight of component A to component A, and adding water to make the volume 5-20 times the total weight of the raw medicinal materials to obtain a keratinocyte function protective agent; or, concentrating and freeze-drying the component A, and adding 0.1%-10% of component B by weight of the total weight of the raw medicinal materials, and mixing evenly to obtain a keratinocyte function protective agent; Or, comprising the steps of: (a): Weigh the raw medicinal materials according to the mass percentage, add 3-5 times the total mass of the raw medicinal materials with water, and cool to room temperature after sterilization; (b): 3-8 ml of liquid Agaricus bisporus spawn is inoculated per 100 g of raw medicinal materials, fermented and cultured for 3-10 days, water is added to make the volume 10-20 times of the total mass of the raw medicinal materials, and the mixture is subjected to slight boiling reflux extraction for 1.5-3 hours, then filtered to clarify, and the small molecule filtrate portion less than 3000 Da is taken, concentrated and freeze-dried to obtain a keratinocyte function protective agent; alternatively, the small molecule filtrate portion less than 3000 Da is taken, water is added to make the volume 5-20 times of the total mass of the raw medicinal materials to obtain a keratinocyte function protective agent.

2. The keratinocyte function protective agent according to claim 1, characterized in that The mass percentages of the components of the raw medicinal materials are: 20%-35% of wild chrysanthemum, 20%-35% of oriental arborvitae leaves, 20%-35% of sophora flavescens and 15%-30% of liquorice.

3. The keratinocyte function protective agent according to any one of claims 1 to 2, characterized in that The keratinocyte function protective agent also contains an appropriate amount of preservatives.

4. The method for preparing the keratinocyte function protective agent according to any one of claims 1 to 3, characterized in that: The steps include: (1): Weigh the raw medicinal materials according to the mass percentage, add water 10-20 times the total mass of the raw medicinal materials, extract at 105°C-115°C in a high-pressure extraction tank for 1.5-3h, filter until clear, and take the small molecule filtrate less than 3000Da to obtain component A; (2): Take Agaricus bisporus mycelium in the logarithmic growth period of fermentation, add water 3-5 times the mass of the Agaricus bisporus mycelium, perform cell disruption treatment, centrifuge to obtain the supernatant to remove protein, and then filter, take the small molecule filtrate with a molecular weight less than 3000Da, concentrate and freeze-dry to obtain component B; (3): Add 0.01%-1% of component B by weight of component A to component A, and add water to make the volume 5-20 times the total weight of the raw medicinal materials to obtain a keratinocyte function protective agent; or, concentrate and freeze-dry the component A, and add 0.1%-10% of component B by weight of the total weight of the raw medicinal materials, mix well, and obtain a keratinocyte function protective agent.

5. The method for preparing the keratinocyte function protective agent according to claim 4, characterized in that: In the step (1), the extraction time is 2 hours; in the steps (1) and (2), the extract is filtered until clarified, filtered through a 2000Da molecular cutoff membrane, and the small molecule filtrate portion is taken.

6. The method for preparing the keratinocyte function protective agent according to claim 4, characterized in that: In the step (3), 0.5% of 1,2-hexanediol, 0.5% of p-hydroxyacetophenone and 1% of component B are added to component A, mixed and then water is added to make the volume 10-15 times the total mass of the raw medicinal materials to obtain a keratinocyte function protective agent.

7. The method for preparing the keratinocyte function protective agent according to any one of claims 1 to 3, characterized in that: The steps include: (a): Weigh the raw medicinal materials according to the mass percentage, add 3-5 times the total mass of the raw medicinal materials with water, and cool to room temperature after sterilization; (b): 3-8 ml of liquid Agaricus bisporus spawn is inoculated per 100 g of raw medicinal materials, fermented and cultured for 3-10 days, water is added to make the volume 10-20 times of the total mass of the raw medicinal materials, and the mixture is subjected to slight boiling reflux extraction for 1.5-3 hours, then filtered to clarify, and the small molecule filtrate portion less than 3000 Da is taken, concentrated and freeze-dried to obtain a keratinocyte function protective agent; alternatively, the small molecule filtrate portion less than 3000 Da is taken, water is added to make the volume 5-20 times of the total mass of the raw medicinal materials to obtain a keratinocyte function protective agent.

8. The method for preparing the keratinocyte function protective agent according to claim 7, characterized in that: In the step (a), the mass percentages of the raw medicinal materials are: 20%-35% wild chrysanthemum, 20%-35% orientalis leaves, 20%-35% sophora flavescens and 15%-30% liquorice; in the step (b), the fermentation culture time is 7-9 days.

9. The method for preparing the keratinocyte function protective agent according to claim 7, characterized in that: In the step (a), water of about 4 times the total mass of the raw medicinal materials is added; in the step (b), the fermentation culture time is 8 days, the water is added to make the volume 16 times the total mass of the raw medicinal materials, and the micro-boiling reflux extraction time is 2 hours; the extract is filtered to clarify, filtered through a 2000Da molecular retention membrane, and the small molecule filtrate portion is taken.

10. The method for preparing the keratinocyte function protective agent according to any one of claims 7 to 9, characterized in that: The steps include: (1) Weigh 22% wild chrysanthemum, 20% oriental arborvitae leaves, 30% sophora flavescens, and 28% liquorice by weight, chop them roughly, and mix them evenly; (2) Add water about 4 times the total mass of the raw medicinal materials, cool to room temperature after sterilization, inoculate 5 ml or 8 ml of Agaricus bisporus liquid culture per 100 g of the raw medicinal materials, and ferment for 8 days; (3) Add water about 16 times the total mass of the raw medicinal materials and reflux for extraction at a low boiling temperature for 2 h; (4) After removing the drug residue, the liquid portion is filtered until it is clear, filtered through a 2000Da molecular cutoff membrane, and the small molecule filtrate portion is taken. After concentration and freeze-drying, a keratinocyte function protectant is obtained.

11. Use of the keratinocyte function-protecting agent according to any one of claims 1 to 3 or the keratinocyte function-protecting agent prepared by the preparation method according to any one of claims 4 to 10 in the preparation of a skin external preparation.

12. Use of the keratinocyte function-protecting agent according to any one of claims 1 to 3 or the keratinocyte function-protecting agent prepared by the preparation method according to any one of claims 4 to 10 in preparing a drug for repairing keratinocyte damage caused by SDS.

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