Erythrinine compound and application thereof in product for repairing skin barrier injury

Abstract

The invention relates to the technical field of skin repair, in particular to an erythrinine compound and application of the erythrinine compound in skin barrier injury repair products. The erythrinine compound disclosed by the invention is beneficial to expanding the application range of erythrinine. The weight ratio of the erythrinine compound to the peach gum polysaccharide hydrolysate in the composition is (0.1-0.5): (0.5-3.5), the molar ratio of the erythrinine to the cysteine to the glycyrrhizic acid in the erythrinine compound is 1: 1: 1, and the cysteine can be replaced by cystine, selenocystine or selenocysteine. The composition compounded by the erythrinine compound and the peach gum polysaccharide hydrolysate can inhibit secretion of macrophage inflammatory factors IL-1, TNF-alpha and IL-18 induced by propionibacterium acnes, inhibit proliferation of the propionibacterium acnes and promote repair of epidermal cell tissues, has the effect of removing inflammatory cells near wounds, and can be used for treating wound diseases. And by inhibiting inflammatory cells from gathering to wounds, human skin keratinocyte wound healing is promoted, and the effect of repairing skin barriers is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of skin repair, and specifically relates to an erythrina alkaloid complex and application thereof in products for repairing skin barrier damage. Background Art

[0002] The structural basis of the skin barrier is the stratum corneum formed by the keratinocytes on the surface, the lipids between the keratinocytes, and the natural protective factors. Its main function is to prevent the loss of water and electrolytes in the body, prevent the invasion of the external environment, and help maintain the body's homeostasis. On the one hand, once the skin barrier is damaged, it will cause the skin surface cells to secrete more oil to repair and protect themselves, which will lead to clogged pores, acne, and acne; on the other hand, after the skin barrier is damaged, the metabolic capacity is reduced, and the aging keratin and cosmetic residues cannot be metabolized and removed in time, which will further aggravate the pore blockage and acne. In addition, the excessive fatty acid stimulation produced by sebum metabolism and the over-proliferation of Propionibacterium acnes in a closed environment cause the skin to produce an inflammatory response. Therefore, the manifestations of acne from mild to severe are whiteheads and blackheads, inflammatory papules and pustules, cysts and nodules, and further develop into acne.

[0003] As a small molecule indole alkaloid, erythrinaine has anti-inflammatory, anti-tumor, anti-anxiety, antispasmodic and antioxidant effects. However, due to its poor solubility in water, erythrinaine is usually dissolved in organic solvents such as ethanol in the prior art to exert its anti-inflammatory effect. However, organic solvents such as ethanol may cause uncomfortable reactions such as redness, itching, and tingling in areas where the skin barrier is damaged, aggravating the symptoms of skin barrier damage and limiting the application of erythrinaine. Summary of the invention

[0004] In view of the above technical problems, the present invention provides an erythrina alkaloid complex and its application in products for repairing skin barrier damage. The present invention changes the physical properties of erythrina alkaloid to make it have good solubility in water, and achieves the effect of repairing damaged skin barrier by promoting wound healing of human skin keratinocytes.

[0005] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0006] The present invention provides an erythrinaine complex, and the preparation method of the erythrinaine complex comprises:

[0007] Dissolve cysteine ​​and glycyrrhizic acid in water, add erythrinaine to the aqueous solution, isolate oxygen and perform heating reaction; the reaction temperature is 60-100°C, the rotation speed is 300-3000rpm, and the reaction is concentrated after 1-8h. The concentrated solution is placed in an organic solvent for cooling, a brown precipitate is precipitated, and the erythrinaine complex is obtained by filtering and drying.

[0008] As an embodiment, the molar ratio of erythrine, cysteine ​​and glycyrrhizic acid is 1:1:1.

[0009] In one embodiment, the cysteine ​​is replaced with cystine, selenocystine or selenocysteine.

[0010] The present invention also provides a composition for repairing skin barrier damage, wherein the composition comprises the above-mentioned erythrinain complex and peach gum polysaccharide hydrolyzate, and the weight ratio of the erythrinain complex to the peach gum polysaccharide hydrolyzate is 0.1-0.5:0.5-3.5.

[0011] As an embodiment, the preparation method of the peach gum polysaccharide hydrolyzate comprises:

[0012] The peach gum is swollen in water, and then placed in anhydrous ethanol to precipitate a white precipitate, thereby obtaining a crude peach gum extract;

[0013] The peach gum crude extract is dissolved in water, mixed with chloroform-n-butanol solution and shaken, and the upper layer solution is taken to obtain a macromolecular peach gum polysaccharide solution;

[0014] The macromolecular peach gum polysaccharide solution is subjected to alkaline hydrolysis, decolorization and dialysis to obtain peach gum polysaccharide hydrolyzate.

[0015] In one embodiment, the molecular weight range of the small molecular weight polysaccharide in the peach gum polysaccharide hydrolyzate is 1×10 4 -5×10 5 Da.

[0016] The present invention also provides a product for repairing skin barrier damage, comprising the composition for repairing skin barrier damage and an auxiliary agent. As an embodiment, the auxiliary agent comprises a preservative and water.

[0017] The present invention also provides a preparation method of a product for repairing skin barrier damage, comprising dissolving erythrina alkaloids complex and peach gum polysaccharide hydrolyzate in water, heating to 70-90° C. and mixing, and adding a preservative and mixing after cooling to obtain the product.

[0018] The present invention also provides a composition or product for repairing skin barrier damage and its use in promoting the migration and repair of skin keratinocytes.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. The present invention prepares a synthetic erythrinaine complex by combining erythrinaine with cysteine ​​and glycyrrhizic acid, thereby changing the original physicochemical properties of erythrinaine. The obtained erythrinaine complex is easily soluble in water, which helps to improve the absorption rate and bioavailability of erythrinaine and expand the application range of erythrinaine. Cysteine ​​is a sulfur-containing amino acid that has antioxidant and detoxifying effects and can protect cells from oxidative stress damage; glycyrrhizic acid is a triterpenoid saponin extracted from licorice and has detoxifying, anti-inflammatory and immunomodulatory effects. Both cysteine ​​and glycyrrhizic acid are natural ingredients with good mildness and low irritation. The addition of cysteine ​​and glycyrrhizic acid improves the biological activity and stability of the erythrinaine complex and has a wider range of applications.

[0021] 2. The composition obtained by compounding the erythrine complex with peach gum polysaccharide hydrolyzate of the present invention has the effect of repairing skin barrier damage. It has been verified that the composition of the present invention can inhibit the secretion of macrophage inflammatory factors IL-1, TNF-α, and IL-18 induced by Propionibacterium acnes, inhibit the proliferation of Propionibacterium acnes, promote epidermal cell tissue repair, have the effect of clearing inflammatory cells near the wound, and promote human skin keratinocyte wound healing by inhibiting the aggregation of inflammatory cells to the wound, thereby achieving the effect of repairing damaged skin barrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The effect of the composition of the present invention on the proliferation of Propionibacterium acnes in a model of co-culture of HaCaT cells and Propionibacterium acnes;

[0023] Figure 2 The effect of the composition of the present invention on the secretion of inflammatory factors of macrophages induced by Propionibacterium acnes;

[0024] Figure 3 The effect of the composition of the present invention on wound healing of human skin keratinocytes;

[0025] Figure 4 The effect of the composition of the present invention on wound healing in zebrafish;

[0026] Figure 5 The effect of the composition of the present invention on the aggregation of inflammatory cells to wounds in zebrafish;

[0027] Figure 6 The present invention shows the effect of the composition of the present invention on clearing inflammatory cells near the wound in zebrafish. DETAILED DESCRIPTION

[0028] The present invention provides an erythrinaine complex, and the preparation method of the erythrinaine complex comprises:

[0029] Dissolve cysteine ​​and glycyrrhizic acid in water, add erythrinaine to the aqueous solution, isolate oxygen and perform heating reaction; the reaction temperature is 60-100°C, the rotation speed is 300-3000rpm, and the reaction is concentrated after 1-8h. The concentrated solution is placed in an organic solvent, cooled to precipitate a brown precipitate, and filtered and dried to obtain the erythrinaine complex.

[0030] In the present invention, the molar ratio of erythrine, cysteine ​​and glycyrrhizic acid is 1:1:1. As an embodiment, the cysteine ​​is replaced by cystine, selenocystine or selenocysteine. The present invention has no particular limitation on the specific sources of erythrine, cysteine ​​and glycyrrhizic acid, and commercially available products can be used.

[0031] In the present invention, the isolated oxygen is one or more of nitrogen or inert gas. As one embodiment, the inert gas is helium, neon, argon, krypton, xenon. The reaction temperature is 60-100°C, in one embodiment, the reaction temperature is 70-90°C; the reaction speed is 300-3000rpm, in one embodiment, the reaction speed is 500-1000rpm; the reaction time is 1-8h, in one embodiment, the reaction time is 3-6h.

[0032] In the present invention, the organic solvent is preferably acetone, and the solid-liquid ratio of precipitate to acetone during the precipitation process is preferably 0.1-0.5 mol precipitate: 0.5-1 L acetone. The cooling temperature is 2-8°C. In one embodiment, the cooling temperature is 4°C; the cooling time is 5-24h. In one embodiment, the cooling time is 8-12h.

[0033] In the present invention, the filtration is preferably reduced pressure filtration, the drying is preferably vacuum drying, and the temperature of the vacuum drying is 40-60° C. Unless otherwise specified, the present invention adopts conventional preparation methods.

[0034] The present invention also provides a composition for repairing skin barrier damage. In the present invention, the weight ratio of the erythrina alkaloid complex to the peach gum polysaccharide hydrolyzate in the composition is 0.1-0.5:0.5-3.5.

[0035] In the present invention, the preparation method of the peach gum polysaccharide hydrolyzate comprises:

[0036] The peach gum is swollen in water, and then placed in anhydrous ethanol to precipitate a white precipitate, thereby obtaining a crude peach gum extract;

[0037] The peach gum crude extract is dissolved in water, mixed with chloroform-n-butanol solution and shaken, and the upper layer solution is taken to obtain a macromolecular peach gum polysaccharide solution;

[0038] The macromolecular peach gum polysaccharide solution is subjected to alkaline hydrolysis, decolorization and dialysis to obtain peach gum polysaccharide hydrolyzate.

[0039] In the present invention, the swelling is preferably heating swelling, the heating temperature is 80-100° C., and the heating time is 3-6 hours.

[0040] In the present invention, the liquid ratio of peach gum to anhydrous ethanol is preferably 10g peach gum: 1-1.5L anhydrous ethanol. In the present invention, the volume ratio of the chloroform-n-butanol mixed solution is 4:1, which is used to remove protein from the peach gum crude extract.

[0041] In the present invention, the alkaline hydrolysis is preferably sodium carbonate or sodium bicarbonate, the pH of the alkaline hydrolysis is 8-12, and in one embodiment, the pH of the alkaline hydrolysis is 9.5-11.5. The temperature of the alkaline hydrolysis is 60-100° C., and in one embodiment, the temperature of the alkaline hydrolysis is 75-90° C. The time of the alkaline hydrolysis is 2-8 hours, and in one embodiment, the time of the alkaline hydrolysis is 2-4 hours.

[0042] In the present invention, the preferred decolorizing agent is activated carbon, the decolorizing temperature is 30-70°C, and in one embodiment, the decolorizing temperature is 40-60°C; the decolorizing time is 1-5h, and in one embodiment, the decolorizing time is 1-3h.

[0043] In the present invention, the molecular weight range of the small molecular weight polysaccharide in the peach gum polysaccharide hydrolyzate is 1×10 4 -5×10 5 Da, the small molecular weight polysaccharide includes arabinose (Ara), galactose (Gal), xylose (Xyl), mannose (Man), rhamnose and uronic acid.

[0044] The present invention also provides a product for repairing skin barrier damage, comprising the above composition for repairing skin barrier damage and an auxiliary agent. In the present invention, the auxiliary agent comprises a preservative and water. In one embodiment, the preservative is one or more of methylparaben, propylparaben, phenoxyethanol, ethylhexylglycerin, potassium sorbate, sodium dehydroacetate, benzyl alcohol, p-chloro-meta-xylenol, and methylisothiazolinone.

[0045] The present invention also provides a method for preparing the above-mentioned product for repairing skin barrier damage, comprising dissolving the erythrina alkaloid complex and peach gum polysaccharide hydrolyzate in water, heating to 70-90° C. and mixing evenly, and adding a preservative and mixing evenly after cooling to obtain the product.

[0046] The composition or product for repairing skin barrier damage of the present invention can be used to promote the migration and repair of skin keratinocytes.

[0047] After the zebrafish tail fin wound was treated with the aqueous solution of the skin barrier damage repairing composition provided by the present invention for 72 hours, the relative growth rate of the tail fin was 111.97±2.00%, which was 11.97% higher than that of the blank control group (100.00±2.34%) (p<0.001), indicating that the composition of the present invention has the effect of promoting the healing of human skin keratinocyte wounds.

[0048] After the zebrafish caudal fin wound was treated with the aqueous solution of the composition for repairing skin barrier damage provided by the present invention for 6 hours, the relative aggregation rate of inflammatory cells at the zebrafish caudal fin wound was 89.42±2.05%, which was reduced by 10.58% (p<0.01) compared with the blank control group, indicating that the composition of the present invention has an inhibitory effect on the aggregation of inflammatory cells at the zebrafish caudal fin wound.

[0049] After the zebrafish caudal fin wound was treated with the aqueous solution of the composition for repairing skin barrier damage provided by the present invention for 6 hours, the relative clearance rate of inflammatory cells at the zebrafish caudal fin wound was 9.74±2.01%, which was increased by 9.74% (p<0.01) compared with the blank control group, indicating that the composition of the present invention has a promoting effect on the clearance of inflammatory cells at the zebrafish caudal fin wound.

[0050] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0051] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0052] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0053] Example 1 Erythrinain Complex

[0054] S1. At room temperature, add 0.3 mol of cysteine ​​to a round-bottom flask, add 0.3 L of sterile distilled water and stir thoroughly to dissolve; dissolve 0.3 mol of glycyrrhizic acid in 0.3 L of sterile distilled water, then slowly add dropwise to the above round-bottom flask and stir thoroughly to mix;

[0055] S2. After adding 0.3 L of sterile distilled water to 0.3 mol of erythrine, add it into a round-bottom flask, introduce argon to isolate oxygen, then set the temperature to 70°C, the speed to 500 rpm, and react for 6 hours;

[0056] S3. After the reaction is completed, the reaction solution is placed in a rotary evaporator for concentration until the solution volume no longer decreases and reaches saturation, 0.8 L of acetone is added to the concentrated solution, and after sufficient mixing, it is placed in a 4°C refrigerator for cooling overnight to precipitate a large amount of brown precipitate, and the precipitate is filtered under reduced pressure, and the filtered precipitate is placed in a vacuum drying oven at 40°C for drying overnight to obtain the erythrina alkaloid complex.

[0057] Example 2 Erythrina alkaloid complex

[0058] S1. At room temperature, add 0.5 mol of selenocystine to a round-bottom flask, add 0.5 L of sterile distilled water and stir thoroughly to dissolve; dissolve 0.5 mol of glycyrrhizic acid in 0.5 L of sterile distilled water, then slowly add dropwise to the above round-bottom flask and stir thoroughly to mix;

[0059] S2. After adding 0.5 L of sterile distilled water to 0.5 mol of erythrine, add it into a round-bottom flask, introduce argon to isolate oxygen, then set the temperature to 90°C, the speed to 1000 rpm, and react for 3 hours;

[0060] S3. After the reaction is completed, the reaction solution is placed in a rotary evaporator for concentration until the solution volume no longer decreases and reaches saturation, 0.8 L of acetone is added to the concentrated solution, and after sufficient mixing, it is placed in a 4°C refrigerator for cooling overnight to precipitate a large amount of brown precipitate, and the precipitate is filtered under reduced pressure, and the filtered precipitate is placed in a vacuum drying oven at 60°C for drying overnight to obtain the erythrina alkaloid complex.

[0061] Example 3 Composition for repairing skin barrier damage

[0062] By weight, it contains 0.1 parts of the erythrina alkaloid complex of Example 1 and 0.5 parts of peach gum polysaccharide hydrolyzate.

[0063] The above-mentioned peach gum polysaccharide hydrolyzate is prepared according to the following steps:

[0064] S1. Weigh 10g peach gum, put it into a 1L beaker, add 0.8L sterile distilled water, heat and stir at 80℃ for 6h, wait until the peach gum is fully swollen and becomes a clear and transparent viscous solution, and cool it at room temperature;

[0065] S2. After the peach gum aqueous solution is cooled to room temperature, pour it into a large beaker filled with 1.5L of anhydrous ethanol and stir it thoroughly. Place the peach gum ethanol mixture in a 4°C refrigerator to cool overnight, and a large amount of white precipitate is precipitated. The precipitate is filtered under reduced pressure, and the filtered precipitate is placed in a vacuum drying oven at 40°C and dried overnight to obtain a crude peach gum extract.

[0066] S3, put the peach gum crude extract into a 1L beaker, add 0.6L of sterile distilled water, heat and stir at 80℃ for 1h, until the peach gum crude extract is fully dissolved, and then cool at room temperature; add a 4:1 volume chloroform-n-butanol mixed solution to the cooled peach gum crude extract aqueous solution, shake it thoroughly, centrifuge and discard the protein precipitate, and obtain a pure macromolecular peach gum polysaccharide solution;

[0067] S4, adding sodium carbonate or sodium bicarbonate solid powder to the macromolecular peach gum polysaccharide solution, stirring fully, adjusting the pH value of the polysaccharide solution to 9.5, and then heating the polysaccharide solution to 75° C., stirring fully to react for 2-4 hours to obtain a hydrolyzate;

[0068] S5. After the hydrolyzate is cooled to room temperature, 40 g of activated carbon is added for decolorization, and the decolorization is fully stirred at 40 ° C for 3 h. After the reaction solution is cooled, the precipitate is filtered under reduced pressure and the filtrate is collected;

[0069] S6. Place the collected filtrate in a dialysis bag and dialyze with 2L of distilled water for 24 hours, replacing the distilled water every 3 hours. Place the dialyzed hydrolyzate in a freeze vacuum dryer at -45°C and vacuum dry overnight to obtain a white flocculent, which is peach gum polysaccharide hydrolyzate.

[0070] The preparation method of the above composition for repairing skin barrier damage is as follows: weigh the above-mentioned parts by weight of the erythrina alkaloid complex and peach gum polysaccharide hydrolyzate, add an appropriate amount of sterile distilled water, heat to 70° C., stir and mix to obtain the composition.

[0071] Example 4 Composition for repairing skin barrier damage

[0072] By weight, it contains 0.5 parts of the erythrina alkaloid complex of Example 1 and 3.5 parts of peach gum polysaccharide hydrolyzate.

[0073] The above-mentioned peach gum polysaccharide hydrolyzate is prepared according to the following steps:

[0074] S1. Weigh 10g peach gum, put it into a 1L beaker, add 0.8L sterile distilled water, heat and stir at 100℃ for 3h, wait until the peach gum is fully swollen and becomes a clear and transparent viscous solution, and cool it at room temperature;

[0075] S2. After the peach gum aqueous solution is cooled to room temperature, it is poured into a large beaker containing 1.5L of anhydrous ethanol and stirred thoroughly. The peach gum ethanol mixture is placed in a 4°C refrigerator to cool overnight, and a large amount of white precipitate is precipitated. The precipitate is filtered under reduced pressure, and the filtered precipitate is placed in a vacuum drying oven at 60°C and dried overnight to obtain a crude peach gum extract.

[0076] S3, put the peach gum crude extract into a 1L beaker, add 0.6L of sterile distilled water, heat and stir at 100°C for 0.5h, until the peach gum crude extract is fully dissolved, and then cool at room temperature; add a 4:1 volume chloroform-n-butanol mixed solution to the cooled peach gum crude extract aqueous solution, shake it thoroughly, centrifuge and discard the protein precipitate, and obtain a pure macromolecular peach gum polysaccharide solution;

[0077] S4, adding sodium carbonate or sodium bicarbonate solid powder to the macromolecular peach gum polysaccharide solution, stirring fully, adjusting the pH value of the polysaccharide solution to 11.5, and then heating the polysaccharide solution to 90° C., stirring fully to react for 2 hours to obtain a hydrolyzate;

[0078] S5. After the hydrolyzate is cooled to room temperature, 40 g of activated carbon is added for decolorization, and the decolorization is fully stirred at 60° C. for 1 h. After the reaction solution is cooled, the precipitate is filtered under reduced pressure and the filtrate is collected;

[0079] S6. Place the collected filtrate in a dialysis bag and dialyze with 2L of distilled water for 24 hours, replacing the distilled water every 6 hours. Place the dialyzed hydrolyzate in a freeze vacuum dryer at -45°C and vacuum dry overnight to obtain a white flocculent, which is peach gum polysaccharide hydrolyzate.

[0080] The preparation method of the above composition for repairing skin barrier damage is as follows: weigh the above-mentioned parts by weight of the erythrina alkaloid complex and peach gum polysaccharide hydrolyzate, add an appropriate amount of sterile distilled water, heat to 90° C., stir and mix to obtain the composition.

[0081] Example 5 Composition for repairing skin barrier damage

[0082] By weight, it contains 0.3 parts of the erythrina alkaloid complex of Example 1 and 2 parts of peach gum polysaccharide hydrolyzate.

[0083] The preparation method of the composition for repairing skin barrier damage is the same as that in Example 3.

[0084] Example 6 Composition for repairing skin barrier damage

[0085] By weight, it contains 0.4 parts of the erythrina alkaloid complex of Example 1 and 3 parts of peach gum polysaccharide hydrolyzate.

[0086] The preparation method of the composition for repairing skin barrier damage is the same as that in Example 3.

[0087] Example 7 Composition for repairing skin barrier damage

[0088] By weight, it contains 0.2 parts of the erythrina alkaloid complex of Example 1 and 1 part of peach gum polysaccharide hydrolyzate.

[0089] The preparation method of the composition for repairing skin barrier damage is the same as that in Example 3.

[0090] Test Example 1: Inhibitory effect of the composition on the proliferation of Propionibacterium acnes

[0091] HaCaT cells in the logarithmic growth phase were collected and divided into 4 groups, namely blank control group, treatment group 1, treatment group 2 and treatment group 3. The above cells were inoculated into 6-well plates containing DMEM medium, 2 mL per well, and cultured in a 37°C, 5% CO2 cell culture incubator.

[0092] Preparation of Propionibacterium acnes bacterial suspension: Cultivate Propionibacterium acnes and centrifuge the cultured Propionibacterium acnes at 5000r / min for 10min. After discarding the supernatant, take the bacterial precipitate and wash it with PBS for 3 times, and then resuspend the bacteria in 3mL PBS to a density of OD 595 is 0.8.

[0093] 5 μL of the above-mentioned Propionibacterium acnes bacterial suspension was added to the blank control group and each treatment group, and then a 0.005% aqueous solution of the composition of Example 3 was added to treatment group 1, a 0.01% aqueous solution of the composition of Example 3 was added to treatment group 2, and a 0.02% aqueous solution of the composition of Example 3 was added to treatment group 3. Water was added to the blank control group, and the added amount was 2 ml. After treating HaCaT cells in a cell culture incubator at 37°C and 5% CO2 for 24 hours in all four groups, the morphological changes of HaCaT cells and the changes in the number of Propionibacterium acnes were observed under an inverted microscope.

[0094] like Figure 1 As shown, a large number of rod-shaped bacteria can be observed attached to the surface of HaCaT cells in the blank control group, and a certain number of HaCaT cells are observed to shrink and die; compared with the blank control group, the number of rod-shaped bacteria attached to the surface of HaCaT cells in treatment groups 1 and 2 is significantly reduced, and the number of shrunken and dead HaCaT cells is reduced; in treatment group 3, it can be observed that there are basically no rod-shaped bacteria on the surface of HaCaT cells, and the HaCaT cells have clear outlines and good growth conditions. The above results show that the composition for repairing skin barrier damage in the present invention can effectively inhibit the growth of Propionibacterium acnes HaCaT cells on the keratin surface of human skin, and has the potential to inhibit the proliferation of Propionibacterium acnes.

[0095] Test Example 2: Inhibitory effect of the composition on the secretion of inflammatory factors IL-1, TNF-α, and IL-18 in macrophages induced by Propionibacterium acnes

[0096] HaCaT cells in the logarithmic growth phase were collected and divided into 5 groups, namely blank control group, model control group, treatment group 1, treatment group 2 and treatment group 3. The above cells were inoculated into 6-well plates containing DMEM medium, 2 mL per well, and cultured in a 37°C, 5% CO2 cell culture incubator.

[0097] Preparation of Propionibacterium acnes bacterial suspension: Cultivate Propionibacterium acnes and centrifuge the cultured Propionibacterium acnes at 5000r / min for 10min. After discarding the supernatant, take the bacterial precipitate and wash it with PBS for 3 times, and then resuspend the bacteria in 3mL PBS to a density of OD 595 is 0.8.

[0098] Treatment group 1 added an aqueous solution of the composition of Example 3 at a concentration of 0.005%, treatment group 2 added an aqueous solution of the composition of Example 3 at a concentration of 0.01%, treatment group 3 added an aqueous solution of the composition of Example 3 at a concentration of 0.02%, and the blank control group and model control group added water, and the added amount was 2 ml. 5 μL of the bacterial suspension of Propionibacterium acnes of the above density was added to the model control group and each treatment group, respectively, and no Propionibacterium acnes bacterial suspension was added to the blank treatment group. After the five groups were treated with HaCaT cells in a cell culture incubator at 37°C and 5% CO2 for 24 hours, the cells were collected to extract RNA. Real-time fluorescence quantitative nucleic acid amplification (RT-qPCR) was used to detect the expression levels of different inflammatory factors (IL-6, IL-18 and TNF-α) in HaCaT cells in different treatment groups.

[0099] like Figure 2 As shown, compared with the blank control group, the expression levels of inflammatory factors IL-6, IL-18 and TNF-α in HaCaT cells in the model control group were significantly increased, indicating that Propionibacterium acnes can stimulate and induce HaCaT cells to produce obvious inflammatory responses. The expression levels of inflammatory factors IL-6, IL-18 and TNF-α in HaCaT cells in treatment groups 1, 2 and 3 were significantly decreased, indicating that the composition for repairing skin barrier damage of the present invention can effectively inhibit the inflammatory response of Propionibacterium acnes to HaCaT cells and has good anti-inflammatory effects.

[0100] Test Example 3: Effect of the composition on epidermal cell tissue repair

[0101] HaCaT cells in the logarithmic growth phase were collected and divided into 4 groups, which were respectively recorded as blank control group, treatment group 1, treatment group 2 and treatment group 3. The above cells were inoculated into 6-well plates containing DMEM medium, 2 mL per well, and placed in a 37°C, 5% CO2 cell culture incubator for culture. After the cells were fully grown, scratches were made with a 10 μL gun tip, and after washing twice with PBS, water was added to the well plate of the blank control group, and an aqueous solution with a concentration of 0.005% of the composition of Example 3 was added to treatment group 1, and an aqueous solution with a concentration of 0.01% of the composition of Example 3 was added to treatment group 2, and an aqueous solution with a concentration of 0.02% of the composition of Example 3 was added to treatment group 3, and the amount added was 2 ml. After 72 hours, the same field of view of the 4 groups was photographed and recorded.

[0102] like Figure 3 As shown, the scratch spacing of the blank control group was significantly shortened after 72 hours; the scratch spacing of the HaCaT cells in treatment group 1 and treatment group 2 was significantly smaller than that of the blank group after 72 hours; the scratches of the HaCaT cells in treatment group 3 basically disappeared after 72 hours, and the cell wounds were completely healed. This shows that with the increase in concentration, the promoting effect of the composition of the present invention on the healing of HaCaT cell scratches increased significantly. The above results show that the composition of the present invention has a good effect of promoting the healing of scratches on HaCaT cells, and the composition of the present invention can significantly promote skin tissue repair.

[0103] Test Example 4: Effect of the composition on wound healing of human skin keratinocytes

[0104] 72hpf zebrafish fry were selected and set up into 4 groups, 10 in each group. The tail fin of the zebrafish was removed with a scalpel. After the tail fin was removed, different treatment solutions were applied to the wound: the blank control group was treated with water, the treatment group 1 was treated with an aqueous solution of the composition of Example 3 at a concentration of 0.005%, the treatment group 2 was treated with an aqueous solution of the composition of Example 3 at a concentration of 0.01%, and the treatment group 3 was treated with an aqueous solution of the composition of Example 3 at a concentration of 0.02%. The volume of the treatment solution was 2 ml. After incubation for 72 hours, the tail fin repair rate was counted by taking photos.

[0105] The results are as follows Figure 4 As shown, the relative growth rate of the tail fin of each group after 72 hours was calculated (relative growth rate = difference in tail fin length of each group / difference in tail fin length of blank group × 100%). Under the same conditions, after 72 hours of treatment of zebrafish in treatment group 1, the relative growth rate of the tail fin was 107.86±1.46%, which was 7.86% higher than that of the blank control group (100.00±2.34%) (p<0.05), indicating that the composition in treatment group 1 has the effect of promoting the repair of zebrafish tail fin (significant); under the same conditions, after 72 hours of treatment of zebrafish in treatment group 2, the relative growth rate of the tail fin was 111.97±2.00%, which was 7.86% higher than that of the blank control group (100.00±2.3 4%) increased by 11.97% (p<0.001), indicating that the composition in treatment group 2 has the effect of promoting the repair of zebrafish tail fin (very significant); under the same conditions, after 72 hours of treatment of zebrafish in treatment group 3, the relative growth rate of the tail fin was 111.78±2.44%, which increased by 11.78% (p<0.001) compared with the blank control group (100.00±2.34%), indicating that the composition in treatment group 3 has the effect of promoting the repair of zebrafish tail fin (very significant). The above results show that the composition of the present invention has the effect of promoting tissue repair under this experimental condition and has the effect of promoting wound healing.

[0106] Test Example 5: Effect of the composition on inhibiting the aggregation of inflammatory cells to wounds

[0107] Normally developed 72hpf Tg (corola: EGFP) transgenic zebrafish embryos were selected and placed in 6-well plates. Four groups of 10 were set up, and 5.0mL of aquaculture water was used for pretreatment for 1 hour. After cutting off 50% of the tail fin, 5.0mL of different treatment solutions were replaced and applied to the wound: the blank control group was treated with water, treatment group 1 was treated with an aqueous solution of the composition of Example 3 at a concentration of 0.005%, treatment group 2 was treated with an aqueous solution of the composition of Example 3 at a concentration of 0.01%, and treatment group 3 was treated with an aqueous solution of the composition of Example 3 at a concentration of 0.02%. After incubation at 28°C for 6 hours, the FITC channel was selected under a fluorescence microscope to observe the aggregation of inflammatory cells in the wound of the zebrafish tail fin, and the number of inflammatory cells was counted.

[0108] like Figure 5 As shown, the relative aggregation rate of inflammatory cells at the caudal fin wound of each group after 6 hours was calculated (relative aggregation rate = (number of inflammatory cells aggregated in each group / number of inflammatory cells aggregated in the blank group) × 100%). After being treated with standard diluted water for 6 hours, the relative aggregation rate of inflammatory cells at the caudal fin wound of zebrafish in the blank control group was 100.00±1.71%; at a concentration of 0.005%, the relative aggregation rates of inflammatory cells at the caudal fin wound of zebrafish in treatment group 1, treatment group 2 and treatment group 3 after 6 hours of treatment were 90.87±2.31%, 89.42±2.05% and 92.79±1.89%, respectively, which were reduced by 9.13% (p<0.01), 10.58% (p<0.01) and 7.21% (p<0.05) respectively compared with the blank control group, indicating that the composition of the present invention has an inhibitory effect on the aggregation of inflammatory cells at the caudal fin injury of zebrafish.

[0109] Test Example 6: Effect of the composition on clearing inflammatory cells at skin lesions

[0110] Normally developed 72hpf Tg (corola: EGFP) transgenic zebrafish embryos were selected and placed in 6-well plates. Four groups of 10 were set up in each group. After 50% of the caudal fin was cut off, the embryos were pretreated with 5.0mL of aquaculture water for 6 hours, and then replaced with 5.0mL of different treatment solutions and applied to the wound: the blank control group was treated with water, the treatment group 1 was treated with an aqueous solution of the composition of Example 3 at a concentration of 0.005%, the treatment group 2 was treated with an aqueous solution of the composition of Example 3 at a concentration of 0.01%, and the treatment group 3 was treated with an aqueous solution of the composition of Example 3 at a concentration of 0.02%. After incubation at 28°C for 6 hours, the FITC channel was selected under a fluorescence microscope to observe the clearance of inflammatory cells in the zebrafish caudal fin wound, and the number of inflammatory cells was counted.

[0111] like Figure 6As shown, the relative clearance rate of inflammatory cells at the caudal fin wound of each group after 6 hours was calculated (relative clearance rate = [(number of inflammatory cells aggregated in the blank group - number of inflammatory cells aggregated in each group) / number of inflammatory cells aggregated in the blank group] × 100%). The relative clearance rate of inflammatory cells at the caudal fin wound of zebrafish in the blank control group after 6 hours of treatment with standard diluted water was 0.00 ± 3.69%; under the same conditions, the relative clearance rates of inflammatory cells in treatment group 1, treatment group 2 and treatment group 3 after treatment were 5.84 ± 2.79%, 5.84 ± 3.25%, and 9.74 ± 2.01%, respectively, which were increased by 5.84%, 5.84% and 9.74% compared with the blank control group, respectively, indicating that the composition of the present invention has a promoting effect on the clearance of inflammatory cells at the caudal fin injury of zebrafish.

[0112] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. An erythrine complex, characterized in that: The preparation method of the erythrina alkaloid complex comprises: Dissolve cysteine ​​and glycyrrhizic acid in water, add erythrinaine to the aqueous solution, isolate oxygen and perform heating reaction; the reaction temperature is 60-100°C, the rotation speed is 300-3000rpm, and the reaction is concentrated after 1-8h. The concentrated solution is placed in an organic solvent for cooling, a brown precipitate is precipitated, and the erythrinaine complex is obtained by filtering and drying.

2. The erythrina alkaloid complex according to claim 1, characterized in that: The molar ratio of erythrine, cysteine ​​and glycyrrhizic acid is 1:1:

1.

3. The erythrina alkaloid complex according to claim 1 or 2, characterized in that: The cysteine ​​is replaced with cystine, selenocystine or selenocysteine.

4. A composition for repairing skin barrier damage, characterized in that: The invention comprises the erythrina alkaloid complex according to any one of claims 1 to 3 and peach gum polysaccharide hydrolysate, wherein the weight ratio of the erythrina alkaloid complex to the peach gum polysaccharide hydrolysate is 0.1-0.5:0.5-3.

5.

5. The composition according to claim 4, characterized in that The preparation method of the peach gum polysaccharide hydrolyzate comprises: The peach gum is swollen in water, and then placed in anhydrous ethanol to precipitate a white precipitate, thereby obtaining a crude peach gum extract; The peach gum crude extract is dissolved in water, mixed with chloroform-n-butanol solution and shaken, and the upper layer solution is taken to obtain a macromolecular peach gum polysaccharide solution; The macromolecular peach gum polysaccharide solution is subjected to alkaline hydrolysis, decolorization and dialysis to obtain peach gum polysaccharide hydrolyzate.

6. The composition according to claim 4 or 5, characterized in that The molecular weight range of the small molecular weight polysaccharide in the peach gum polysaccharide hydrolysate is 1×10 4 -5×10 5 Da.

7. A product for repairing skin barrier damage, characterized in that: The invention comprises the composition for repairing skin barrier damage as described in any one of claims 4 to 6 and an auxiliary agent.

8. The product according to claim 7, characterized in that The adjuvants include preservatives and water.

9. The method for preparing the product according to claim 7 or 8, characterized in that: Dissolve the erythrina alkaloid complex and peach gum polysaccharide hydrolyzate in water, heat to 70-90°C and mix evenly, add preservatives after cooling and mix evenly to obtain the product.

10. Use of the composition for repairing skin barrier damage according to any one of claims 4 to 6, or the product for repairing skin barrier damage according to any one of claims 7 to 8 in promoting migration and repair of skin keratinocytes.

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