Composition for repairing damaged skin cells and preparation method thereof

Abstract

The invention provides a composition for repairing damaged skin cells and a preparation method thereof, and belongs to the technical field of skin repair, the composition comprises a glycyrrhiza glabra root extract, cordyceps cicadae extract peptide, a ganoderic acid-exopolysaccharide compound and a citrus aurantium extract. The glycyrrhiza glabra root extract is obtained by performing ultrasonic extraction and purification on root core coarse powder of glycyrrhiza glabra through a mixed solution containing choline chloride and lactic acid. The isaria cicadae miquel extract peptide is obtained by sequentially carrying out enzymolysis and purification on isaria cicadae miquel dry powder through chitinase, fig protease and serrapeptase. The ganoderic acid-exopolysaccharide compound is obtained by fermenting antrodia camphorata in a culture medium containing olive squalane to obtain a water phase and mycelia, performing enzymolysis on the mycelia through chitinase and beta-glucanase to obtain enzymatic hydrolysate, combining the enzymatic hydrolysate with the water phase, and performing purification. The citrus aurantium flower extract is obtained by performing water extraction and purification on citrus aurantium flower powder. All the components are matched and can accurately act on key pathological links of damaged skin cells, and the safe, mild and efficient repairing function is achieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of skin repair, and particularly relates to a composition for repairing damaged skin cells and a preparation method thereof. BACKGROUND

[0002] In the medical field, some patients have developed dependence on hormones when treating skin diseases such as eczema and seborrheic dermatitis due to improper medication or long-term abuse of glucocorticoid ointments, and the original skin disease will recur and worsen once the medication is stopped.

[0003] The damage caused by hormone face to the skin is multi-faceted and severe. From the cellular level, hormones inhibit the metabolism of skin cells, causing the proliferation rate of keratinocytes to slow down and differentiation to be abnormal, resulting in a thinning of the stratum corneum and a loss of the skin's original tough protection. The activity of fibroblasts in the dermis is also inhibited, and the synthesis of collagen and elastic fibers is reduced, causing the skin to gradually lose elasticity and become loose and prematurely aged. From the perspective of skin barrier function, damaged skin cells cannot be tightly arranged, and intercellular lipids are lost, resulting in impaired skin barrier function and an inability to effectively resist physical, chemical, and biological stimuli from the outside world, such as ultraviolet light, cold air, and chemical ingredients in cosmetics, which in turn leads to frequent allergic reactions.

[0004] In terms of clinical manifestations, patients with hormone face often have abnormal skin appearance, such as thin and transparent facial skin, with clearly visible subcutaneous dilated capillaries, presenting obvious red blood vessels; large areas of facial erythema with bright red or dark red color, and severe swelling; and dense small papules and pustules on the skin surface, resembling acne but different from ordinary acne, with a distribution often related to the area of hormone use, and symptoms worsening after stopping hormone use. At the same time, patients also have strong discomfort, with a burning and tight sensation on the skin, with symptoms significantly exacerbated by heat; repeated itching that severely affects daily life, and scratching that can easily cause skin damage and infection, further exacerbating inflammation. In addition, hormone face has the characteristic of repeated symptoms that are difficult to heal, with a "rebound" phenomenon occurring after stopping hormone use, with the original symptoms suddenly worsening, and the use of hormones temporarily alleviating symptoms, leading to a vicious cycle of prolonged and recurrent disease, which can also cause irreversible damage to the skin such as atrophy and pigmentation, causing great physical and mental suffering to patients.

[0005] Traditional treatment methods for hormone face have many limitations. Early on, simple hormone reduction therapy was used, which gradually reduced the amount of hormones used, but this method can easily cause severe withdrawal symptoms, and the patient's skin symptoms will worsen dramatically in the short term, which is difficult to tolerate, and the recurrence rate is high. Later developed drug treatments, such as the use of tacrolimus ointment and other immunomodulators, have some effect, but can also cause adverse reactions and safety problems. Some physical treatment methods, such as laser therapy, can improve some symptoms such as red blood vessels, but can cause greater damage to the skin, which can exacerbate the destruction of the skin barrier and is not suitable for all patients with hormone face.

[0006] With the cross-fusion and development of skin science, cell biology, bioengineering and other disciplines, new opportunities and hopes have been brought for the development of more effective facial damaged skin cell repair products for hormone face; it is necessary to continuously focus on cell-level precision repair technology, to deeply explore the molecular mechanism of damaged skin cells in hormone face, to extract and prepare active ingredients with high repair function by using advanced bio-fermentation, genetic engineering and other technologies, to precisely act on damaged cells, to activate the self-repair mechanism of cells, to promote cell proliferation and differentiation, and to accelerate the renewal of skin cells, so as to safely, gently and effectively repair damaged skin cells. SUMMARY

[0007] In view of the problems of great repair difficulty, hormone dependence and side effects of existing damaged skin cells, the present application provides a composition for repairing damaged skin cells and a preparation method thereof. The glycyrrhiza glabra root extract, chrysochroa rhodostoma extract peptide, ganoderma acid-extracellular polysaccharide complex and dali flower extract are prepared by a special method, and each component is used in a specific ratio to precisely act on the key pathological link of damaged skin cells, realize safe, gentle and efficient repair function, promote fibroblast synthesis of collagen, repair damaged cells, restore barrier function, break the vicious cycle of hormone dependence and improve hormone face. The specific technical scheme is as follows: A composition for repairing damaged skin cells, the composition comprises glycyrrhiza glabra root extract, chrysochroa rhodostoma extract peptide, ganoderma acid-extracellular polysaccharide complex and dali flower extract in a mass ratio of (4-6):(2-3):(3.5-5):(1-2); The preparation of the glycyrrhiza glabra root extract comprises ultrasonic extraction of the root core coarse powder of glycyrrhiza glabra in a mixed solution containing choline chloride and lactic acid, and adsorption purification of the extraction liquid on an A21 macroporous adsorption resin column to obtain a product; The preparation of the chrysochroa rhodostoma extract peptide comprises sequential enzymolysis of the chrysochroa rhodostoma dry powder by chitinase, ficin and serratia peptidase, and adsorption purification of the enzyme hydrolysate on a G-15 dextran gel column to obtain a product; The ganoderic acid-exopolysaccharide complex preparation includes fermentation of T. sinense in a culture medium containing squalane to obtain a water phase and mycelium, enzymatic hydrolysis of the mycelium by chitinase and beta-glucanase to obtain an enzymatic hydrolysis liquid, combination of the enzymatic hydrolysis liquid and the water phase, ultrasonic activation, and loading onto an AB-8 macroporous resin column for adsorption purification to obtain the product. The preparation of the Dalbergia hupeana extract includes water extraction of Dalbergia hupeana pollen at a temperature of 110-120 DEG C and a pressure of 0.3-0.4 MPa, adsorption purification of the extract liquid by a D101 macroporous resin column to obtain the product.

[0008] The preparation method of the Glycyrrhiza glabra root extract includes: taking root core coarse powder of Glycyrrhiza glabra, soaking in hot water, filtering to obtain filter cake; preparing a mixed solution containing 5wt%-6wt% choline chloride and 7wt%-8wt% lactic acid by using an ethanol aqueous solution; adding the filter cake into the mixed solution at a solid-liquid ratio of 1g:15mL-20mL, ultrasonic treatment, centrifugation, taking the liquid, removing ethanol by vacuum concentration, loading onto an A21 macroporous adsorption resin column, washing with deionized water for 1-2 column volumes to remove impurities, eluting with 20%-30% volume concentration of an ethanol aqueous solution for 2-3 column volumes, collecting the eluate, removing ethanol by vacuum concentration, and freeze-drying to obtain the Glycyrrhiza glabra root extract.

[0009] In the preparation method of the Glycyrrhiza glabra root extract, the particle size of the root core coarse powder is below 40-60 mesh; the amount of the hot water is 6-8 times the mass of the root core coarse powder; the temperature of the hot water is 80-90 DEG C; the soaking time is 20-30 min; the volume ratio of ethanol to water in the ethanol aqueous solution is (5-6):(4-5); the ultrasonic treatment is performed at 50-60 DEG C and 200-250 W for 20-30 min; the centrifugation is performed at 6000-8000 rpm for 10-15 min; and the temperature of the vacuum concentration is 50-60 DEG C.

[0010] In the composition, the preparation method of the extract peptide of Chrysanthemum morifolium Ramat. comprises the following steps: adding dry powder of Chrysanthemum morifolium Ramat. into phosphate buffer solution with 10 times to 12 times of the mass of the dry powder of Chrysanthemum morifolium Ramat. and with pH of 5.0 to 6.0, then adding chitinase with 0.5% to 1% of the mass of the dry powder of Chrysanthemum morifolium Ramat., and carrying out enzymolysis at 40 to 45 for 1 to 2 hours; then increasing the temperature to 60 to 65, adding ficin with 0.5% to 1% of the mass of the dry powder of Chrysanthemum morifolium Ramat., and carrying out enzymolysis at 60 to 65 for 1.5 to 2 hours; increasing the temperature to inactivate the enzyme, decreasing the temperature to 45 to 50, adjusting the pH to 7.0 to 7.5, adding serratia peptidase with 0.5% to 1% of the mass of the dry powder of Chrysanthemum morifolium Ramat., and carrying out enzymolysis at 45 to 50 for 1 to 1.5 hours; increasing the temperature to inactivate the enzyme, decreasing the temperature to room temperature, centrifuging, taking supernatant, reducing pressure and concentrating, loading onto a G-15 dextran gel column, eluting with deionized water for 3 to 4 column volumes, collecting the eluate, reducing pressure and concentrating at 50 to 55, and freeze-drying to obtain the extract peptide of Chrysanthemum morifolium Ramat.

[0011] In the preparation method of the extract peptide of Chrysanthemum morifolium Ramat., the particle size of the dry powder of Chrysanthemum morifolium Ramat. is below 60 to 80 mesh; the temperature for inactivating the enzyme is increased to 85 to 90 for 10 to 15 minutes; the centrifugation is carried out at 6000 to 8000 rpm for 10 to 20 minutes; the supernatant is reduced pressure and concentrated to 30% to 50% of the volume before loading; and the temperature for reducing pressure and concentrating is 50 to 55.

[0012] In the composition, the preparation method of the ganoderic acid-extracellular polysaccharide complex comprises the following steps: inoculating Ganoderma multiflorum spore liquid into liquid medium with pH of 4.5 to 5.0, and carrying out culture and aeration at 25 to 28 and 200 to 250 rpm for 4 to 6 days; adding squalane, and continuing to culture for 2 to 3 days to obtain fermentation liquor; centrifuging the fermentation liquor to collect water phase and mycelium precipitate; adding deionized water to the mycelium precipitate, adjusting the pH to 5.0 to 5.5, adding chitinase with 0.8% to 1.2% of the mass of the mycelium precipitate and β-glucanase with 0.8% to 1.2% of the mass of the mycelium precipitate, and carrying out enzymolysis at 40 to 45 for 3 to 4 hours; inactivating the enzyme, decreasing the temperature to room temperature to obtain enzyme hydrolysate, centrifuging the enzyme hydrolysate, and taking supernatant; combining the supernatant with the water phase, and ultrasonicating to obtain activated liquid; loading the activated liquid onto an AB-8 macroporous resin column, eluting with 10% to 15% volume concentration of ethanol aqueous solution for 2 to 3 column volumes to remove impurities, eluting with 50% to 60% volume concentration of ethanol aqueous solution for 2 to 3 column volumes, collecting the eluate, removing ethanol by reducing pressure and concentrating, and freeze-drying to obtain the ganoderic acid-extracellular polysaccharide complex.

[0013] In the preparation method of the ganoderic acid-exopolysaccharide complex, the liquid culture of the T. sinensis is obtained by culturing mycelium of the T. sinensis in a PDA slant medium and then culturing the mycelium in a liquid medium; the inoculation amount of the liquid culture of the T. sinensis is 8% to 12% of the volume of the liquid medium; the olive squalane is sterilized; the addition amount of the olive squalane is 6% to 10% of the volume of the liquid medium; the centrifugation of the fermentation broth is first performed at 3000 rpm to 4000 rpm for 10 min to 15 min, and then performed at 5500 rpm to 6000 rpm for 15 min to 20 min; the amount of the deionized water is 10 times to 12 times of the mass of the mycelium precipitate; the enzyme inactivation is performed at 85°C to 90°C for 10 min to 15 min; the centrifugation of the enzyme solution is performed at 5500 rpm to 6000 rpm for 15 min to 20 min; the ultrasonic treatment is performed at 4°C to 6°C, 200 W to 250 W and 20 kHz to 25 kHz for 3 min to 5 min; and the temperature of the reduced-pressure concentration is 45°C to 50°C.

[0014] In the composition, the preparation method of the Dalbergia hupeana flower extract includes the following steps: adding Dalbergia hupeana flower powder and deionized water into a high-pressure reaction kettle at a solid-liquid ratio of 1 g: 15 mL to 20 mL, extracting at a temperature of 110°C to 120°C, a pressure of 0.3 MPa to 0.4 MPa and a stirring speed of 100 rpm to 150 rpm for 30 min to 50 min to obtain an extraction liquid, centrifuging the extraction liquid, taking the supernatant, reducing the pressure and concentrating, loading the concentrated solution onto a D101 macroporous resin column, washing the column with deionized water to remove impurities, eluting the column with 65% to 70% ethanol aqueous solution, collecting the eluate, removing ethanol by reducing the pressure and concentrating, and freeze-drying to obtain the Dalbergia hupeana flower extract.

[0015] In the preparation method of the Dalbergia hupeana flower extract, the particle size of the Dalbergia hupeana flower powder is less than 40 mesh to 60 mesh; the centrifugation is performed at 4000 rpm to 5000 rpm for 15 min to 20 min; the supernatant is concentrated to 30% to 50% of the volume by reducing the pressure before loading; and the temperature of the reduced-pressure concentration is 50°C to 55°C.

[0016] The preparation method of the composition for repairing damaged skin cells includes the following steps: The G. glabra root extract, the Cicindela chinensis flower extracted peptide, the ganoderic acid-exopolysaccharide complex and the Dalbergia hupeana flower extract are mixed at a mass ratio to obtain the composition.

[0017] The composition for repairing damaged skin cells is compounded with ingredients that are pharmaceutically or cosmetically acceptable to prepare a product for repairing damaged skin cells.

[0018] The application provides a composition for repairing damaged skin cells and a preparation method thereof, and beneficial effects include: Firstly, the composition of the application can precisely act on the key pathological link of damaged skin cells to achieve safe, mild and efficient repair function. The core mechanism includes: activating the self-repair signal pathway of damaged cells (such as keratinocytes and fibroblasts), promoting normal cell proliferation and differentiation, accelerating skin cell renewal to restore the structure of the stratum corneum and the dermis; inhibiting the transcription and secretion of inflammatory factors (such as TNF-α and IL-6), reducing the inflammatory reaction caused by hormone face; promoting fibroblasts to synthesize collagen, repairing damaged cells, restoring barrier function, breaking the vicious cycle of hormone dependence, and improving hormone face.

[0019] Secondly, the root extract of Glycyrrhiza glabra: During the preparation process, the root core powder ensures sufficient release of active ingredients, hot water is used for preliminary removal of water-soluble impurities, and an ultrasonic high-efficiency extraction of active ingredients is carried out by using an ethanol aqueous solution containing choline chloride and lactic acid, and A21 macroporous adsorption resin is used for precise purification to improve the purity of active ingredients. These parameters lay a foundation for the extract to have efficient anti-inflammatory and protective effects on cells against oxidative damage.

[0020] Thirdly, the extract peptide of Bauhinia championii: Chitinase is used for preliminary enzymolysis to release more active substances, no flower fruit protease is used for proteolysis, and Serratia peptidase is used for depth enzymolysis to cooperatively generate small molecule active peptides; G-15 dextran gel is used for precise retention of active peptides and removal of impurity peptides. The process ensures that the active peptides can efficiently bind to cell surface receptors, activate proliferation signals and inhibit the transcription of inflammatory factors.

[0021] Fourthly, the ganoderic acid-extracellular polysaccharide complex: After the T. sinense is cultured in a liquid medium, olive squalane is added for induction to promote the synthesis of ganoderic acid and the secretion of extracellular polysaccharides; chitinase and β-glucanase are used for co-enzymolysis of mycelium to release active ingredients, and AB-8 macroporous resin is used for balanced adsorption of ganoderic acid and polysaccharides. These steps ensure that the proportion of each component in the complex is appropriate, and the complex can synergistically play the roles of anti-inflammatory, stabilization of extracellular matrix and promotion of collagen synthesis.

[0022] Fifthly, the extract of Bauhinia championii flower: High-pressure extraction is used to efficiently extract volatile oil and flavonoids, and D101 macroporous resin is used for purification to retain effective ingredients; an appropriate amount of extract can improve skin microcirculation, assist in enhancing the anti-inflammatory effect, and does not damage the cell structure.

[0023] Six, the four components complement and synergize each other in function, rather than the superposition of single component effect. The three of Glycyrrhiza glabra L. root extract, Dalbergia hupeana extract and Cicada superantigen peptide synergize to create a good microenvironment for cell proliferation and repair. Cicada superantigen peptide activates the proliferation signal of keratinocytes and fibroblasts, in the Ganoderma lucidum acid-extracellular polysaccharide complex, extracellular polysaccharide provides stable matrix support for cell proliferation, and ganoderma acid helps to reduce inflammation to protect cell activity; Glycyrrhiza glabra L. root extract protects cells from oxidative damage, realizes the complete repair chain of "anti-inflammatory-proliferation-repair", and the four components are used in a specific ratio to significantly improve the repair effect, and avoid the imbalance of the overall function caused by the excess or deficiency of a single component. DETAILED DESCRIPTION

[0024] The application will be further described below in combination with specific implementation examples, but the application is not limited to these examples.

[0025] Example 1 A composition for repairing damaged skin cells, the composition has a mass ratio of 5:2.5:4.2:1.5 of Glycyrrhiza glabra L. root extract, Cicada superantigen peptide, Ganoderma lucidum acid-extracellular polysaccharide complex and Dalbergia hupeana extract.

[0026] In this embodiment, the preparation method of Glycyrrhiza glabra L. root extract includes: taking the root core coarse powder (50 mesh undersize) of Glycyrrhiza glabra L., adding 7 times the mass of 85℃ hot water to soak for 25 min, filtering to take the filter cake; using an ethanol aqueous solution (the volume ratio of ethanol to water is 5.5:4.5) to prepare a mixed solution containing 5.5wt% choline chloride and 7.5wt% lactic acid; adding the filter cake into the mixed solution according to the solid-liquid ratio of 1g:18mL, ultrasonic at 55℃ for 25 min, centrifuging at 7000rpm for 12 min, taking the liquid, and concentrating and removing ethanol at 55℃, and loading to an A21 macroporous adsorption resin column, washing with deionized water for 1.5 column volumes, then eluting with 25% volume concentration ethanol aqueous solution for 2.5 column volumes, collecting the eluate, concentrating and removing ethanol at 55℃, and freeze-drying to obtain Glycyrrhiza glabra L. root extract.

[0027] In this embodiment, the preparation method of the extract peptide of F. formosanum includes: adding the dry powder of F. formosanum (60-mesh undersize) into 11 times of the mass of pH 5.5 phosphate buffer solution of the dry powder of F. formosanum, then adding 0.8% of the mass of chitinase of the dry powder of F. formosanum, and carrying out enzymolysis at 42°C for 1.5 h; then increasing the temperature to 62°C, adding 0.8% of the mass of bromelain of the dry powder of F. formosanum, and carrying out enzymolysis at 62°C for 1.5 h; increasing the temperature to 88°C to inactivate the enzyme for 12 min, decreasing to 48°C, adjusting the pH to 7.3, adding 0.8% of the mass of serratia peptidase of the dry powder of F. formosanum, and carrying out enzymolysis at 48°C for 1 h; increasing the temperature to 88°C to inactivate the enzyme for 12 min, and then decreasing to room temperature; centrifuging at 7000 rpm for 15 min, and taking the supernatant; concentrating the supernatant to 40% of the volume at 52°C under reduced pressure, and then loading onto a G-15 dextran gel column; eluting with 3.5 column volumes of deionized water, collecting the eluate, concentrating at 52°C under reduced pressure, and then freeze-drying to obtain the extract peptide of F. formosanum.

[0028] In this embodiment, the preparation method of the ganoderic acid-exopolysaccharide complex includes: inoculating the activated culture of the liquid medium of the culture of N. sinense with 10% of the volume of the liquid medium, and then inoculating the sterilized liquid medium in a liquid medium (after sterilization treatment) at 27°C, 220 rpm stirring and aeration for 5 days; adding 8% of the volume of the sterilized liquid medium of the squalane, and continuing to culture for 2.5 days to obtain a fermentation broth; centrifuging the fermentation broth at 3500 rpm for 12 min, and then centrifuging at 5800 rpm for 18 min to collect the water phase (supernatant) and mycelium precipitate; adding 11 times of the mass of deionized water to the mycelium precipitate, adjusting the pH to 5.2, adding 1.0% of the mass of chitinase and 1.0% of the mass of β-glucanase of the mycelium precipitate, and carrying out enzymolysis at 42°C for 3.5 h, and then inactivating the enzyme at 88°C for 12 min, and then decreasing to room temperature to obtain an enzymolysis liquid; centrifuging the enzymolysis liquid at 5800 rpm for 18 min, and then taking the supernatant; combining the supernatant with the water phase, and then ultrasonicating at 5°C, 200 W and 20 kHz for 4 min to obtain an activation liquid; loading the activation liquid onto an AB-8 macroporous resin column, eluting with 2.5 column volumes of 12% volume concentration of ethanol aqueous solution to remove impurities, and then eluting with 2.5 column volumes of 55% volume concentration of ethanol aqueous solution, collecting the eluate, removing ethanol at 48°C under reduced pressure, and then freeze-drying to obtain the ganoderic acid-exopolysaccharide complex.

[0029] In the formula, the formula of the liquid medium includes: 40 g / L of glucose, 8 g / L of proteose peptone, 3 g / L of yeast extract powder, 1.5 g / L of potassium dihydrogen phosphate, and 0.8 g / L of magnesium sulfate heptahydrate, the solvent is deionized water, and the pH value is adjusted to 4.8; the liquid medium is sterilized (121°C high-pressure sterilization for 25 min) before use.

[0030] The activation culture method of the Guanacaste croaker spawn liquid comprises the following steps: 210 g of peeled and diced potatoes are added into 0.7 L of water and boiled for 25 min, and then filtered; the filtrate is taken out, 21 g of glucose and 18 g of agar are added, and then heated and melted; after being diluted to 1 L with deionized water, the pH value is adjusted to 4.8, and then the mixture is divided into test tubes and sterilized at 121 ℃ for 25 min; a slope is prepared, and then cooled and solidified to obtain a PDA slope culture medium; the Guanacaste croaker spawn is inoculated on the PDA slope culture medium, and then cultured at 27 ℃ in the dark for 6 days to obtain mycelium; the mycelium is inoculated into a triangular flask, each triangular flask is filled with 200 mL of the above-mentioned formula liquid culture medium which has been sterilized in advance, and then cultured at 27 ℃ and 180 rpm for 3.5 days to obtain the Guanacaste croaker spawn liquid after activation culture.

[0031] In the embodiment, the preparation method of the Dalbergia hupeana flower extract comprises the following steps: Dalbergia hupeana flower powder (50-mesh undersize) and deionized water are added into a high-pressure reaction kettle at a solid-liquid ratio of 1 g:18 mL, and then extracted at a temperature of 115 ℃, a pressure of 0.35 MPa and a stirring speed of 120 rpm for 40 min to obtain an extraction liquid; the extraction liquid is centrifuged at 4500 rpm for 18 min, and then the supernatant is taken out and concentrated to 40% of the volume at 52 ℃ under reduced pressure; the supernatant is loaded onto a D101 macroporous resin column, eluted with deionized water for 2.5 column volumes to remove impurities, and then eluted with 68% volume concentration ethanol aqueous solution for 3.5 column volumes; the eluate is collected, the ethanol is removed at 52 ℃ under reduced pressure, and then freeze-dried to obtain the Dalbergia hupeana flower extract.

[0032] Embodiment 2 A composition for repairing damaged skin cells, the composition comprising glycyrrhiza glabra root extract, chrysochroa rhodostoma flower extract peptide, ganoderma acid-extracellular polysaccharide complex and dalbergia hupeana flower extract at a mass ratio of 4:3:3.5:2.

[0033] In the embodiment, the preparation method of the glycyrrhiza glabra root extract comprises the following steps: the core powder (40-mesh undersize) of the roots of glycyrrhiza glabra is soaked in 8 times the mass of hot water at 80 ℃ for 30 min, and then filtered to obtain the filter cake; a mixed solution containing 5 wt% choline chloride and 8 wt% lactic acid is prepared by using an ethanol aqueous solution (the volume ratio of ethanol to water is 5:5); the filter cake is added into the mixed solution at a solid-liquid ratio of 1 g:15 mL, and then ultrasonically treated at 60 ℃ and 200 W for 30 min; the liquid is obtained by centrifuging at 6000 rpm for 15 min, and then concentrated to remove ethanol at 50 ℃ under reduced pressure; the liquid is loaded onto an A21 macroporous adsorption resin column, eluted with deionized water for 2 column volumes, and then eluted with 20% volume concentration ethanol aqueous solution for 3 column volumes; the eluate is collected, the ethanol is removed at 50 ℃ under reduced pressure, and then freeze-dried to obtain the glycyrrhiza glabra root extract.

[0034] In this embodiment, the preparation method of the peptide extracted from Flos Cnidi includes: adding the Flos Cnidi dry powder (80 mesh undersize) into the 10 times mass of pH 6.0 phosphate buffer solution of the Flos Cnidi dry powder, then adding 0.5% chitinase of the mass of the Flos Cnidi dry powder, and carrying out enzymolysis at 45℃ for 1 h; then increasing the temperature to 65℃, adding 0.5% bromelain of the mass of the Flos Cnidi dry powder, and carrying out enzymolysis at 65℃ for 1.5 h; increasing the temperature to 90℃ to inactivate the enzyme for 10 min, decreasing to 50℃, adjusting the pH to 7.0, adding 1% serratia peptidase of the mass of the Flos Cnidi dry powder, and carrying out enzymolysis at 50℃ for 1 h; increasing the temperature to 90℃ to inactivate the enzyme for 10 min, and then centrifuging at 8000 rpm for 10 min after decreasing to room temperature; taking the supernatant; concentrating the supernatant to 30% of the volume at 55℃ under reduced pressure; loading onto a G-15 dextran gel column; eluting with 4 column volumes of deionized water; collecting the eluate; concentrating at 50℃ under reduced pressure; and freeze-drying to obtain the peptide extracted from Flos Cnidi.

[0035] In this embodiment, the preparation method of the ganoderic acid-extracellular polysaccharide complex includes: inoculating the activated culture of the T. sinense strain liquid into a liquid culture medium (after sterilization treatment) at 12% of the volume of the liquid culture medium, and culturing at 25℃ under 250 rpm stirring and aeration for 4 days; adding 10% of the volume of the liquid culture medium of the sterilized squalane, and continuing to culture for 2 days to obtain a fermentation broth; centrifuging the fermentation broth at 4000 rpm for 10 min, and then centrifuging at 6000 rpm for 15 min to collect the water phase (supernatant) and mycelium precipitate; adding 12 times the mass of deionized water to the mycelium precipitate, adjusting the pH to 5.0, adding 1.2% chitinase and 0.8% β-glucanase of the mass of the mycelium precipitate, and carrying out enzymolysis at 45℃ for 3 h, inactivating the enzyme at 90℃ for 10 min, and then decreasing to room temperature to obtain an enzymolysis liquid; centrifuging the enzymolysis liquid at 6000 rpm for 15 min, and taking the supernatant; combining the supernatant with the water phase, and ultrasonicating at 6℃, 200 W, and 25 kHz for 3 min to obtain an activation liquid; loading the activation liquid onto an AB-8 macroporous resin column, eluting with 2 column volumes of 15% volume concentration ethanol aqueous solution to remove impurities, and then eluting with 2 column volumes of 60% volume concentration ethanol aqueous solution, collecting the eluate, removing ethanol at 50℃ under reduced pressure, and freeze-drying to obtain the ganoderic acid-extracellular polysaccharide complex.

[0036] The formula of the liquid culture medium includes: 30 g / L of glucose, 5 g / L of proteose peptone, 2 g / L of yeast extract, 1 g / L of potassium dihydrogen phosphate, and 0.5 g / L of magnesium sulfate heptahydrate, the solvent is deionized water, and the pH value is adjusted to 4.5; and the liquid culture medium is sterilized (121℃ high-pressure sterilization for 20 min) before use.

[0037] The activation culture method of the Guanacaste croaker spawn liquid comprises the following steps: 200 g of peeled and diced potatoes are added into 0.6 L of water and boiled for 20 min, and then filtered; the filtrate is taken out, 20 g of glucose and 15 g of agar are added, and then heated and melted; the melted solution is diluted to 1 L with deionized water, and the pH value is adjusted to 4.5; the solution is divided into test tubes and sterilized at 121 ℃ for 20 min; the slope culture medium is prepared; the Guanacaste croaker spawn is inoculated on the PDA slope culture medium and cultured at 25 ℃ in the dark for 5 days; and the mycelium is obtained.

[0038] In the embodiment, the preparation method of the Dalbergia hupeana flower extract comprises the following steps: Dalbergia hupeana flower powder (40-mesh undersize) and deionized water are added into a high-pressure reaction kettle at a solid-liquid ratio of 1 g:20 mL, and extracted at a temperature of 110 ℃, a pressure of 0.4 MPa and a stirring speed of 100 rpm for 50 min to obtain an extraction liquid; the extraction liquid is centrifuged at 4000 rpm for 20 min, and the supernatant is taken out and concentrated to 50% of the volume at 50 ℃ under reduced pressure; the concentrated solution is loaded onto a D101 macroporous resin column, eluted with deionized water for 2 column volumes to remove impurities, and then eluted with 70% volume concentration ethanol aqueous solution for 3 column volumes; the eluate is collected, and the ethanol is removed under reduced pressure at 55 ℃; and the Dalbergia hupeana flower extract is obtained by freeze-drying.

[0039] Example 3 A composition for repairing damaged skin cells, the composition comprising glycyrrhiza glabra root extract, chrysalidescu flower extract peptide, ganoderma acid-extracellular polysaccharide complex and dalbergia hupeana flower extract at a mass ratio of 6:2:5:1.

[0040] In the embodiment, the preparation method of the glycyrrhiza glabra root extract comprises the following steps: the core powder (60-mesh undersize) of the roots of glycyrrhiza glabra is soaked in hot water at 90 ℃ for 20 min at a solid-liquid ratio of 1 g:20 mL, and then filtered to obtain the filter cake; an ethanol aqueous solution (ethanol and water at a volume ratio of 6:4) containing 6 wt% of choline chloride and 7 wt% of lactic acid is prepared; the filter cake is added into the mixed solution at a solid-liquid ratio of 1 g:20 mL, and then ultrasonically treated at 50 ℃ and 250 W for 20 min; the liquid is obtained by centrifuging at 8000 rpm for 10 min, and then concentrated to remove ethanol at 60 ℃ under reduced pressure; and the glycyrrhiza glabra root extract is obtained by loading the concentrated solution onto an A21 macroporous adsorption resin column, eluting with deionized water for 1 column volume, eluting with 30% volume concentration ethanol aqueous solution for 2 column volumes, collecting the eluate, removing ethanol under reduced pressure at 60 ℃, and freeze-drying.

[0041] In this embodiment, the preparation method of the extract peptide of Chrysochameliidae includes: adding the dry powder of Chrysochameliidae (60 mesh undersize) into the phosphate buffer solution with pH 5.0 at 12 times the mass of the dry powder of Chrysochameliidae, then adding chitinase with 1% of the mass of the dry powder of Chrysochameliidae, and carrying out enzymolysis at 40℃ for 2 hours; then increasing the temperature to 60℃, adding the bromelain with 1% of the mass of the dry powder of Chrysochameliidae, and carrying out enzymolysis at 60℃ for 2 hours; increasing the temperature to 85℃ to inactivate the enzyme for 15 minutes, decreasing to 45℃, adjusting the pH to 7.5, adding the serratia peptidase with 0.5% of the mass of the dry powder of Chrysochameliidae, and carrying out enzymolysis at 45℃ for 1.5 hours; increasing the temperature to 85℃ to inactivate the enzyme for 15 minutes, and then decreasing to room temperature; centrifuging at 6000 rpm for 20 minutes; taking the supernatant; concentrating the supernatant at 50℃ under reduced pressure to 50% of the volume; loading onto a G-15 dextran gel column; eluting with deionized water for 3 column volumes; collecting the eluate; concentrating at 55℃ under reduced pressure; and freeze-drying to obtain the extract peptide of Chrysochameliidae.

[0042] In this embodiment, the preparation method of the ganoderic acid-extracellular polysaccharide complex includes: inoculating the activated culture of the Antrodia camphorata strain liquid into the liquid culture medium (after sterilization) at 8% of the volume of the liquid culture medium, and culturing at 28℃ under 200 rpm stirring and aeration for 6 days; adding the sterilized squalane with 6% of the volume of the liquid culture medium, and continuing to culture for 3 days to obtain a fermentation broth; centrifuging the fermentation broth at 3000 rpm for 15 minutes, and then at 5500 rpm for 20 minutes to collect the water phase (supernatant) and mycelium precipitate; adding deionized water with 10 times the mass of the mycelium precipitate, adjusting the pH to 5.5, adding chitinase with 0.8% of the mass of the mycelium precipitate and β-glucanase with 1.2% of the mass of the mycelium precipitate, and carrying out enzymolysis at 40℃ for 4 hours; inactivating the enzyme at 85℃ for 15 minutes, and then decreasing to room temperature to obtain an enzymolysis liquid; centrifuging the enzymolysis liquid at 5500 rpm for 20 minutes, and taking the supernatant; combining the supernatant with the water phase, and ultrasonicating at 4℃, 250 W, and 20 kHz for 5 minutes to obtain an activation liquid; loading the activation liquid onto an AB-8 macroporous resin column, eluting with 10% volume concentration of ethanol aqueous solution for 3 column volumes to remove impurities, and then eluting with 50% volume concentration of ethanol aqueous solution for 3 column volumes; collecting the eluate, removing ethanol at 45℃ under reduced pressure, and freeze-drying to obtain the ganoderic acid-extracellular polysaccharide complex.

[0043] In the formula, the formula of the liquid culture medium includes: 50 g / L of glucose, 10 g / L of protein peptone, 5 g / L of yeast extract powder, 2 g / L of potassium dihydrogen phosphate, and 1 g / L of magnesium sulfate heptahydrate, the solvent is deionized water, and the pH value is adjusted to 5.0; the liquid culture medium is sterilized (121℃ high-pressure sterilization for 30 minutes) before use.

[0044] The activation culture method of the Guanacaste crinum bulbifera strain liquid comprises the following steps: 220 g of peeled and diced potatoes are added into 0.8 L of water and boiled for 30 min, then filtered, and the filtrate is obtained; 22 g of glucose and 20 g of agar are added into the filtrate, and after being heated and melted, the volume is adjusted to 1 L with deionized water, the pH is adjusted to 5.0, and then the mixture is divided into test tubes and sterilized at 121 ℃ for 30 min; the test tubes are arranged into slopes, and after being cooled and solidified, PDA slope culture medium is obtained; the Guanacaste crinum bulbifera strain is inoculated on the PDA slope culture medium, and the culture is carried out at 28 ℃ in the dark for 7 days to obtain mycelium; the mycelium is inoculated into a triangular flask, each triangular flask contains 250 mL of the above-mentioned formula liquid culture medium which has been sterilized in advance, and the culture is carried out at 28 ℃ and 200 rpm for 4 days to obtain the Guanacaste crinum bulbifera strain liquid after activation culture.

[0045] In the embodiment, the preparation method of the Dalbergia hupeana flower extract comprises the following steps: Dalbergia hupeana flower powder (60-mesh undersize) and deionized water are added into a high-pressure reaction kettle at a solid-liquid ratio of 1 g:15 mL, and extraction is carried out at a temperature of 120 ℃, a pressure of 0.3 MPa and a stirring speed of 150 rpm for 30 min to obtain an extraction liquid; the extraction liquid is centrifuged at 5000 rpm for 15 min, the supernatant is taken, and the volume is reduced to 30% at 55 ℃ under reduced pressure; the supernatant is loaded onto a D101 macroporous resin column, impurities are removed by elution with deionized water for 3 column volumes, and then the Dalbergia hupeana flower extract is obtained by eluting with 65% (volume concentration) ethanol aqueous solution for 4 column volumes, removing ethanol under reduced pressure at 50 ℃ and freeze-drying.

[0046] The preparation method of the composition for repairing damaged skin cells in each of the above embodiments comprises the following steps: The Glycyrrhiza glabra root extract, the Cicada peptin, the Ganoderma acid-extracellular polysaccharide complex and the Dalbergia hupeana flower extract are mixed according to the mass ratio of the components in each embodiment to obtain the composition of each embodiment.

[0047] The composition for repairing damaged skin cells in each of the above embodiments is compounded with ingredients that are pharmaceutically or cosmetically acceptable to prepare a product for repairing damaged skin cells.

[0048] Comparative Example 1 The difference from Example 1 is that the mass ratio of the Glycyrrhiza glabra root extract, the Cicada peptin, the Ganoderma acid-extracellular polysaccharide complex and the Dalbergia hupeana flower extract is changed to 7:0.5:4.2:1.5.

[0049] Comparative Example 2 The difference from Example 1 is that the mass ratio of the Glycyrrhiza glabra root extract, the Cicada peptin, the Ganoderma acid-extracellular polysaccharide complex and the Dalbergia hupeana flower extract is changed to 2:5.5:4.2:1.5.

[0050] Comparative Example 3 The difference from Example 1 is that the mass ratio of the Radix Glycyrrhizae extract, the Antheraea yamamai extract peptide, the Ganoderic acid-exopolysaccharide complex and the Dalbergia hupeana extract is changed to 1.5:2.5:4.2:5.

[0051] Comparative Example 4 The difference from Example 1 is that the mass ratio of the Radix Glycyrrhizae extract, the Antheraea yamamai extract peptide, the Ganoderic acid-exopolysaccharide complex and the Dalbergia hupeana extract is changed to 5:5.5:1.2:1.5.

[0052] Comparative Example 5 The difference from Example 1 is that the mass ratio of the Radix Glycyrrhizae extract, the Antheraea yamamai extract peptide, the Ganoderic acid-exopolysaccharide complex and the Dalbergia hupeana extract is changed to 5:0.5:6.2:1.5.

[0053] Comparative Example 6 The difference from Example 1 is that in the preparation method of the Radix Glycyrrhizae extract, the AB-8 macroporous resin is replaced by the A21 macroporous adsorption resin.

[0054] Comparative Example 7 The difference from Example 1 is that in the preparation method of the Antheraea yamamai extract peptide, the ficin is replaced by the papain.

[0055] Comparative Example 8 The difference from Example 1 is that in the preparation method of the Antheraea yamamai extract peptide, the serratiopeptidase is replaced by the bromelain.

[0056] Comparative Example 9 The difference from Example 1 is that in the preparation method of the Antheraea yamamai extract peptide, the G-15 dextran gel is replaced by the LH-60 dextran gel.

[0057] Comparative Example 10 The difference from Example 1 is that in the preparation method of the Ganoderic acid-exopolysaccharide complex, the squalane is not added.

[0058] Comparative Example 11 The difference from Example 1 is that in the preparation method of the Ganoderic acid-exopolysaccharide complex, the AB-8 macroporous resin is replaced by the D101 macroporous resin.

[0059] Comparative Example 12 The difference from Example 1 is that in the preparation method of the Dalbergia hupeana extract, the D101 macroporous resin is replaced by the A21 macroporous adsorption resin.

[0060] The raw materials used in the above examples and comparative examples are as follows: choline chloride is from Hebei Taohai Biotechnology Co., Ltd., food grade; lactic acid is from Anhui Wimao Biotechnology Co., Ltd., food grade lactic acid 80; A21 macroporous adsorption resin is from Shanghai Yuanye Biotechnology Co., Ltd., model S24709; kachnar flower is from Bozhou Huajunzi Biotechnology Co., Ltd.; chitinase is from Jiangsu Juren Biotechnology Co., Ltd., enzyme activity 100,000 U / g; ficin is from Nanjing Songuan Biotechnology Co., Ltd., enzyme activity 100,000 U / g; serralysin is from Shandong Aicai Biotechnology Co., Ltd., enzyme activity 100,000 U / g; G-15 dextran gel is from Shanghai Yuanye Biotechnology Co., Ltd., model S14030; amorphous antrodia camphorata is from Northeast Food and Drug Fungus Research Institute; olive squalane is from Guangzhou Haoting Fine Chemical Co., Ltd.; β-glucanase is from Guangdong Ousman Biotechnology Co., Ltd., enzyme activity 100,000 U / g; AB-8 macroporous resin is from Shanghai Yuanye Biotechnology Co., Ltd., model S30931; D101 macroporous resin is from Shanghai Yuanye Biotechnology Co., Ltd., model S14161; peptone and yeast extract powder are from Shandong Liangshan Peptone Bioproducts Co., Ltd.; agar is from Huizhou Ketian Biotechnology Co., Ltd.; papain is from Xi'an Kangpulais Biotechnology Co., Ltd., enzyme activity 100,000 U / g; bromelain is from Peizuo Biotechnology (Xi'an) Co., Ltd., enzyme activity 100,000 U / g; LH-60 dextran gel is from Shanghai Yuanye Biotechnology Co., Ltd., model S14038.

[0061] I. Barrier cell proliferation detection: Logarithmic growth phase HaCaT cells (human keratinocytes) and HDF cells (human dermal fibroblasts) were selected, counted after trypsin digestion at 0.25%, and inoculated into 96-well plates at a density of 5×10 3 cells per well, 100 μL of DMEM high glucose complete culture medium containing 10% FBS and 1% penicillin-streptomycin was added to each well. The culture plate was placed in a 37°C, 5% CO2 incubator for pre-culture for 24 hours to ensure that the cell adhesion rate was more than 85%.

[0062] Prepare test solution in advance: prepare 10 mg / mL composition stock solution (magnetic stirring for 30 minutes to completely dissolve) with the above complete culture medium, filter sterilize, and dilute to 200 μg / mL working solution. At the same time, set up three types of controls: negative control group containing only complete culture medium (with cells and no composition), blank control group containing only complete culture medium (no cells), and positive control group containing 10 ng / mL EGF.

[0063] Discard the old culture medium in the 96-well plate, add 100 μL of 200 μg / mL working solution to each well of the experimental group, add the corresponding culture medium to the negative control group and the positive control group, and add complete culture medium without cells to the blank control group, and set 6 replicate wells for each group. After returning to the incubator for continuous culture for 48 hours, add 10 μL of CCK-8 solution to each well, incubate in the dark for 2 hours, and measure the absorbance (OD value) of each well at 450 nm wavelength (reference wavelength 630 nm) using a microplate reader. Calculate the cell proliferation rate by the formula: cell proliferation rate (%) = [(OD experimental group-OD blank control group) / (OD negative control group-OD blank control group)] x 100%. If the proliferation rate exceeds 100%, it indicates that the composition can promote cell proliferation, and the higher the value, the stronger the promotion effect.

[0064] CCK-8 detection can indirectly reflect cell metabolic activity and quantity, but not direct cell counting. Therefore, the proliferation rate is a relative index, which is usually positively correlated with cell number. It can also reflect whether the ingredients are toxic to cells and whether they are safe and reliable.

[0065] II. Inflammatory factor expression detection: RAW264.7 macrophages were used as the research object and inoculated into a 24-well plate at 5 x 10 4 cells per well, and 1 mL of DMEM culture medium containing 10% FBS was added. The cells were cultured in a 37°C, 5% CO2 incubator for 24 hours.

[0066] Prepare the test solution in advance: prepare a 10 mg / mL composition stock solution (magnetic stirring for 30 minutes to dissolve) with DMEM culture medium containing 10% FBS, filter sterilize, and dilute to a 200 μg / mL working solution. The experiment is divided into three groups: a blank control group containing only culture medium (without LPS and composition), an inflammation model group containing 1 μg / mL LPS (without composition), and an experimental group containing 1 μg / mL LPS and 200 μg / mL test solution, with 3 replicate wells for each group.

[0067] Discard the old culture medium in the 24-well plate, add the corresponding solution according to the grouping, and put it back into the incubator for continuous culture for 24 hours. Then collect the supernatant of each well, centrifuge at 3000 rpm (centrifugal radius 8 cm) for 5 minutes, and take the supernatant for standby. According to the operation instruction of TNF-α, IL-6 ELISA kit, add the supernatant and standard to the reaction well, incubate at 37°C for 60 minutes, wash after incubation, add enzyme-labeled second antibody for incubation for 30 minutes, add substrate for color development for 15 minutes, add stop solution to terminate the reaction, and determine the absorbance at 450 nm wavelength (reference wavelength 570 nm) by enzyme-labeled instrument. According to the standard curve, calculate the concentration of each well of inflammatory factor, and evaluate the anti-inflammatory effect by the formula inflammatory factor inhibition rate (%) = [1-(experimental group factor concentration-blank control group factor concentration) / (LPS inflammatory model group factor concentration-blank control group factor concentration)]x100%. The higher the inhibition rate, the stronger the anti-inflammatory effect of the composition.

[0068] III. Collagen synthesis detection (Sirius Red staining method): Take human dermal fibroblasts (HDF) as the research object, inoculate them into a 24-well plate, 5x10 4 4 cells per well, add 1 mL of DMEM culture medium containing 10% FBS, replace it with culture medium containing 1 μM Dex after 24 hours of culture, and continue to culture for 24 hours to establish a hormone damage model.

[0069] Preparation of test solution: 10 mg / mL composition stock solution was prepared with DMEM medium containing 10% FBS (magnetic stirring for 30 minutes to dissolve), and then diluted to 200 μg / mL working solution after sterilization by filtration. After the model was established, the Dex-containing medium was discarded, 1 mL of working solution was added to the experimental group, and a model control group (only fresh medium was added after Dex injury without composition) and a normal control group (no Dex injury, fresh medium without composition was added) were set up, 3 replicates per group, and the culture was continued for 72 hours. After the culture ended, the cells were gently washed twice with PBS, 1 mL of 4% paraformaldehyde was added to each well for room temperature fixation for 30 minutes; after fixation, the cells were washed with deionized water for 3 times, 1 mL of 0.1% Sirius Red dye (dissolved in a saturated solution of picric acid with pH 2.0) was added, and the cells were dyed at room temperature for 1 hour in the dark; the dye was removed, the cells were washed with 0.1M hydrochloric acid for 3 times (5 minutes each time) to remove the unbound dye, and then 1 mL of 0.1M NaOH / methanol solution (volume ratio 1:1) was added, and the cells were shaken at room temperature for 10 minutes to desorb the dye bound to the collagen. The desorption solution was transferred to a 96-well plate (3 measurements were made for each sample, 100 μL each time), and the absorbance (OD value) was measured at 540 nm wavelength by using an enzyme-labeled instrument. The amount of collagen synthesis was evaluated by comparing the OD values: normal control group > composition group > model control group, which indicated that the composition could promote the synthesis of type I collagen (COL-I) in the injured HDF cells, and the higher the OD value, the stronger the repair effect.

[0070] Table 1 test results (average value)

[0071] From the above results, the data results of Example 1 to Example 3 are better, and the core reason is that they strictly follow the "optimal component ratio + effective preparation process", realize the synergistic effect of each active ingredient, and avoid the functional imbalance caused by the excess or deficiency of a single component, so as to ensure the efficient play of the three core indicators of cell proliferation, inflammation inhibition and collagen synthesis. The effective preparation and purification process ensures high purity and high activity of each active ingredient.

[0072] Comparative Example 1: The core function of the Chrysoecia flava extract peptide is to activate the signal pathway, up-regulate the expression of proliferation-related proteins, and reduce the release of inflammatory factors by inhibiting transcription factors. When the proportion decreases, the active peptide concentration is not enough to effectively bind to the cell surface receptor, the conversion is blocked, the proliferation rate decreases; at the same time, the transcription and secretion of TNF-α and IL-6 are enhanced, and the inflammation inhibition rate is greatly reduced. In addition, the insufficient activity of fibroblasts directly leads to a decrease in the expression of genes required for collagen synthesis, and the OD value decreases.

[0073] The proportion of the main antioxidant and anti-inflammatory active substances of the extract of Radix Glycyrrhizae in the comparative example 2 is reduced, and the amount of intracellular ROS accumulation increases. On the one hand, the apoptosis rate of cells increases, and the proliferation rate decreases; on the other hand, the activity of key enzymes for collagen synthesis in cells is inhibited, leading to a blockage of the synthesis of collagen precursors. At the same time, the lack of anti-inflammatory ingredients weakens the ability to remove inflammatory factors, and the concentrations of TNF-α and IL-6 increase, and the inhibition rate decreases, and the synergistic effect of the whole composition is insufficient.

[0074] The volatile oil and flavonoid ingredients in the extract of Daidai flower in the comparative example 3 can improve microcirculation and assist in anti-inflammation when the amount is appropriate, but when the amount is excessive, the volatile oil can destroy the phospholipid bilayer structure of the cell membrane, leading to an increase in cell permeability and a decrease in cell adhesion ability, and the metabolism and proliferation of HaCaT and HDF cells are inhibited. At the same time, excessive flavonoids can competitively bind to cell surface receptors, blocking the signal pathway, and reducing the amount of collagen synthesis. In addition, the stress response caused by cell damage slightly promotes the secretion of inflammatory factors, further reducing the inflammation inhibition rate.

[0075] The ganoderic acid-extracellular polysaccharide complex in the comparative example 4 promotes the expression of genes related to collagen synthesis in cells, and the polysaccharide component can also enhance the structural stability of the extracellular matrix, providing support for cell proliferation. When the proportion is reduced, the transcription of collagen synthesis genes decreases; and the synergistic effect with other components is insufficient, leading to a decrease in various performances.

[0076] The lack of cell proliferation activation caused by the insufficient extract peptide of Jinchanhua in the comparative example 5 cannot be compensated by the excessive ganoderic acid-extracellular polysaccharide. Excessive extracellular polysaccharide can form a viscous polysaccharide film on the cell surface, hindering the entry of nutrients such as amino acids and glucose into the cells, leading to a blockage of energy metabolism and material synthesis of cells, and a decrease in the proliferation rate. At the same time, excessive ganoderic acid slightly stimulates cells, slightly increases the secretion of inflammatory factors, and reduces the inflammation inhibition rate. In addition, the insufficient supply of nutrients also leads to a decrease in the efficiency of cell collagen synthesis, and a low OD value.

[0077] The adsorption selectivity of A21 macroporous adsorption resin for active ingredients such as glycyrrhizic acid and flavonoids in Radix Glycyrrhizae is higher than that of AB-8 resin. AB-8 resin has a larger pore size, and is easy to adsorb polysaccharides, small molecule sugars and other impurities, leading to a low purity of glycyrrhizic acid in the final extract. After the purity of active ingredients decreases, the synergistic effect is weakened, and the impurities are harmful, the ability to remove ROS and inhibit the activation of NF-κB is weakened, the apoptosis rate of HaCaT cells increases, and the proliferation rate decreases; the activity of proline hydroxylase in HDF cells is insufficient, and the collagen synthesis decreases; and the inflammation inhibition rate also decreases due to the decrease in the concentration of anti-inflammatory ingredients.

[0078] Example 7: The enzyme cleavage site of ficin has high matching degree with the amino acid sequence of the protein of Flos Cervi, and can generate effective small molecule active peptides. These peptides can efficiently enter cells to play a role. However, the specific active peptides in the papain enzyme hydrolysate are insufficient, and the impurity peptides also have interference hazards. The decrease in the concentration and effective activity of active peptides leads to insufficient cell proliferation activation, weakened inflammation factor inhibition, and lower indicators.

[0079] Example 8: The secondary enzyme hydrolysis of serratiopeptidase combined with ficin has good enzyme hydrolysis synergy and can obtain effective polypeptides. Bromelain can also hydrolyze proteins and peptides, but it has poor synergy with ficin for the enzyme hydrolysis of Flos Cervi extracted peptides. The change in the secondary enzyme hydrolysis cleavage site leads to a decrease in effective polypeptides and the generation of impurity peptides, resulting in changes in active peptides in the enzyme hydrolysis product. Therefore, the cell proliferation rate decreases slightly due to insufficient active peptides, and the inflammation inhibition rate and other indicators further decrease due to different peptide effects and the lack of double anti-inflammatory mechanism.

[0080] Example 9: G-15 dextran gel can accurately retain active peptides in Flos Cervi while removing macromolecular impurities. The separation range of LH-60 gel is 1000-100000 Da, and the separation range is different, resulting in a lower retention rate of small molecule active peptides, a decrease in the purity of active peptides in the final extract, and an increase in the content of macromolecular peptides and impurities. Although the decrease in the purity of active peptides does not change the structure and activity of the peptides, the decrease in the concentration leads to a decrease in the efficiency of cell signal activation, and the indicators are lower.

[0081] Example 10: Olive squalane, as a secondary metabolic inducer of Antrodia cinnamomea, can promote ganoderic acid synthesis; at the same time, it can improve the permeability of the cell membrane and increase the secretion of extracellular polysaccharides. Without olive squalane, the yield and purity of ganoderic acid and the yield of extracellular polysaccharides decrease, leading to a decrease in the anti-inflammatory activity and collagen synthesis promotion activity of the complex. Therefore, the inflammation inhibition rate decreases significantly due to insufficient ganoderic acid, and the collagen synthesis OD value decreases due to insufficient polysaccharide support.

[0082] Example 11: AB-8 resin has balanced adsorption selectivity for lipid-soluble ganoderic acid and water-soluble extracellular polysaccharides, and can realize the synergistic purification of the two. D101 resin is more prone to adsorb water-soluble impurities such as monosaccharides and amino acids, leading to a decrease in the elution rate of ganoderic acid and an imbalance in the ratio of ganoderic acid to polysaccharides in the complex (excessive polysaccharides). Insufficient ganoderic acid leads to a decrease in anti-inflammatory activity and inflammation inhibition rate; excessive polysaccharides have limited promotion effect on collagen synthesis and cannot compensate for the functional defects of ganoderic acid, resulting in a decrease in cell proliferation rate and collagen synthesis OD value.

[0083] Comparative Example 12: The adsorption capacity of D101 resin for flavonoids and volatile oil in Gardenia jasminoides Ellis is better than that of A21 resin. The adsorption rate of A21 resin for volatile oil with lower polarity is low, the effective components are lost, the composition of the product changes after purification, and the indicators decrease.

Claims

1. A composition for repairing damaged skin cells, characterized in that, The composition comprises the glycyrrhiza inflata root extract, the chrysochroa rhodostoma extract peptide, the ganoderma lucidum acid-extracellular polysaccharide complex and the malus halliana flower extract in a mass ratio of (4-6):(2-3):(3.5-5):(1-2); ​ The preparation of the glycyrrhiza inflata root extract comprises ultrasonic extraction of the root core coarse powder of the glycyrrhiza inflata in a mixed solution containing choline chloride and lactic acid, and adsorption purification of the extraction liquid on an A21 macroporous adsorption resin column to obtain the product. The preparation of the chrysochroa rhodostoma extract peptide comprises sequential enzymolysis of the chrysochroa rhodostoma dry powder by chitinase, ficin and serralysin, and adsorption purification of the enzymolysis liquid on a G-15 sephadex gel column to obtain the product. The preparation of the ganoderma lucidum acid-extracellular polysaccharide complex comprises fermentation of the annamocota lucida in a culture medium containing squalane to obtain a water phase and mycelium, co-enzymolysis of the mycelium by chitinase and beta-glucanase to obtain an enzymolysis liquid, combination of the enzymolysis liquid with the water phase, ultrasonic activation, and adsorption purification of the product on an AB-8 macroporous resin column. The preparation of the malus halliana flower extract comprises water extraction of the malus halliana pollen under the conditions of a temperature of 110-120 DEG C and a pressure of 0.3-0.4 MPa, and adsorption purification of the extraction liquid on a D101 macroporous resin column to obtain the product.

2. The composition for repairing damaged skin cells according to claim 1, wherein The preparation method of the glycyrrhiza inflata root extract comprises the following steps: taking the root core coarse powder of the glycyrrhiza inflata, soaking the powder in hot water, filtering to obtain a filter cake, preparing a mixed solution containing 5wt%-6wt% choline chloride and 7wt%-8wt% lactic acid by using an ethanol aqueous solution, adding the filter cake into the mixed solution according to a solid-liquid ratio of 1g:15-20 mL, ultrasonic treatment, centrifugation, taking the liquid, removing ethanol by concentration under reduced pressure, and loading the liquid onto an A21 macroporous adsorption resin column, washing the column with deionized water for 1-2 column volumes to remove impurities, eluting the column with an ethanol aqueous solution with a concentration of 20%-30% for 2-3 column volumes, collecting the eluate, removing ethanol by concentration under reduced pressure, and freeze-drying to obtain the glycyrrhiza inflata root extract.

3. A composition for repairing damaged skin cells according to claim 2, wherein The particle size of the root core coarse powder is below 40-60 mesh; the hot water is used in an amount of 6-8 times the mass of the root core coarse powder; the temperature of the hot water is 80-90 DEG C; the soaking time is 20-30 min; the volume ratio of ethanol to water in the ethanol aqueous solution is (5-6):(4-5); the ultrasonic treatment is performed at 50-60 DEG C and 200-250 W for 20-30 min; the centrifugation is performed at 6000-8000 rpm for 10-15 min; and the concentration temperature under reduced pressure is 50-60 DEG C.

4. The composition for repairing damaged skin cells according to claim 1, wherein The preparation method of the extract peptide of the Chrysochroina pruinosa comprises the following steps: adding the dry powder of the Chrysochroina pruinosa into 10-12 times of mass of phosphate buffer solution with pH 5.0-6.0, then adding 0.5-1% of chitinase of the mass of the dry powder of the Chrysochroina pruinosa, and carrying out enzymolysis at 40-45 DEG C for 1-2 hours; then increasing the temperature to 60-65 DEG C, adding 0.5-1% of bromelain of the mass of the dry powder of the Chrysochroina pruinosa, and carrying out enzymolysis at 60-65 DEG C for 1.5-2 hours; increasing the temperature to inactivate the enzyme, decreasing the temperature to 45-50 DEG C, adjusting the pH to 7.0-7.5, adding 0.5-1% of serratia peptidase of the mass of the dry powder of the Chrysochroina pruinosa, and carrying out enzymolysis at 45-50 DEG C for 1-1.5 hours; increasing the temperature to inactivate the enzyme, decreasing the temperature to room temperature, centrifuging, taking the supernatant, reducing-pressure concentrating, loading onto a G-15 dextran gel column, eluting with 3-4 column volumes of deionized water, collecting the eluate, reducing-pressure concentrating at 50-55 DEG C, and freeze-drying to obtain the extract peptide of the Chrysochroina pruinosa.

5. A composition for repairing damaged skin cells according to claim 4, wherein The particle size of the dry powder of the Chrysochroina pruinosa is below 60-80 mesh; the temperature for inactivating the enzyme is increased to 85-90 DEG C for 10-15 minutes; the centrifugation is carried out at 6000-8000 rpm for 10-20 minutes; the supernatant is reduced-pressure concentrated to 30-50% of the volume before loading; and the temperature for reducing-pressure concentrating is 50-55 DEG C.

6. The composition for repairing damaged skin cells according to claim 1, wherein The preparation method of the ganoderic acid-extracellular polysaccharide complex comprises the following steps: inoculating Ganoderma multiflorum spore liquid into liquid medium with pH 4.5-5.0, and culturing at 25-28 DEG C and 200-250 rpm for 4-6 days; adding squalane, and continuing to culture for 2-3 days to obtain a fermentation liquor; centrifuging the fermentation liquor to collect water phase and mycelium precipitate; adding deionized water to the mycelium precipitate, adjusting the pH to 5.0-5.5, adding 0.8-1.2% of chitinase and 0.8-1.2% of beta-glucanase of the mass of the mycelium precipitate, and carrying out enzymolysis at 40-45 DEG C for 3-4 hours; inactivating the enzyme, decreasing the temperature to room temperature to obtain an enzymolysis liquor, and centrifuging the enzymolysis liquor to take the supernatant; combining the supernatant with the water phase, and ultrasonicating to obtain an activation liquor; loading the activation liquor onto an AB-8 macroporous resin column, eluting 2-3 column volumes of 10-15% volume concentration of ethanol aqueous solution to remove impurities, and then eluting 2-3 column volumes of 50-60% volume concentration of ethanol aqueous solution, collecting the eluate, removing ethanol by reducing-pressure concentrating, and freeze-drying to obtain the ganoderic acid-extracellular polysaccharide complex.

7. A composition for repairing damaged skin cells according to claim 6, wherein The Ganoderma cochlear liquid culture is activated after culture, and is obtained by culturing mycelium of Ganoderma cochlear on PDA slant medium and then culturing the mycelium on liquid medium; the inoculation amount of the Ganoderma cochlear liquid culture is 8% to 12% of the volume of the liquid medium; the olive squalane is sterilized; the addition amount of the olive squalane is 6% to 10% of the volume of the liquid medium; the centrifugation of the fermentation liquid is first centrifuged at 3000 rpm to 4000 rpm for 10 min to 15 min, and then centrifuged at 5500 rpm to 6000 rpm for 15 min to 20 min; the amount of the deionized water is 10 times to 12 times of the mass of the mycelium precipitate; the enzyme inactivation is performed at 85°C to 90°C for 10 min to 15 min; the centrifugation of the enzyme solution is performed at 5500 rpm to 6000 rpm for 15 min to 20 min; the ultrasonic treatment is performed at 4°C to 6°C, 200 W to 250 W and 20 kHz to 25 kHz for 3 min to 5 min; and the temperature for the vacuum concentration is 45°C to 50°C.

8. The composition for repairing damaged skin cells according to claim 1, wherein The preparation method of the Dalbergia hupeana flower extract includes: adding Dalbergia hupeana flower powder and deionized water according to a solid-liquid ratio of 1g: 15mL to 20mL into a high-pressure reaction kettle, extracting at a temperature of 110°C to 120°C, a pressure of 0.3MPa to 0.4MPa and a stirring speed of 100rpm to 150rpm for 30min to 50min to obtain an extraction liquid, centrifuging, taking supernatant, vacuum concentrating, loading onto a D101 macroporous resin column, washing with deionized water for 2 to 3 column volumes to remove impurities, eluting with 65% to 70% volume concentration of an ethanol aqueous solution for 3 to 4 column volumes, collecting the eluate, vacuum concentrating to remove ethanol, and freeze-drying to obtain the Dalbergia hupeana flower extract.

9. A composition for repairing damaged skin cells according to claim 8, wherein The particle size of the Dalbergia hupeana flower powder is below 40 mesh to 60 mesh; the centrifugation is performed at 4000rpm to 5000rpm for 15min to 20min; the supernatant is vacuum concentrated to 30% to 50% of the volume before loading; and the temperature for the vacuum concentration is 50°C to 55°C.

10. A method of preparing a composition for repairing damaged skin cells according to claim 1, wherein, The method comprises the following steps: The licorice root extract, the Cicindela chinensis flower extraction peptide, the ganoderic acid-exopolysaccharide complex and the Dalbergia hupeana flower extract are mixed according to a mass ratio to obtain a composition.

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