A composition for repairing damaged skin cells and a method for preparing the same

Abstract

The application provides a kind of composition for repairing damaged skin cells and its preparation method, the skin repair technical field, the composition includes liquorice root extract, golden cicada flower extraction peptide, ganoderic acid-extracellular polysaccharide complex and dalbergia hupeana flower extract.Liquorice root extract is the core powder of liquorice root by ultrasonic extraction in mixed solution containing choline chloride and lactic acid, and is obtained by purification.Golden cicada flower extraction peptide is golden cicada flower dry powder by chitinase, ficin and serratia peptidase in turn enzymolysis, and is obtained by purification.Ganoderic acid-extracellular polysaccharide complex is the water phase and mycelium obtained by fermentation of t. sinensis in the medium containing squalane, the mycelium is co-enzymolysis by chitinase and beta-glucanase to obtain enzymolysis solution, and the enzymolysis solution is combined with water phase, and is obtained by purification.Dalbergia hupeana flower extract is dalbergia hupeana pollen extracted by water, and is obtained by purification.Each component can precisely act on the key pathological link of damaged skin cells, and realize safe, mild and efficient repair function.

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 rapidly in the short term, which is difficult to tolerate, and the recurrence rate is high. Later developed drug therapy, such as the use of tacrolimus ointment and other immunomodulators, has 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 of 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, cicada flower extraction 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:

[0008] A composition for repairing damaged skin cells, the composition comprises glycyrrhiza glabra root extract, cicada flower extraction peptide, ganoderma acid-extracellular polysaccharide complex and dali flower extract in a mass ratio of (4-6):(2-3):(3.5-5):(1-2);

[0009] 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 through an A21 macroporous adsorption resin column to obtain a product;

[0010] The preparation of the cicada flower extraction peptide comprises sequential enzymolysis of the cicada flower dry powder by chitinase, ficin and serratia peptidase, and adsorption purification of the enzymolysis liquid through a G-15 dextran gel column to obtain a product;

[0011] The ganoderic acid-exopolysaccharide complex is prepared by fermenting the T. sinense in a culture medium containing squalane to obtain a water phase and mycelium, subjecting the mycelium to co-enzymolysis by chitinase and beta-glucanase to obtain an enzymolysis liquid, combining the enzymolysis liquid with the water phase, subjecting the combination to ultrasonic activation, and then subjecting the combination to AB-8 macroporous resin column adsorption purification to obtain the product.

[0012] The preparation of the Dalbergia hupeana extract includes water extraction of Dalbergia hupeana 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 extract by a D101 macroporous resin column to obtain the product.

[0013] In the composition, the preparation method of the Glycyrrhiza glabra root extract includes: taking root core coarse powder of Glycyrrhiza glabra, soaking the root core coarse powder in hot water, and filtering to obtain a filter cake; preparing a mixed solution containing 5-6 wt% choline chloride and 7-8 wt% lactic acid by using an ethanol aqueous solution; adding the filter cake into the mixed solution at a solid-liquid ratio of 1 g: 15-20 mL, subjecting the mixture to ultrasonic treatment, centrifuging, taking the liquid, removing ethanol under reduced pressure, and subjecting the liquid to A21 macroporous adsorption resin column adsorption purification, washing the column with deionized water to remove impurities, eluting the column with 20-30% ethanol aqueous solution, collecting the eluate, removing ethanol under reduced pressure, and freeze-drying to obtain the Glycyrrhiza glabra root extract.

[0014] 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 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 temperature for the reduced pressure concentration is 50-60 DEG C.

[0015] The preparation method of the Cordyceps militaris extract peptide in the above composition includes: adding Cordyceps militaris dried powder to a pH 5.0-6.0 phosphate buffer solution at 10-12 times its mass (by weight) of the Cordyceps militaris dried powder, then adding 0.5%-1% (by weight) of chitinase (by weight) of the Cordyceps militaris dried powder, and enzymatically hydrolyzing at 40-45°C for 1-2 hours; then raising the temperature to 60-65°C, adding 0.5%-1% (by weight) of fig protease (by weight) of the Cordyceps militaris dried powder, and enzymatically hydrolyzing at 60-65°C for 1.5-2 hours. The enzyme was inactivated by heating, then cooled to 45℃~50℃, and the pH was adjusted to 7.0~7.5. 0.5%~1% of Serratia marcescens peptidase (by weight of dried Cordyceps militaris powder) was added, and the enzyme was hydrolyzed at 45℃~50℃ for 1h~1.5h. The enzyme was inactivated by heating again, and after cooling to room temperature, the mixture was centrifuged, the supernatant was collected, concentrated under reduced pressure, and loaded onto a G-15 dextran gel column. The column was eluted with deionized water for 3~4 column volumes, the eluent was collected, concentrated under reduced pressure at 50℃~55℃, and then freeze-dried to obtain Cordyceps militaris extract peptides.

[0016] In the above-mentioned method for preparing Cordyceps militaris extract peptides, the particle size of the Cordyceps militaris powder is sieve-sized material passing through a 60-80 mesh sieve; the enzyme inactivation by heating is performed at 85-90℃ for 10-15 minutes; the centrifugation is performed at 6000-8000 rpm for 10-20 minutes; the supernatant is concentrated under reduced pressure to 30%-50% of its volume before loading; the temperature of the reduced pressure concentration is 50-55℃.

[0017] The preparation method of the ganoderic acid-extracellular polysaccharide complex in the above composition includes: inoculating Antrodia camphorata mycelium solution into a liquid culture medium with a pH of 4.5-5.0, and culturing it at 25℃-28℃ and 200rpm-250rpm with stirring and aeration for 4-6 days; adding olive squalane and continuing to culture for 2-3 days to obtain a fermentation broth; centrifuging the fermentation broth and collecting the aqueous phase and mycelial precipitate; adding deionized water to the mycelial precipitate, adjusting the pH to 5.0-5.5, and adding 0.8%-1.2% (by weight of the mycelial precipitate) of chitinase and 0.8% (by weight of the mycelial precipitate) of... ~1.2% β-glucanase was used to hydrolyze the enzyme at 40℃~45℃ for 3h~4h, then the enzyme was inactivated and cooled to room temperature to obtain the hydrolysate. The hydrolysate was centrifuged and the supernatant was collected. The supernatant was combined with the aqueous phase and sonicated to obtain the activated solution. The activated solution was loaded onto an AB-8 macroporous resin column and washed with 10%~15% v / v ethanol aqueous solution for 2~3 column volumes to remove impurities. Then it was eluted with 50%~60% v / v ethanol aqueous solution for 2~3 column volumes. The eluent was collected, concentrated under reduced pressure to remove ethanol, and freeze-dried to obtain the ganoderic acid-extracellular polysaccharide complex.

[0018] In the above method for preparing the ganoderic acid-extracellular polysaccharide complex, the *Antrodia camphorata* inoculum is an activated and cultured *Antrodia camphorata* inoculum. The inoculum is prepared by first culturing *Antrodia camphorata* on a PDA slant medium to obtain mycelia, and then culturing the mycelia in a liquid medium. The inoculum volume is 8%–12% of the liquid medium volume. The oleuropein squalane is sterilized. The amount of oleuropein squalane added is 6%–10% of the liquid medium volume. The fermentation broth is centrifuged at 3000 rpm–4000 rpm for 10 min. Centrifuge at 5500 rpm to 6000 rpm for 15 to 20 minutes, followed by centrifugation at 5500 rpm to 6000 rpm for 15 to 20 minutes; the amount of deionized water used is 10 to 12 times the mass of the mycelial precipitate; the enzyme inactivation is performed at 85℃ to 90℃ for 10 to 15 minutes; the enzymatic hydrolysate is centrifuged at 5500 rpm to 6000 rpm for 15 to 20 minutes; the ultrasonication is performed at 4℃ to 6℃, 200W to 250W, and 20kHz to 25kHz for 3 to 5 minutes; the vacuum concentration temperature is 45℃ to 50℃.

[0019] The preparation method of the bitter orange flower extract in the above composition includes: adding bitter orange flower pollen and deionized water at a solid-liquid ratio of 1g:15mL-20mL to a high-pressure reactor, extracting for 30min-50min at a temperature of 110℃-120℃, a pressure of 0.3MPa-0.4MPa, and a stirring speed of 100rpm-150rpm to obtain the extract, centrifuging, collecting the supernatant, concentrating under reduced pressure, loading the extract onto a D101 macroporous resin column, rinsing with deionized water for 2-3 column volumes to remove impurities, then eluting with 65%-70% ethanol aqueous solution for 3-4 column volumes, collecting the eluent, concentrating under reduced pressure to remove ethanol, and freeze-drying to obtain the bitter orange flower extract.

[0020] In the above-mentioned method for preparing bitter orange flower extract, the particle size of the bitter orange pollen is sieve size of 40-60 mesh; the centrifugation is performed at 4000-5000 rpm for 15-20 minutes; the supernatant is concentrated under reduced pressure to 30%-50% of its volume before loading; and the temperature of the reduced pressure concentration is 50℃-55℃.

[0021] The preparation method of the above-mentioned composition for repairing damaged skin cells includes the following steps:

[0022] The root extract of Glycyrrhiza glabra, peptides extracted from Cordyceps militaris, ganoderic acid-extracellular polysaccharide complex, and extract of Citrus aurantium were mixed in a specific mass ratio to obtain the composition.

[0023] The above-mentioned composition for repairing damaged skin cells is combined with pharmaceutically or cosmetically available ingredients to prepare a product for repairing damaged skin cells.

[0024] The present invention provides a composition for repairing damaged skin cells and a method for preparing the same, the beneficial effects of which include:

[0025] I. The composition of this invention achieves safe, gentle, and highly effective repair by precisely targeting key pathological processes in damaged skin cells. Its core mechanisms include: activating the self-repair signaling pathways of damaged cells (such as keratinocytes and fibroblasts), promoting normal cell proliferation and differentiation, and accelerating skin cell renewal to restore the structure of the stratum corneum and dermis; inhibiting the transcription and secretion of inflammatory factors (such as TNF-α and IL-6), reducing the inflammatory response caused by hormone-induced rosacea; promoting collagen synthesis by fibroblasts, repairing damaged cells, restoring barrier function, breaking the vicious cycle of hormone dependence, and improving hormone-induced rosacea.

[0026] II. Licorice Root Extract: During preparation, coarse root powder ensures full release of active ingredients; hot water soaking initially removes water-soluble impurities; an ethanol-water solution containing choline chloride and lactic acid is used for efficient ultrasonic extraction of active ingredients; and A21 macroporous adsorption resin is used for precise purification to improve the purity of active ingredients. These parameters lay the foundation for the extract's highly effective anti-inflammatory properties and its ability to protect cells from oxidative damage.

[0027] III. Cordyceps militaris extract peptides: Initial enzymatic hydrolysis with chitinase releases more active substances, followed by proteolytic hydrolysis with fig protease and deep enzymatic hydrolysis with Serratia marcescens enzyme, synergistically generating small-molecule active peptides; G-15 dextran gel precisely retains the active peptides and removes impurity peptides. This process ensures that the active peptides can efficiently bind to cell surface receptors, activating proliferation signals and inhibiting the transcription of inflammatory factors.

[0028] IV. Ganoderic Acid-Extracellular Polysaccharide Complex: After culturing Antrodia camphorata in liquid culture medium, olive squalane is added to induce and promote the synthesis of ganoderic acid and the secretion of extracellular polysaccharides. Chitinase and β-glucanase jointly enzymatically hydrolyze the mycelium to release active ingredients, and AB-8 macroporous resin evenly adsorbs ganoderic acid and polysaccharides. These steps ensure that the proportions of each component in the complex are appropriate, synergistically exerting anti-inflammatory, extracellular matrix stabilizing, and collagen synthesis-promoting effects.

[0029] V. Bitter Orange Flower Extract: High-pressure extraction efficiently extracts volatile oils and flavonoids, while D101 macroporous resin purification preserves the active ingredients. Appropriate amounts of the extract can improve skin microcirculation, enhance anti-inflammatory effects, and do not damage cell structure.

[0030] VI. The four components complement and synergize functionally, rather than being the sum of the effects of a single component. The licorice root extract, bitter orange flower extract, and Cordyceps militaris extract peptide work synergistically to create a favorable microenvironment for cell proliferation and repair. Cordyceps militaris extract peptide activates proliferation signals in keratinocytes and fibroblasts; in the ganoderic acid-extracellular polysaccharide complex, extracellular polysaccharides provide stable matrix support for cell proliferation, while ganoderic acid helps reduce inflammation to ensure cell viability; licorice root extract protects cells from oxidative damage, achieving a complete repair chain of "anti-inflammatory-proliferation-repair." The four components, used in specific proportions, significantly enhance the repair effect and avoid imbalances in overall functional effects caused by excessive or insufficient amounts of any single component. Detailed Implementation

[0031] The present invention will be further described below with reference to specific implementation examples, but the present invention is not limited to these embodiments.

[0032] Example 1

[0033] A composition for repairing damaged skin cells, wherein the composition comprises, by mass ratio of licorice root extract, cicada flower extract peptide, ganoderic acid-extracellular polysaccharide complex, and bitter orange flower extract in a ratio of 5:2.5:4.2:1.5.

[0034] In this embodiment, the preparation method of Glycyrrhiza glabra root extract includes: taking coarse root core powder (50-mesh sieve material) of Glycyrrhiza glabra, soaking it in hot water at 85°C for 25 min with 7 times its weight of the coarse root core powder, filtering and collecting the filter cake; preparing a mixture containing 5.5 wt% choline chloride and 7.5 wt% lactic acid with an ethanol-water solution (ethanol to water volume ratio of 5.5:4.5); adding the filter cake to the mixture at a solid-liquid ratio of 1 g:18 mL, sonicating at 55°C and 200 W for 25 min, centrifuging at 7000 rpm for 12 min, collecting the liquid, concentrating under reduced pressure at 55°C to remove ethanol, loading the sample onto an A21 macroporous adsorption resin column, rinsing with deionized water for 1.5 column volumes, then eluting with 25% ethanol-water solution for 2.5 column volumes, collecting the eluent, concentrating under reduced pressure at 55°C to remove ethanol, and freeze-drying to obtain Glycyrrhiza glabra root extract.

[0035] In this embodiment, the preparation method of Cordyceps militaris extract peptides includes: adding Cordyceps militaris dried powder (60-mesh sieve material) to 11 times its weight of pH 5.5 phosphate buffer, then adding 0.8% (by weight of Cordyceps militaris dried powder) of chitinase, and hydrolyzing at 42°C for 1.5 h; then raising the temperature to 62°C, adding 0.8% (by weight of Cordyceps militaris dried powder) of fig protease, and hydrolyzing at 62°C for 1.5 h; raising the temperature to 88°C to inactivate the enzyme for 12 min, then lowering the temperature to 48°C and adjusting... The pH was adjusted to 7.3, and 0.8% (by weight of) Serratia marcescens peptidase from the dried Cordyceps militaris powder was added. The mixture was enzymatically hydrolyzed at 48℃ for 1 hour. The enzyme was then inactivated at 88℃ for 12 minutes, cooled to room temperature, and centrifuged at 7000 rpm for 15 minutes. The supernatant was collected. The supernatant was concentrated under reduced pressure at 52℃ to 40% of its volume and then loaded onto a G-15 dextran gel column. The column was eluted with deionized water for 3.5 column volumes. The eluent was collected, concentrated under reduced pressure at 52℃, and then freeze-dried to obtain the Cordyceps militaris extract peptide.

[0036] In this embodiment, the preparation method of the ganoderic acid-extracellular polysaccharide complex includes: inoculating the activated and cultured Antrodia camphorata mycelium solution into a sterilized liquid culture medium at 10% of the liquid culture medium volume, and culturing it at 27°C and 220 rpm with stirring and aeration for 5 days; adding 8% of the liquid culture medium volume of sterilized olive squalane, and continuing to culture for 2.5 days to obtain the fermentation broth; centrifuging the fermentation broth at 3500 rpm for 12 min, and then centrifuging it at 5800 rpm for 18 min, collecting the aqueous phase (supernatant) and mycelial precipitate; adding 11 times its mass of deionized water to the mycelial precipitate, adjusting the pH to 5.2, and adding 1.0% of the mycelial precipitate mass. Chitinase and 1.0% β-glucanase were hydrolyzed at 42℃ for 3.5 h, then inactivated at 88℃ for 12 min, and cooled to room temperature to obtain the hydrolysate. The hydrolysate was centrifuged at 5800 rpm for 18 min, and the supernatant was collected. The supernatant was combined with the aforementioned aqueous phase and sonicated at 5℃, 200 W, and 20 kHz for 4 min to obtain the activated solution. The activated solution was loaded onto an AB-8 macroporous resin column and washed with 12% (v / v) ethanol aqueous solution for 2.5 column volumes to remove impurities. Then, it was eluted with 55% (v / v) ethanol aqueous solution for 2.5 column volumes. The eluent was collected, concentrated under reduced pressure at 48℃ to remove ethanol, and then freeze-dried to obtain the ganoderic acid-extracellular polysaccharide complex.

[0037] The liquid culture medium formula includes: 40 g / L glucose, 8 g / L peptone, 3 g / L yeast extract, 1.5 g / L potassium dihydrogen phosphate and 0.8 g / L magnesium sulfate heptahydrate, with deionized water as the solvent and pH adjusted to 4.8; the liquid culture medium is sterilized before use (autoclave at 121℃ for 25 min).

[0038] The activation culture method of Antrodia camphorata inoculum includes: peeling and cutting 210g of potato into pieces, adding 0.7L of water and boiling for 25min, filtering, taking the filtrate, adding 21g of glucose and 18g of agar, heating to melt, and then adjusting the volume to 1L with deionized water, adjusting the pH to 4.8, dispensing into test tubes, autoclaving at 121℃ for 25min, arranging into slant agar, cooling and solidifying to obtain PDA slant culture medium; picking Antrodia camphorata inoculum and inoculating it onto the PDA slant culture medium, placing it at 27℃ in the dark for 6 days to obtain mycelium; inoculating the mycelium into Erlenmeyer flasks, each Erlenmeyer flask containing 200mL of pre-sterilized liquid culture medium of the above formula, and culturing at 27℃ and 180rpm on a shaker for 3.5 days to obtain the activated culture of Antrodia camphorata inoculum.

[0039] In this embodiment, the preparation method of bitter orange flower extract includes: adding bitter orange flower pollen (50-mesh sieve) and deionized water at a solid-liquid ratio of 1g:18mL to a high-pressure reactor, extracting for 40min at a temperature of 115℃, a pressure of 0.35MPa, and a stirring speed of 120rpm to obtain an extract; centrifuging the extract at 4500rpm for 18min, taking the supernatant, concentrating it under reduced pressure at 52℃ to 40% of its volume, loading it onto a D101 macroporous resin column, rinsing with deionized water for 2.5 column volumes to remove impurities, then eluting with 68% ethanol aqueous solution for 3.5 column volumes, collecting the eluent, concentrating it under reduced pressure at 52℃ to remove ethanol, and freeze-drying to obtain bitter orange flower extract.

[0040] Example 2

[0041] A composition for repairing damaged skin cells, wherein the composition comprises, by mass ratio, licorice root extract, cicada flower extract peptide, ganoderic acid-extracellular polysaccharide complex, and bitter orange flower extract.

[0042] In this embodiment, the preparation method of Glycyrrhiza glabra root extract includes: taking coarse powder of Glycyrrhiza glabra root core (40-mesh sieve), soaking it in 8 times its weight of hot water at 80°C for 30 min, filtering and taking the filter cake; preparing a mixed solution containing 5 wt% choline chloride and 8 wt% lactic acid with an ethanol-water solution (ethanol to water volume ratio of 5:5); adding the filter cake to the mixed solution at a solid-liquid ratio of 1 g:15 mL, sonicating at 60°C and 200 W for 30 min, centrifuging at 6000 rpm for 15 min, taking the liquid, concentrating under reduced pressure at 50°C to remove ethanol, loading the sample onto an A21 macroporous adsorption resin column, rinsing with deionized water for 2 column volumes, then eluting with 20% volume concentration ethanol-water solution for 3 column volumes, collecting the eluent, concentrating under reduced pressure at 50°C to remove ethanol, and freeze-drying to obtain Glycyrrhiza glabra root extract.

[0043] In this embodiment, the preparation method of Cordyceps militaris extract peptides includes: adding Cordyceps militaris dried powder (80-mesh sieve residue) to 10 times its weight of pH 6.0 phosphate buffer, then adding 0.5% (by weight of Cordyceps militaris dried powder) of chitinase, and hydrolyzing at 45°C for 1 hour; then raising the temperature to 65°C, adding 0.5% (by weight of Cordyceps militaris dried powder) of fig protease, and hydrolyzing at 65°C for 1.5 hours; then raising the temperature to 90°C to inactivate the enzyme for 10 minutes, lowering the temperature to 50°C, and adjusting... Adjust the pH to 7.0, add 1% (by weight) of Serratia marcescens peptidase from the dried Cordyceps militaris powder, and enzymatically hydrolyze at 50℃ for 1 h; heat to 90℃ to inactivate the enzyme for 10 min, cool to room temperature, centrifuge at 8000 rpm for 10 min, and collect the supernatant; concentrate the supernatant under reduced pressure at 55℃ to 30% of its volume, load it onto a G-15 dextran gel column, elute with deionized water for 4 column volumes, collect the eluent, concentrate under reduced pressure at 50℃, and freeze-dry to obtain Cordyceps militaris extract peptides.

[0044] In this embodiment, the preparation method of the ganoderic acid-extracellular polysaccharide complex includes: inoculating the activated and cultured Antrodia camphorata mycelium solution into a sterilized liquid culture medium at 12% of the liquid culture medium volume, and culturing it at 25°C and 250 rpm with stirring and aeration for 4 days; adding 10% of the liquid culture medium volume of sterilized olive squalane, and continuing to culture for 2 days to obtain the fermentation broth; centrifuging the fermentation broth at 4000 rpm for 10 min, and then centrifuging it at 6000 rpm for 15 min, collecting the aqueous phase (supernatant) and mycelial precipitate; adding 12 times the mass of deionized water to the mycelial precipitate, adjusting the pH to 5.0, and adding 1 part by mass of the mycelial precipitate. 2% chitinase and 0.8% β-glucanase were hydrolyzed at 45℃ for 3 h, then inactivated at 90℃ for 10 min, and cooled to room temperature to obtain the hydrolysate. The hydrolysate was centrifuged at 6000 rpm for 15 min, and the supernatant was collected. The supernatant was combined with the aforementioned aqueous phase and sonicated at 6℃, 200 W, and 25 kHz for 3 min to obtain the activation solution. The activation solution was loaded onto an AB-8 macroporous resin column, eluted with 15% (v / v) ethanol aqueous solution for 2 column volumes to remove impurities, and then eluted with 60% (v / v) ethanol aqueous solution for 2 column volumes. The eluent was collected, concentrated under reduced pressure at 50℃ to remove ethanol, and then freeze-dried to obtain the ganoderic acid-extracellular polysaccharide complex.

[0045] The liquid culture medium formula includes: 30 g / L glucose, 5 g / L peptone, 2 g / L yeast extract, 1 g / L potassium dihydrogen phosphate and 0.5 g / L magnesium sulfate heptahydrate, with deionized water as the solvent and pH adjusted to 4.5; the liquid culture medium is sterilized before use (autoclave at 121℃ for 20 min).

[0046] The activation culture method of Antrodia camphorata inoculum includes: peeling and cutting 200g of potato into pieces, adding 0.6L of water and boiling for 20min, filtering, taking the filtrate, adding 20g of glucose and 15g of agar, heating to melt, and then adjusting the volume to 1L with deionized water, adjusting the pH to 4.5, dispensing into test tubes, autoclaving at 121℃ for 20min, arranging into slant agar, cooling and solidifying to obtain PDA slant culture medium; picking Antrodia camphorata inoculum and inoculating it onto the PDA slant culture medium, placing it at 25℃ in the dark for 5 days to obtain mycelium; inoculating the mycelium into Erlenmeyer flasks, each Erlenmeyer flask containing 100mL of pre-sterilized liquid culture medium of the above formula, and culturing at 25℃ and 150rpm on a shaker for 3 days to obtain activated Antrodia camphorata inoculum.

[0047] In this embodiment, the preparation method of bitter orange flower extract includes: adding bitter orange flower pollen (40-mesh sieve material) and deionized water at a solid-liquid ratio of 1g:20mL to a high-pressure reactor, extracting for 50min at a temperature of 110℃, a pressure of 0.4MPa, and a stirring speed of 100rpm to obtain an extract; centrifuging the extract at 4000rpm for 20min, taking the supernatant, concentrating it under reduced pressure at 50℃ to 50% of its volume, loading it onto a D101 macroporous resin column, rinsing with deionized water for 2 column volumes to remove impurities, then eluting with 70% ethanol aqueous solution for 3 column volumes, collecting the eluent, concentrating it under reduced pressure at 55℃ to remove ethanol, and freeze-drying to obtain bitter orange flower extract.

[0048] Example 3

[0049] A composition for repairing damaged skin cells, wherein the composition comprises, by mass ratio, licorice root extract, cicada flower extract peptide, ganoderic acid-extracellular polysaccharide complex, and bitter orange flower extract.

[0050] In this embodiment, the preparation method of Glycyrrhiza glabra root extract includes: taking coarse root core powder (60-mesh sieve material) of Glycyrrhiza glabra, soaking it in hot water at 90°C for 20 min with 6 times its weight of the coarse root core powder, filtering and taking the filter cake; preparing a mixed solution containing 6 wt% choline chloride and 7 wt% lactic acid with an ethanol-water solution (ethanol to water volume ratio of 6:4); adding the filter cake to the mixed solution at a solid-liquid ratio of 1 g: 20 mL, sonicating at 50°C and 250 W for 20 min, centrifuging at 8000 rpm for 10 min, taking the liquid, concentrating under reduced pressure at 60°C to remove ethanol, loading the sample onto an A21 macroporous adsorption resin column, rinsing with deionized water for 1 column volume, then eluting with 30% volume concentration ethanol-water solution for 2 column volumes, collecting the eluent, concentrating under reduced pressure at 60°C to remove ethanol, and freeze-drying to obtain Glycyrrhiza glabra root extract.

[0051] In this embodiment, the preparation method of Cordyceps militaris extract peptides includes: adding Cordyceps militaris dried powder (60-mesh sieve material) to 12 times its weight of pH 5.0 phosphate buffer, then adding 1% (by weight of Cordyceps militaris dried powder) of chitinase, and enzymatically hydrolyzing at 40°C for 2 hours; then raising the temperature to 60°C, adding 1% (by weight of Cordyceps militaris dried powder) of fig protease, and enzymatically hydrolyzing at 60°C for 2 hours; then raising the temperature to 85°C to inactivate the enzyme for 15 minutes, lowering the temperature to 45°C, and adjusting the pH to 7. 5. Add 0.5% (by weight of dried Cordyceps militaris) of Serratia marcescens enzyme to the coptis chinensis powder and hydrolyze at 45℃ for 1.5 h; heat to 85℃ to inactivate the enzyme for 15 min, cool to room temperature, centrifuge at 6000 rpm for 20 min, and collect the supernatant; concentrate the supernatant to 50% of its volume under reduced pressure at 50℃, load it onto a G-15 dextran gel column, elute with deionized water for 3 column volumes, collect the eluent, concentrate under reduced pressure at 55℃, and freeze-dry to obtain Cordyceps militaris extract peptides.

[0052] In this embodiment, the preparation method of the ganoderic acid-extracellular polysaccharide complex includes: inoculating the activated and cultured Antrodia camphorata mycelium solution into a sterilized liquid culture medium at 8% of the liquid culture medium volume, and culturing it at 28°C and 200 rpm with stirring and aeration for 6 days; adding 6% of sterilized olive squalane (by volume of the liquid culture medium), and continuing to culture for 3 days to obtain the fermentation broth; centrifuging the fermentation broth at 3000 rpm for 15 min, and then centrifuging it at 5500 rpm for 20 min, collecting the aqueous phase (supernatant) and mycelial precipitate; adding 10 times its mass of deionized water to the mycelial precipitate to adjust the pH to 5.5, and adding 0.8% of the mycelial precipitate mass. % chitinase and 1.2% β-glucanase were hydrolyzed at 40℃ for 4 h, then inactivated at 85℃ for 15 min, and cooled to room temperature to obtain the hydrolysate. The hydrolysate was centrifuged at 5500 rpm for 20 min, and the supernatant was collected. The supernatant was combined with the aforementioned aqueous phase and sonicated at 4℃, 250 W, and 20 kHz for 5 min to obtain the activation solution. The activation solution was loaded onto an AB-8 macroporous resin column, eluted with 10% (v / v) ethanol aqueous solution for 3 column volumes to remove impurities, and then eluted with 50% (v / v) ethanol aqueous solution for 3 column volumes. The eluent was collected, concentrated under reduced pressure at 45℃ to remove ethanol, and then freeze-dried to obtain the ganoderic acid-extracellular polysaccharide complex.

[0053] The liquid culture medium formula includes: 50 g / L glucose, 10 g / L peptone, 5 g / L yeast extract, 2 g / L potassium dihydrogen phosphate and 1 g / L magnesium sulfate heptahydrate, with deionized water as the solvent and pH adjusted to 5.0; the liquid culture medium is sterilized before use (autoclave at 121℃ for 30 min).

[0054] The activation culture method of Antrodia camphorata inoculum includes: peeling and cutting 220g of potato into pieces, adding 0.8L of water and boiling for 30min, filtering, taking the filtrate, adding 22g of glucose and 20g of agar, heating to melt, and then adjusting the volume to 1L with deionized water, adjusting the pH to 5.0, dispensing into test tubes, autoclaving at 121℃ for 30min, arranging into slant agar, cooling and solidifying to obtain PDA slant culture medium; picking Antrodia camphorata inoculum and inoculating it onto the PDA slant culture medium, placing it at 28℃ in the dark for 7 days to obtain mycelium; inoculating the mycelium into Erlenmeyer flasks, each Erlenmeyer flask containing 250mL of pre-sterilized liquid culture medium of the above formula, and culturing at 28℃ and 200rpm on a shaker for 4 days to obtain activated Antrodia camphorata inoculum.

[0055] In this embodiment, the preparation method of bitter orange flower extract includes: adding bitter orange flower pollen (60-mesh sieve material) and deionized water at a solid-liquid ratio of 1g:15mL to a high-pressure reactor, extracting for 30min at a temperature of 120℃, a pressure of 0.3MPa, and a stirring speed of 150rpm to obtain an extract; centrifuging the extract at 5000rpm for 15min, taking the supernatant, concentrating it under reduced pressure at 55℃ to 30% of its volume, loading it onto a D101 macroporous resin column, rinsing with deionized water for 3 column volumes to remove impurities, then eluting with 65% ethanol aqueous solution for 4 column volumes, collecting the eluent, concentrating it under reduced pressure at 50℃ to remove ethanol, and freeze-drying to obtain bitter orange flower extract.

[0056] A method for preparing a composition for repairing damaged skin cells according to the above embodiments includes the following steps:

[0057] The extracts of Glycyrrhiza glabra root, Cordyceps militaris extract peptide, Ganoderma lucidum acid-extracellular polysaccharide complex, and Citrus aurantium flower extract were mixed according to the mass ratio of the components in each embodiment to obtain the compositions of each embodiment.

[0058] The above-described compositions for repairing damaged skin cells are combined with pharmaceutically or cosmetically available ingredients to prepare products for repairing damaged skin cells.

[0059] Comparative Example 1

[0060] The difference from Example 1 is that the mass ratio of Glycyrrhiza glabra root extract, Cordyceps militaris extract peptide, Ganoderma lucidum acid-extracellular polysaccharide complex and Citrus aurantium flower extract is changed to 7:0.5:4.2:1.5.

[0061] Comparative Example 2

[0062] The difference from Example 1 is that the mass ratio of Glycyrrhiza glabra root extract, Cordyceps militaris extract peptide, Ganoderma lucidum acid-extracellular polysaccharide complex and Citrus aurantium flower extract is changed to 2:5.5:4.2:1.5.

[0063] Comparative Example 3

[0064] The difference from Example 1 is that the mass ratio of Glycyrrhiza glabra root extract, Cordyceps militaris extract peptide, Ganoderma lucidum acid-extracellular polysaccharide complex and Citrus aurantium flower extract is changed to 1.5:2.5:4.2:5.

[0065] Comparative Example 4

[0066] The difference from Example 1 is that the mass ratio of Glycyrrhiza glabra root extract, Cordyceps militaris extract peptide, Ganoderma lucidum acid-extracellular polysaccharide complex and Citrus aurantium flower extract is changed to 5:5.5:1.2:1.5.

[0067] Comparative Example 5

[0068] The difference from Example 1 is that the mass ratio of Glycyrrhiza glabra root extract, Cordyceps militaris extract peptide, Ganoderma lucidum acid-extracellular polysaccharide complex and Citrus aurantium flower extract is changed to 5:0.5:6.2:1.5.

[0069] Comparative Example 6

[0070] The difference from Example 1 is that in the preparation method of Glycyrrhiza glabra root extract, A21 macroporous adsorption resin is replaced with AB-8 macroporous resin.

[0071] Comparative Example 7

[0072] The difference from Example 1 is that in the preparation method of Cordyceps militaris extract peptides, fig protease is replaced with papain.

[0073] Comparative Example 8

[0074] The difference from Example 1 is that in the preparation method of the peptide extracted from Cordyceps militaris, serrata peptides are replaced with bromelain.

[0075] Comparative Example 9

[0076] The difference from Example 1 is that in the preparation method of Cordyceps militaris extract peptides, G-15 dextran gel is replaced with LH-60 dextran gel.

[0077] Comparative Example 10

[0078] The difference from Example 1 is that olive squalane is not added in the preparation method of the ganoderic acid-extracellular polysaccharide complex.

[0079] Comparative Example 11

[0080] The difference from Example 1 is that in the preparation method of the ganoderic acid-extracellular polysaccharide complex, AB-8 macroporous resin is replaced with D101 macroporous resin.

[0081] Comparative Example 12

[0082] The difference from Example 1 is that in the preparation method of bitter orange flower extract, D101 macroporous resin is replaced with A21 macroporous adsorption resin.

[0083] The raw materials used in the above embodiments and comparative examples were sourced as follows: Choline chloride was sourced from Hebei Tuohai Biotechnology Co., Ltd., food grade. Lactic acid was supplied by Anhui Weimao Biotechnology Co., Ltd., food grade lactic acid 80. A21 macroporous adsorption resin was sourced from Shanghai Yuanye Biotechnology Co., Ltd., model S24709. Cordyceps militaris was sourced from Bozhou Huajunzi Biotechnology Co., Ltd. Chitinase was sourced from Jiangsu Juchuang Bioengineering Co., Ltd., enzyme activity 100,000 U / g. Fig protease was sourced from Nanjing Songguan Biotechnology Co., Ltd., enzyme activity 100,000 U / g. Serratia peptides were sourced from Shandong Aicai Biotechnology Co., Ltd., enzyme activity 100,000 U / g. G-15 dextran gel was sourced from Shanghai Yuanye Biotechnology Co., Ltd., model S14030. Antrodia camphorata strain was sourced from the Northeast Institute of Food and Medicinal Fungi. Olive squalane was sourced from Guangzhou Haoting Fine Chemical Co., Ltd. β-glucanase was sourced from Guangdong Ousman Biotechnology Co., Ltd., enzyme activity 100,000 U / g. AB-8 macroporous resin was sourced from Shanghai Yuanye Biotechnology Co., Ltd., model S30931. D101 macroporous resin was sourced from Shanghai Yuanye Biotechnology Co., Ltd., model S14161. Peptone and yeast extract were sourced from Shandong Liangshan Peptone Biological Products Co., Ltd. Agar was sourced from Huizhou Ketian Biotechnology Co., Ltd. Papain was sourced from Xi'an Compress Biotechnology Co., Ltd., with an enzyme activity of 100,000 U / g. Bromelain was sourced from Peptide Biotechnology (Xi'an) Co., Ltd., with an enzyme activity of 100,000 U / g. LH-60 dextran gel was sourced from Shanghai Yuanye Biotechnology Co., Ltd., model S14038.

[0084] I. Barrier cell proliferation detection:

[0085] HaCaT cells (human keratinocytes) and HDF cells (human dermal fibroblasts) in the logarithmic growth phase were selected, digested with 0.25% trypsin, and counted at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of 100 μL into 96-well plates, with 100 μL of DMEM high-glucose complete medium containing 10% FBS and 1% penicillin-streptomycin added to each well. The plates were pre-cultured at 37°C in a 5% CO2 incubator for 24 hours to ensure cell adhesion exceeded 85%.

[0086] Prepare the test solution in advance: Prepare a 10 mg / mL stock solution of the composition using the above complete culture medium (stir magnetically for 30 minutes until completely dissolved), filter to sterilize, and then dilute to a working solution of 200 μg / mL. Simultaneously, set up three types of controls: a negative control group containing only complete culture medium (cells present but no composition), a blank control group containing only complete culture medium (cell-free), and a positive control group containing 10 ng / mLEGF.

[0087] Discard the old culture medium from 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 and positive control groups, respectively. Add complete culture medium without cells to the blank control group. Set up 6 replicates for each group. After returning to the incubator and continuing to incubate for 48 hours, add 10 μL of CCK-8 solution to each well and incubate in the dark for 2 hours. Measure the absorbance (OD value) of each well using a microplate reader at a wavelength of 450 nm (reference wavelength 630 nm). Calculate the cell proliferation rate using the formula: Cell proliferation rate (%) = [(OD experimental group - OD blank control group) / (OD negative control group - OD blank control group)] × 100%. A proliferation rate exceeding 100% indicates that the composition can promote cell proliferation, and the higher the value, the stronger the promoting effect.

[0088] CCK-8 assays indirectly reflect cellular metabolic activity and quantity, rather than direct cell counts. Therefore, proliferation rate is a relative indicator, usually positively correlated with cell number. It can also reflect whether a component is toxic to cells and whether it is safe and reliable.

[0089] II. Detection of inflammatory factor expression:

[0090] RAW264.7 macrophages were used as the research subject and seeded into 24-well plates at 5 × 10⁶ cells per well. 4 Add 1 mL of DMEM medium containing 10% FBS to each cell and incubate at 37°C and 5% CO2 for 24 hours.

[0091] Pre-preparation of test solution: Prepare a 10 mg / mL stock solution of the composition using DMEM medium containing 10% FBS (dissolve by magnetic stirring for 30 minutes), filter to sterilize, and then dilute to a working solution of 200 μg / mL. The experiment was divided into three groups: a blank control group containing only the culture medium (no LPS, no composition), an inflammation model group containing 1 μg / mL LPS (no composition), and an experimental group containing 1 μg / mL LPS and 200 μg / mL test solution. Each group had 3 replicates.

[0092] Discard the old culture medium from the 24-well plate, add the corresponding solutions according to the group, and return to the incubator for 24 hours of further incubation. Then collect the cell supernatant from each well, centrifuge at 3000 rpm (8 cm radius) for 5 minutes, and use the supernatant for later use. Following the instructions of the TNF-α and IL-6 ELISA kit, add the supernatant and standards to the reaction wells, incubate at 37°C for 60 minutes, wash, add enzyme-labeled secondary antibody and incubate for 30 minutes, add substrate for color development for 15 minutes, and terminate the reaction with stop solution. Measure the absorbance at 450 nm (reference wavelength 570 nm) using a microplate reader. Calculate the concentration of inflammatory factors in each well based on the standard curve. Evaluate the anti-inflammatory effect using 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)] × 100%. A higher inhibition rate indicates a stronger anti-inflammatory effect of the composition.

[0093] III. Collagen Synthesis Detection (Sirius Red Staining Method):

[0094] Human dermal fibroblasts (HDF) were used as the research subject and seeded into 24-well plates, with 5 × 10⁶ cells per well. 4 Add 1 mL of DMEM medium containing 10% FBS to cells, culture for 24 hours, then replace with medium containing 1 μM Dex, and continue culturing for another 24 hours to establish a hormone-induced injury model.

[0095] Prepare the test solution in advance: Prepare a 10 mg / mL stock solution of the composition using DMEM medium containing 10% FBS (dissolve by magnetic stirring for 30 minutes), filter to sterilize, and then dilute to a working solution of 200 μg / mL. After the model is established, discard the medium containing Dex, add 1 mL of working solution to the experimental group, and set up a model control group (only fresh medium is added after Dex damage, without the composition) and a normal control group (no Dex damage, fresh medium without the composition is added), with 3 replicates for each group, and continue to incubate for 72 hours. After culture, cells were gently rinsed twice with PBS, and 1 mL of 4% paraformaldehyde was added to each well for fixation at room temperature for 30 minutes. After fixation, cells were rinsed three times with deionized water, and 1 mL of 0.1% Sirius Red dye (dissolved in a saturated picric acid solution at pH 2.0) was added. Cells were stained at room temperature in the dark for 1 hour. The dye was discarded, and cells were rinsed three times (5 minutes each time) with 0.1M hydrochloric acid to remove unbound dye. Then, 1 mL of 0.1M NaOH / methanol solution (volume ratio 1:1) was added, and cells were shaken at room temperature for 10 minutes to desorb the dye bound to collagen. The desorbed solution was transferred to a 96-well plate (each sample was measured independently three times, 100 μL each time), and the absorbance (OD value) was measured at 540 nm using a microplate reader. Collagen synthesis was assessed by comparing OD values: normal control group > composition group > model control group. This indicates that the composition can promote the synthesis of type I collagen (COL-I) in damaged HDF cells, and the higher the OD value, the stronger the repair effect.

[0096] Table 1. Test Results (Average)

[0097]

[0098] The results above show that Examples 1 to 3 yielded better data. The core reason lies in their strict adherence to the "optimal component ratio + effective preparation process," achieving synergistic effects of each active ingredient without functional imbalance caused by excessive or insufficient amounts of any single component. This ensures the efficient performance of the three core indicators: cell proliferation, inflammation suppression, and collagen synthesis. The effective preparation and purification process ensures high purity and strong activity of each active ingredient.

[0099] Comparative Example 1: The core function of peptides extracted from Cordyceps militaris is to activate signaling pathways, upregulate the expression of proliferation-related proteins, and reduce the release of inflammatory factors by inhibiting transcription factors. When their proportion decreases, the concentration of active peptides is insufficient to effectively bind to cell surface receptors, hindering conversion and reducing the proliferation rate; simultaneously, the transcription and secretion of TNF-α and IL-6 are enhanced, significantly reducing the inflammation suppression rate. Furthermore, insufficient fibroblast activity directly leads to reduced expression of genes required for collagen synthesis, resulting in a decrease in OD values.

[0100] Comparative Example 2: The proportion of the main antioxidant and anti-inflammatory active substances in the licorice root extract decreased, and the accumulation of intracellular ROS increased. On the one hand, this induced an increase in cell apoptosis rate and a decrease in cell proliferation rate; on the other hand, it inhibited the activity of key enzymes in collagen synthesis in cells, leading to the inhibition of collagen precursor synthesis. At the same time, the insufficient anti-inflammatory components weakened the ability to clear inflammatory factors, increased the concentration of TNF-α and IL-6, and decreased the inhibition rate, resulting in insufficient synergistic effect of the entire composition.

[0101] Comparative Example 3: The volatile oils and flavonoids in the bitter orange flower extract can improve microcirculation and aid in anti-inflammation when taken in appropriate amounts. However, in excessive amounts, the volatile oils disrupt the phospholipid bilayer structure of the cell membrane, leading to increased cell permeability and decreased cell adhesion, thus inhibiting the metabolism and proliferation of HaCaT and HDF cells. Simultaneously, excessive flavonoids competitively bind to receptors on the cell surface, blocking signaling pathways and reducing collagen synthesis. Furthermore, the stress response triggered by cell damage slightly promotes the secretion of inflammatory factors, further reducing the inflammation suppression rate.

[0102] Comparative Example 4: The ganoderic acid-extracellular polysaccharide complex promotes the expression of genes related to collagen synthesis in cells. Its polysaccharide components also enhance the structural stability of the extracellular matrix, providing support for cell proliferation. When its proportion decreases, the transcription of collagen synthesis genes decreases; insufficient synergy with other components leads to a decline in various properties.

[0103] Comparative Example 5: The cell proliferation activation defect caused by insufficient peptides extracted from Cordyceps militaris cannot be compensated for by excessive ganoderic acid-extracellular polysaccharides. Excessive extracellular polysaccharides form a viscous polysaccharide membrane on the cell surface, hindering the entry of nutrients such as amino acids and glucose into the cell, thus impairing cellular energy metabolism and substance synthesis, and reducing the proliferation rate. At the same time, excessive ganoderic acid produces a slight stimulation to the cells, promoting a slight increase in the secretion of inflammatory factors and reducing the inflammation suppression rate. In addition, insufficient nutrient supply also leads to a decrease in the efficiency of cellular collagen synthesis and a low OD value.

[0104] Comparative Example 6: The A21 macroporous adsorption resin showed higher adsorption selectivity for glycyrrhizic acid, flavonoids, and other active ingredients in *Glycyrrhiza glabra* than the AB-8 resin. Due to its larger pore size, the AB-8 resin easily adsorbed impurities such as polysaccharides and small-molecule sugars, resulting in low purity of glycyrrhizic acid in the final extract. Decreased purity of active ingredients weakened synergistic effects, and the presence of impurities further reduced its ability to scavenge ROS and inhibit NF-κB activation. This led to increased apoptosis and decreased proliferation of HaCaT cells; insufficient proline hydroxylase activity in HDF cells and reduced collagen synthesis; and a decrease in the inhibition rate of inflammatory factors due to the reduced concentration of anti-inflammatory components.

[0105] Comparative Example 7: The cleavage site of fig protease has a high degree of matching with the amino acid sequence of Cordyceps militaris protein, generating effective small-molecule bioactive peptides. These peptides can efficiently enter cells and exert their effects. In contrast, the enzymatic hydrolysis products of papain contain insufficient effective specific bioactive peptides, and impurity peptides can also cause interference and harm. The decreased concentration and effective activity of bioactive peptides lead to insufficient cell proliferation activation, weakened inhibition of inflammatory factors, and lower levels of various indicators.

[0106] Comparative Example 8: The combination of serrata peptiderase and fig protease for secondary enzymatic hydrolysis exhibits good synergistic effects, yielding effective peptides. While bromelain can also hydrolyze proteins and peptides, its synergistic effect with fig protease is poor in the hydrolysis of peptides extracted from Cordyceps militaris. Changes in the cleavage sites during secondary hydrolysis lead to a reduction in effective peptides and the generation of impurity peptides, resulting in alterations in the bioactive peptides in the hydrolysis products. Consequently, cell proliferation rate decreases slightly due to insufficient bioactive peptides, and indicators such as inflammation inhibition rate further decline due to different peptide effects and the lack of a dual anti-inflammatory mechanism.

[0107] Comparative Example 9: G-15 dextran gel accurately retains the active peptides in Cordyceps militaris while removing large molecular impurities. LH-60 gel has a separation range of 1000-100000 Da; different separation ranges result in lower retention rates of small molecule active peptides, leading to reduced purity of active peptides and increased content of large molecular peptides and impurities in the final extract. Although the decreased purity of active peptides did not change their structure or activity, the reduced concentration weakened cell signal activation efficiency, resulting in lower levels of various indicators.

[0108] Comparative Example 10: Olive squalane, as a secondary metabolism inducer of *Antrodia camphorata*, can promote the synthesis of ganoderic acids; it also improves the permeability of the cell membrane and increases the secretion of extracellular polysaccharides. Without olive squalane, the yield and purity of ganoderic acids and the yield of extracellular polysaccharides decrease, leading to a weakening of both the anti-inflammatory activity and collagen synthesis-promoting activity of the complex. Therefore, the inflammation inhibition rate is significantly reduced due to insufficient ganoderic acids, and the OD value of collagen synthesis is reduced due to insufficient polysaccharide support.

[0109] Comparative Example 11: AB-8 resin exhibits balanced adsorption selectivity for both lipid-soluble ganoderic acid and water-soluble extracellular polysaccharides, enabling synergistic purification of both. D101 resin more readily adsorbs water-soluble impurities such as monosaccharides and amino acids, leading to a decreased elution rate of ganoderic acid and an imbalance in the ratio of ganoderic acid to polysaccharides in the complex (with a relative excess of polysaccharides). Insufficient ganoderic acid weakens anti-inflammatory activity and reduces the inflammation inhibition rate; excessive polysaccharides have limited effect on promoting collagen synthesis and cannot compensate for the functional defects of ganoderic acid, thus reducing cell proliferation rate and collagen synthesis OD value.

[0110] Comparative Example 12: D101 resin showed better adsorption capacity for active components such as flavonoids and volatile oils in bitter orange blossoms than A21 resin. A21 resin had a low adsorption rate for volatile oils with low polarity, resulting in loss of effective components, changes in the composition of the purified product, and a decrease in various indicators.

Claims

1. A composition for repairing damaged skin cells, characterized in that, The composition contains, in a mass ratio of (4-6):(2-3):(3.5-5):(1-2) licorice root extract, Cordyceps militaris extract peptide, ganoderic acid-extracellular polysaccharide complex and Citrus aurantium flower extract; The preparation of the licorice root extract includes ultrasonic extraction of crude licorice root core powder by ultrasonic extraction with a mixture containing choline chloride and lactic acid, and purification of the extract by adsorption on an A21 macroporous adsorption resin column to obtain the product. The preparation of the Cordyceps militaris extract peptides includes sequential enzymatic hydrolysis of Cordyceps militaris dry powder by chitinase, fig protease and serrata peptidease, and purification of the hydrolysate by adsorption on a G-15 dextran gel column to obtain the product. The preparation of the Ganoderma lucidum acid-extracellular polysaccharide complex includes fermenting Antrodia camphorata in a medium containing olive squalane to obtain an aqueous phase and mycelium. The mycelium is then enzymatically hydrolyzed by chitinase and β-glucanase to obtain an enzymatic hydrolysate. The enzymatic hydrolysate is combined with the aqueous phase, activated by ultrasonication, and then loaded onto an AB-8 macroporous resin column for adsorption and purification to obtain the product. The preparation of the bitter orange flower extract includes water extraction of bitter orange pollen at a temperature of 110℃~120℃ and a pressure of 0.3MPa~0.4MPa, followed by purification of the extract by adsorption using a D101 macroporous resin column to obtain the product.

2. The composition for repairing damaged skin cells according to claim 1, characterized in that, The preparation method of the licorice root extract includes: taking coarse powder of licorice root core, soaking it in hot water, filtering and obtaining filter cake; preparing a mixed solution containing 5wt%–6wt% choline chloride and 7wt%–8wt% lactic acid with an ethanol aqueous solution; adding the filter cake to the mixed solution at a solid-liquid ratio of 1g:15mL–20mL, sonicating, centrifuging, collecting the liquid, concentrating under reduced pressure to remove ethanol, loading the sample onto an A21 macroporous adsorption resin column, rinsing with deionized water for 1–2 column volumes to remove impurities, then eluting with a 20%–30% volume concentration ethanol aqueous solution for 2–3 column volumes, collecting the eluent, concentrating under reduced pressure to remove ethanol, and freeze-drying to obtain the licorice root extract.

3. The composition for repairing damaged skin cells according to claim 2, characterized in that, The root core powder has a particle size of passing through a 40-60 mesh sieve; the amount of hot water used is 6-8 times the mass of the root core powder; the temperature of the hot water is 80℃-90℃; the soaking time is 20-30 minutes; the volume ratio of ethanol to water in the ethanol-water solution is (5-6):(4-5); the ultrasonication is performed at 50℃-60℃ and 200W-250W for 20-30 minutes; the centrifugation is performed at 6000rpm-8000rpm for 10-15 minutes; and the temperature for vacuum concentration is 50℃-60℃.

4. The composition for repairing damaged skin cells according to claim 1, characterized in that, The preparation method of the Cordyceps militaris extract peptides includes: adding Cordyceps militaris dried powder to a pH 5.0-6.0 phosphate buffer solution at 10-12 times its mass; then adding 0.5%-1% (by weight) chitinase from the Cordyceps militaris dried powder; and hydrolyzing at 40-45℃ for 1-2 hours; then raising the temperature to 60-65℃; adding 0.5%-1% (by weight) fig protease from the Cordyceps militaris dried powder; and hydrolyzing at 60-65℃ for 1.5-2 hours; and then raising the temperature... Enzyme inactivation was performed by cooling the temperature to 45℃~50℃ and adjusting the pH to 7.0~7.

5. 0.5%~1% (by weight of dried Cordyceps militaris powder) of Serratia marcescens enzyme was added, and enzymatic hydrolysis was carried out at 45℃~50℃ for 1h~1.5h. After inactivation, the temperature was raised to room temperature, centrifuged, and the supernatant was collected, concentrated under reduced pressure, and loaded onto a G-15 dextran gel column. The column was eluted with deionized water for 3~4 column volumes, and the eluent was collected. After concentration under reduced pressure at 50℃~55℃, the eluent was freeze-dried to obtain Cordyceps militaris extract peptides.

5. The composition for repairing damaged skin cells according to claim 4, characterized in that, The particle size of the dried Cordyceps militaris powder is that which passes through a 60-80 mesh sieve; the enzyme inactivation is carried out by heating to 85-90℃ for 10-15 minutes; the centrifugation is carried out at 6000-8000 rpm for 10-20 minutes; the supernatant is concentrated under reduced pressure to 30%-50% of its volume before loading the sample; the temperature of the reduced pressure concentration is 50-55℃.

6. The composition for repairing damaged skin cells according to claim 1, characterized in that, The preparation method of the ganoderic acid-extracellular polysaccharide complex includes: inoculating Antrodia camphorata mycelium solution into a liquid culture medium with a pH of 4.5–5.0, and culturing it at 25℃–28℃ and 200 rpm with stirring and aeration for 4–6 days; adding olive squalane and continuing to culture for 2–3 days to obtain a fermentation broth; centrifuging the fermentation broth and collecting the aqueous phase and mycelial precipitate; adding deionized water to the mycelial precipitate, adjusting the pH to 5.0–5.5, and adding 0.8%–1.2% (by weight of the mycelial precipitate) of chitinase and 0.8%–1.2% (by weight of the mycelial precipitate) of chitinase. % β-glucanase was hydrolyzed at 40℃~45℃ for 3h~4h, the enzyme was inactivated, and the solution was cooled to room temperature to obtain the hydrolysate. The hydrolysate was centrifuged and the supernatant was collected. The supernatant was combined with the aqueous phase and sonicated to obtain the activated solution. The activated solution was loaded onto an AB-8 macroporous resin column and washed with 10%~15% v / v ethanol aqueous solution for 2~3 column volumes to remove impurities. Then, it was eluted with 50%~60% v / v ethanol aqueous solution for 2~3 column volumes. The eluent was collected, concentrated under reduced pressure to remove ethanol, and freeze-dried to obtain the ganoderic acid-extracellular polysaccharide complex.

7. The composition for repairing damaged skin cells according to claim 6, characterized in that, The *Antrodia camphorata* inoculum solution is an activated and cultured *Antrodia camphorata* inoculum solution. The inoculum solution is obtained by first culturing *Antrodia camphorata* mycelia on PDA slant medium, and then culturing the mycelia on liquid medium. The inoculum volume is 8%–12% of the liquid medium volume. The olive squalane is sterilized. The amount of olive squalane added is 6%–10% of the liquid medium volume. The fermentation broth is centrifuged at 3000–4000 rpm for 10–15 minutes, and then centrifuged again at 5000 rpm for 5 minutes. Centrifuge at 500 rpm to 6000 rpm for 15 to 20 minutes; the amount of deionized water used is 10 to 12 times the mass of the mycelial precipitate; the enzyme inactivation is performed at 85℃ to 90℃ for 10 to 15 minutes; the enzyme hydrolysate is centrifuged at 5500 rpm to 6000 rpm for 15 to 20 minutes; the sonication is performed at 4℃ to 6℃, 200W to 250W, and 20kHz to 25kHz for 3 to 5 minutes; the vacuum concentration temperature is 45℃ to 50℃.

8. The composition for repairing damaged skin cells according to claim 1, characterized in that, The preparation method of the bitter orange flower extract includes: adding bitter orange flower pollen and deionized water at a solid-liquid ratio of 1g:15mL-20mL to a high-pressure reactor, extracting for 30min-50min at a temperature of 110℃-120℃, a pressure of 0.3MPa-0.4MPa, and a stirring speed of 100rpm-150rpm to obtain the extract, centrifuging, collecting the supernatant, concentrating under reduced pressure, loading the extract onto a D101 macroporous resin column, rinsing with deionized water for 2-3 column volumes to remove impurities, then eluting with 65%-70% ethanol aqueous solution for 3-4 column volumes, collecting the eluent, concentrating under reduced pressure to remove ethanol, and freeze-drying to obtain the bitter orange flower extract.

9. The composition for repairing damaged skin cells according to claim 8, characterized in that, The pollen of the bitter orange tree has a particle size of passing through a sieve of 40-60 mesh; the centrifugation is performed at 4000-5000 rpm for 15-20 minutes; the supernatant is concentrated under reduced pressure to 30%-50% of its volume before loading the sample; the temperature of the reduced pressure concentration is 50℃-55℃.

10. A method for preparing the composition for repairing damaged skin cells according to claim 1, characterized in that, Includes the following steps: The root extract of Glycyrrhiza glabra, peptides extracted from Cordyceps militaris, ganoderic acid-extracellular polysaccharide complex, and extract of Citrus aurantium were mixed in a specific mass ratio to obtain the composition.

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