A new application of walnut bark extract

Abstract

This invention discloses a novel application of walnut bark extract. The invention employs a complex enzymatic hydrolysis combined with gradient solvent extraction, supplemented by macroporous resin purification technology, to obtain a walnut bark extract enriched with polyphenolic compounds. It has been demonstrated that this extract can increase the elastin content in human dermal fibroblasts, inhibit the expression of various types of matrix metalloproteinases, and has the effects of improving skin firmness and anti-photoaging. Therefore, it can be applied in the preparation of cosmetics with firming and anti-photoaging functions.

Description

Technical Field

[0001] This invention relates to a walnut bark extract, and more particularly to a new application of the walnut bark extract. Background Technology

[0002] Skin aging is a multifactorial process, with skin laxity and decreased elasticity being the main manifestations. Skin firmness is primarily determined by the content and structure of collagen and elastin in its matrix. Collagen, as an important structural component of the skin, provides support and resilience; while elastin gives the skin elasticity, enabling it to withstand external pressure and return to its original shape. With age, the activity of fibroblasts gradually declines, reducing the synthesis of collagen and elastin. Existing collagen is also damaged by factors such as enzymatic degradation, oxidation, and ultraviolet radiation, leading to skin laxity and loss of elasticity. UVA radiation induces the production of large amounts of reactive oxygen species (ROS) in the skin. These free radicals attack cell structures through oxidation and activate matrix metalloproteinases (MMPs), thereby accelerating the degradation of collagen and elastin. This process directly damages the skin's mechanical properties, manifesting as deepening wrinkles, skin laxity, and other signs of aging.

[0003] Common anti-wrinkle and firming ingredients include active substances such as peptides and vitamins.

[0004] Juglans mandshurica is a deciduous tree belonging to the genus Juglans, mainly distributed in Northeast and North my country. Its unripe fruit husk, bark, and branch bark are traditional Chinese and Manchu medicinal materials. Juglans mandshurica roots mainly contain abundant phenols, quinones, and tannins, as well as organic acids, glycosides, flavonoids, and triterpenoids. Current research on Juglans mandshurica focuses on its anti-tumor, anti-inflammatory, antibacterial, and anti-obesity properties, and it has been proven to possess various biological activities, including anti-inflammatory, whitening, antioxidant, and anti-glycation effects.

[0005] However, existing research has failed to effectively link the chemical composition of walnut with its anti-aging effects on the skin. Systematic research on the effects of walnut on the core mechanisms of skin aging, such as extracellular matrix degradation, collagen loss, and elastic fiber breakage, is still lacking, and the potential anti-wrinkle and firming effects of walnut have not yet been discovered. Summary of the Invention

[0006] The purpose of this invention is to provide a new application for walnut bark extract. This invention investigates the effect of walnut bark in improving skin firmness.

[0007] The technical solution of the present invention is the application of a walnut bark extract in the preparation of cosmetics with at least one of the following effects: firming, anti-wrinkle, and anti-photoaging.

[0008] In the aforementioned applications, the main component of the walnut bark extract is polyphenols, and the polyphenol content is greater than 50%.

[0009] In the aforementioned applications, the walnut bark extract can improve the decrease in the expression levels of elastin, type I collagen, and type III collagen, and the increase in the expression levels of matrix metalloproteinases caused by ultraviolet radiation (especially UVA).

[0010] In the aforementioned applications, the preparation method of the walnut bark extract includes the following steps:

[0011] A. Raw material processing: Preparation of walnut bark powder;

[0012] B. Compound enzymatic hydrolysis: Add deionized water to the walnut bark powder, and add 0.7-1.0% (by weight of raw material) of cellulase and 0.2-0.4% (by weight of raw material) of hemicellulase, and perform enzymatic hydrolysis to obtain walnut enzymatic hydrolysate.

[0013] C. Component extraction: Add 90-95% ethanol (v / v) to the walnut enzymatic hydrolysate, extract and filter at room temperature; add 50-80% ethanol (v / v) to the residue, extract and filter at room temperature; add 50-80% ethanol (v / v) to the residue, heat and extract and filter; combine all extracts to obtain walnut extract containing polyphenols.

[0014] D. Concentration under reduced pressure: The polyphenol-containing walnut extract is concentrated under reduced pressure to obtain walnut concentrate.

[0015] E. Macroporous resin purification: The concentrated walnut extract is loaded onto macroporous resin for adsorption, and then washed with water; after washing with water, it is desorbed and eluted with 40-70% ethanol by volume, and the eluent is collected to obtain the walnut eluent.

[0016] F. Concentration and Drying: The walnut eluent is distilled and concentrated to obtain walnut extract, which is then vacuum dried and pulverized to obtain a brown powdery walnut bark extract.

[0017] In the aforementioned application, during step B, the pH of the enzymatic hydrolysate is adjusted to 4.8±0.5, and the enzymatic hydrolysate is carried out in a water bath with stirring at 40-55℃ for 2.5-3.5 hours.

[0018] In the aforementioned application, in step C, the volume ratio of 90-95% ethanol to walnut enzymatic hydrolysate is 1:1, the material-to-liquid ratio of ethanol extracted at room temperature is 1:10 (w:v), and the material-to-liquid ratio of ethanol extracted by heating is 1:20 (w:v).

[0019] In the aforementioned application, in step D, the conditions for vacuum concentration are: vacuum concentration is carried out at 40-55℃ until the mass is 6-8 times that of the walnut bark powder, the ethanol content is 10-20%, and the solid content is 1-3%.

[0020] In the aforementioned application, in step E, the loading flow rate of the walnut concentrate is 2-4 BV / h, and the flow rate of deionized water during washing is 2-4 BV / h.

[0021] In the aforementioned application, step F specifically involves: distilling and concentrating the walnut eluent at 40-55℃ to a mass equal to 1 / 10 of the walnut bark powder mass to obtain a walnut extract; vacuum drying the walnut extract at 35-45℃ and 15-25 Pa for 40-70 hours; and then pulverizing and grinding the dried solid to obtain a brown powdery walnut bark extract.

[0022] The present invention also provides a firming, anti-wrinkle or anti-photoaging cosmetic containing walnut bark extract.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] This invention employs a combined enzymatic hydrolysis and gradient solvent extraction process, supplemented by macroporous resin purification technology, to efficiently enrich polyphenolic compounds from walnut bark. Human dermal fibroblast experiments have demonstrated that, under UVA conditions, the obtained walnut bark extract can promote collagen and elastin synthesis and inhibit matrix metalloproteinase expression, thereby effectively enhancing skin elasticity, improving skin firmness, and alleviating skin laxity, thus playing an anti-photoaging role. It can be applied to the preparation of cosmetics with firming, anti-wrinkle, and anti-photoaging effects. Detailed Implementation

[0025] The present invention will be further described below, but this should not be construed as limiting the invention.

[0026] In the description of this invention, it should also be noted that, unless otherwise explicitly specified and limited, the terms "set," "install," "connect," and "link" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, a hinged connection, a rotatable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium, or a connection within two components. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0027] Example 1:

[0028] The preparation method of walnut bark extract includes the following steps:

[0029] A. Raw material processing: Crush the dried walnut bark and pass it through a 24-mesh sieve to obtain walnut bark powder for later use.

[0030] B. Compound enzymatic hydrolysis: Add deionized water to the walnut bark powder at a material-to-liquid ratio of 1:10 (w:w), add 0.7% cellulase and 0.2% hemicellulase by weight of the raw materials, adjust the pH of the hydrolysate to 4.8, and perform enzymatic hydrolysis in a water bath at 50℃ for 2.5 hours to obtain walnut enzymatic hydrolysate.

[0031] C. Component Extraction: Add 95% ethanol (v / v) to the walnut enzymatic hydrolysate at a volume ratio of 1:1, stir and extract at room temperature for 1.0 h, filter, and collect the supernatant as the walnut extract enriched with polyphenols; add 75% ethanol (v / v) to the residue at a material-to-liquid ratio of 1:10 (w:v), stir and extract at room temperature for 30 min, filter again, collect the supernatant and combine it with the walnut extract; add 50% ethanol (v / v) to the residue at a material-to-liquid ratio of 1:20 (w:v), heat and reflux at 65℃ and stir and extract for 3 h, filter to obtain the walnut extract, combine all extracts to obtain the walnut extract containing polyphenols.

[0032] D. Vacuum Concentration: The polyphenol-containing walnut extract is concentrated under reduced pressure at 50°C until its mass is 7 times that of the walnut bark powder, the ethanol content is 15%, and the solid content is 1%. After filtration, the walnut concentrate is obtained.

[0033] E. Macroporous resin purification: Take the pretreated LX-T83 resin (the resin volume is 2.5 times the weight of the walnut bark powder), load the walnut concentrate at 3 BV / h for adsorption, and then wash with 5 BV of deionized water at 2 BV / h; after washing, desorb and elute with 5 BV of 40% ethanol, collect the eluent, and obtain the walnut eluent.

[0034] F. Concentration and Drying: The walnut eluent was distilled and concentrated at 50°C to 1 / 10 of the walnut bark powder to obtain walnut extract; the walnut extract was vacuum dried at 40°C and 20 Pa for 40 hours, and the dried solid was pulverized and ground to obtain brown powdered walnut bark extract.

[0035] Example 2:

[0036] The preparation method of walnut bark extract includes the following steps:

[0037] A. Raw material processing: Crush the dried walnut bark and pass it through a 24-mesh sieve to obtain walnut bark powder for later use.

[0038] B. Compound enzymatic hydrolysis: Add deionized water to the walnut bark powder at a material-to-liquid ratio of 1:10 (w:w), add 0.9% cellulase and 0.3% hemicellulase by weight of the raw materials, adjust the pH of the hydrolysate to 4.8, and perform enzymatic hydrolysis in a water bath at 50℃ for 3 hours to obtain walnut enzymatic hydrolysate.

[0039] C. Component Extraction: Add 95% ethanol (v / v) to the walnut enzymatic hydrolysate at a volume ratio of 1:1, stir and extract at room temperature for 1.5 h, filter, and collect the supernatant as the walnut extract enriched with polyphenols; add 50% ethanol (v / v) to the residue at a material-to-liquid ratio of 1:10 (w:v), stir and extract at room temperature for 30 min, filter again, collect the supernatant and combine it with the walnut extract; add 70% ethanol (v / v) to the residue at a material-to-liquid ratio of 1:20 (w:v), heat and reflux at 65℃ and stir and extract for 2 h, filter to obtain the walnut extract, combine all extracts to obtain the walnut extract containing polyphenols.

[0040] D. Vacuum Concentration: The polyphenol-containing walnut extract is concentrated under reduced pressure at 50°C until its mass is 6 times that of the walnut bark powder, with an ethanol content of 15% and a solid content of 2%. After filtration, the walnut concentrate is obtained.

[0041] E. Purification of macroporous resin: Take the pretreated LX-T83 resin (the resin volume is 2.5 times the weight of the walnut bark powder), load the concentrated walnut extract onto the resin at 2 BV / h for adsorption, and then wash with 5 BV of deionized water at 3 BV / h; after washing, desorb and elute with 5 BV of 50% ethanol, collect the eluent, and obtain the walnut eluent.

[0042] F. Concentration and Drying: The walnut eluent was distilled and concentrated at 50°C to 1 / 10 of the walnut bark powder to obtain walnut extract; the walnut extract was vacuum dried at 40°C and 20 Pa for 60 hours, and the dried solid was pulverized and ground to obtain brown powdered walnut bark extract.

[0043] Example 3:

[0044] The preparation method of walnut bark extract includes the following steps:

[0045] A. Raw material processing: Crush the dried walnut bark and pass it through a 24-mesh sieve to obtain walnut bark powder for later use.

[0046] B. Compound enzymatic hydrolysis: Add deionized water to the walnut bark powder at a material-to-liquid ratio of 1:10 (w:w), add 1.0% cellulase and 0.4% hemicellulase by weight of the raw materials, adjust the pH of the hydrolysate to 4.8, and perform enzymatic hydrolysis by stirring in a water bath at 50℃ for 3.5 hours to obtain walnut enzymatic hydrolysate.

[0047] C. Component Extraction: Add 95% ethanol (v / v) to the walnut enzymatic hydrolysate at a volume ratio of 1:1, stir and extract at room temperature for 2.0 h, filter, and collect the supernatant as the walnut extract enriched with polyphenols; add 80% ethanol (v / v) to the residue at a material-to-liquid ratio of 1:10 (w:v), stir and extract at room temperature for 30 min, filter again, collect the supernatant and combine it with the walnut extract; add 80% ethanol (v / v) to the residue at a material-to-liquid ratio of 1:20 (w:v), heat and reflux at 65℃ and stir and extract for 4 h, filter to obtain the walnut extract, combine all extracts to obtain the walnut extract containing polyphenols.

[0048] D. Vacuum Concentration: The polyphenol-containing walnut extract is concentrated under reduced pressure at 50°C until its mass is 8 times that of the walnut bark powder, with an ethanol content of 15% and a solid content of 3%. After filtration, the concentrated walnut extract is obtained.

[0049] E. Purification of macroporous resin: Take the pretreated LX-T83 resin (the resin volume is 2.5 times the weight of the walnut bark powder), load the concentrated walnut extract onto the resin at 4 BV / h for adsorption, and then wash with 5 BV of deionized water at 4 BV / h; after washing, desorb and elute with 5 BV of 70% ethanol, collect the eluent, and obtain the walnut eluent.

[0050] F. Concentration and Drying: The walnut eluent was distilled and concentrated at 50°C to a mass of 1 / 10 of the walnut bark powder to obtain walnut extract; the walnut extract was vacuum dried at 40°C and 20 Pa for 70 hours, and the dried solid was pulverized and ground to obtain brown powdered walnut bark extract.

[0051] Experimental Example 1: Detection of polyphenol content.

[0052] 1-1. Experimental Methods:

[0053] (1) Experimental method: The total polyphenol content was determined by spectrophotometry according to the total polyphenol determination method in the "Announcement of the National Health Commission of the People's Republic of China".

[0054] (2) Experimental steps:

[0055] Preparation of standard working solutions: Gallic acid standard working solution: Accurately pipette 0 mL, 0.04 mL, 0.08 mL, 0.12 mL, 0.16 mL, and 0.20 mL of gallic acid standard stock solution, add water to 0.20 mL respectively, shake well, and prepare a series of standard working solutions with concentrations of 0 mg / L, 40 mg / L, 80 mg / L, 120 mg / L, 160 mg / L, and 200 mg / L. Prepare fresh solutions before use.

[0056] Weigh 1g (accurate to 0.1mg) of the brown powdered walnut bark extract samples from Examples 1-3 into a beaker, dissolve in 10mL of water, transfer to a 25mL volumetric flask, rinse the beaker with 10mL of water, combine the solutions into the volumetric flask, and dilute to volume with water. Transfer 1.0mL to a 100mL volumetric flask, dilute to volume with water, and mix well to obtain the sample solution for analysis.

[0057] Accurately pipette 0.2 mL each of the gallic acid series standard working solution and sample solution into graduated test tubes. Add 1.8 mL of distilled water and 0.2 mL of Folin-Ciocalteu reagent to each test tube, shake well, and react for 5 min. Once the color in the test tube turns light green, add 2 mL of 7% sodium carbonate solution and 0.8 mL of distilled water, and shake well. Let stand at room temperature for 90 min, and then measure the absorbance using a spectrophotometer at 750 nm using a 10 mm cuvette. Construct a standard curve based on the absorbance (A) and concentration of the gallic acid series standard working solutions. Calculate the total polyphenol content (calculated as gallic acid) using the following formula:

[0058] X(%)=(C×V×K×100%) / (m×10 6 );

[0059] In the formula:

[0060] X represents the percentage content of total polyphenols in the sample;

[0061] C represents the concentration of gallic acid in the test solution calculated from the standard curve, in micrograms per milliliter (μg / mL).

[0062] V represents the constant volume, expressed in milliliters (mL).

[0063] K is the sample dilution factor;

[0064] m represents the sample size, measured in grams (g).

[0065] 10 6 Conversion factor for converting units of micrograms (μg) to grams (g);

[0066] 100% converts the result to a percentage.

[0067] The calculation result is rounded to two decimal places.

[0068] 1-2. Experimental Results:

[0069] (1) Plotting the standard curve:

[0070] Fitting equation: y = 2.101144 x - 0.000033;

[0071] R 2 = 0.999536.

[0072] (2) Results of polyphenol content detection in samples:

[0073] The sample test results were substituted into the standard curve equation to calculate the polyphenol content. The test results are shown in Table 1.

[0074] Table 1. Results of Polyphenol Content Detection

[0075]

[0076] The test results of this experiment show that the walnut bark extract prepared using this example has a high content of polyphenol-like substances (≥50%). The main identified components of the walnut bark extracts obtained in Examples 1, 2, and 3 are polyphenols. The results show that there is no significant difference in the polyphenol content of the walnut bark extract products obtained from the three examples (p>0.05), proving that the differences in the components of the products obtained from different examples are small, and their efficacy is mutually referential. Therefore, the following experimental examples are analyzed based primarily on the test results of the sample from Example 2.

[0077] Experimental Example 2: Elastase Inhibition Experiment.

[0078] Human leukocyte elastase is a destructive enzyme present in the body that hydrolyzes certain connective tissue components such as elastin, proteoglycans, and certain types of collagen. Overexpression of elastase leads to a decrease in collagen content in the skin, and the gradual degradation and fragmentation of elastic fibers, resulting in skin wrinkles, aging, and other problems.

[0079] 2-1. Experimental Methods:

[0080] (1) Reaction system:

[0081] Table 2-1. Order of reagent addition and reaction system

[0082]

[0083] (2) Solution preparation:

[0084] 50 mM Tris-HCl: Dilute to 50 mM using 1 M Tris-HCl.

[0085] Elastase preparation: Dissolve 10 mg of elastase powder (30 U / mg) in 50 mM Tris-HCl to obtain a 300 U / mL elastase solution. Continue to dilute with 50 mM Tris-HCl 500 times to 600 mU / mL. This operation is performed on ice.

[0086] Preparation of AAAPAN (N-succinyl-alanine-alanine-alanine-p-nitroaniline) solution: Prepare a 0.4582 g / L solution using 50 mM Tris-HCl.

[0087] Positive control preparation: Prepare EGCG solutions of 0.0625 mg / mL, 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, and 1 mg / mL respectively for testing.

[0088] Sample preparation: Dilute the samples to be tested with water to 0.0625 mg / mL, 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, and 2 mg / mL respectively for detection.

[0089] (3) Experimental steps:

[0090] According to the reaction system in (1), add the corresponding reagents, let stand at 25°C for 15 min, then quickly add 150 μL of AAAPAN solution to each group of EP tubes and mix thoroughly. Then let the reaction tubes stand again for 20 min. This operation is carried out on ice.

[0091] After the reaction, the samples were spotted onto the plate in sequence, and the absorbance was measured at a wavelength of 410 nm using an ELISA reader.

[0092] Calculate the elastase inhibition rate of the sample according to the calculation formula:

[0093] Inhibition rate (%) = (1 − (A2−A3) / (A0−A1)) × 100%;

[0094] In the formula: A0 is the absorbance value of the enzyme tube, A1 is the absorbance value of the non-enzyme tube, A2 is the absorbance value of the sample tube, and A3 is the absorbance value of the sample control tube.

[0095] 2-2. Experimental Results:

[0096] (1) Results of the positive control group:

[0097] Table 2-2. Results of EGCG elastase inhibition test in the positive control group

[0098]

[0099] Based on the results in Table 2-2, the IC50 value of EGCG's elastase inhibitory ability was calculated to be 0.2852 mg / mL, proving that the positive control has reference value.

[0100] (2) Sample test results:

[0101] Table 2-3. Results of elastase inhibition test in sample group Example 2

[0102]

[0103] Based on the calculations in Table 4-3 above, the IC50 value of the elastase inhibition ability of the sample in Example 2 was 0.089 mg / mL, which was lower than that of the positive control group EGCG. This demonstrates that the sample has good elastase inhibition potential in biochemical analysis experiments, and its efficacy can be further verified.

[0104] Experimental Example 3: Human dermal fibroblast toxicity test.

[0105] 3-1. Toxicity test method: MTT method.

[0106] Experimental steps:

[0107] Cell seeding: Human dermal fibroblasts were seeded into 96-well plates (5 × 10⁻⁶ cells per well). 3 (each well) was incubated at 37°C and 5% CO2 for 24 hours.

[0108] Drug administration: The sample groups were added with the corresponding concentrations of the samples in Table 3-1. The blank control and blank solvent groups were replaced with fresh culture medium and incubated at 37℃ for 24h. After incubation, MTT solution was added to each well and cultured for another 4h. The culture medium was then discarded, DMSO was added and shaken to mix, and the absorbance value was measured at 490nm.

[0109] Cell viability (%) = (OD) 490 Sample group - OD 490 Blank solvent group) / (OD 490 Blank control group - OD 490 (blank solvent group) × 100%. Where OD 490 : Absorbance value at 490nm.

[0110] Table 3-1. Grouping Table for Cytotoxicity Experiments

[0111]

[0112] 3-2. Results of human dermal fibroblast cytotoxicity test:

[0113] Table 3-2. Cytotoxicity Results of Samples from Example 2

[0114]

[0115] The results of the cytotoxicity test showed that the sample in Example 2 had no cytotoxicity when the concentration was less than 0.0125%, and 0.002% and 0.01% were subsequently selected as the experimental concentrations.

[0116] Experimental Example 4: Determination of the firming effect of human dermal fibroblasts.

[0117] 4-1. Method for testing firming efficacy:

[0118] Elastin exists in the extracellular matrix and plays an important role in maintaining skin elasticity. The loss of elastin is the main cause of aging phenomena such as skin laxity and sagging.

[0119] Type I and Type III collagen are the main structural proteins of the skin and are essential for maintaining skin elasticity and strength. Type I collagen is the most abundant type of collagen in the skin.

[0120] Ultraviolet (UV) radiation can penetrate the skin, reaching the epidermis and dermis, and UVA is a key factor leading to photoaging of the skin. UV exposure causes the degradation of elastin, type I collagen, and type III collagen, triggering oxidative stress within cells and producing large amounts of reactive oxygen species (ROS). Excessive ROS accumulation stimulates the expression of matrix metalloproteinases (MMPs), which degrade collagen and elastin, leading to decreased synthesis and accelerated breakdown, altering their conformation, affecting the skin's mechanical properties, and resulting in sagging skin and increased wrinkles.

[0121] (1) Detection protocol for the expression levels of elastin (ELN) and Collagen I (COL1) and Collagen III (COL3):

[0122] Table 4-1. Experimental grouping table for ELN, COL1, and COL3 expression levels

[0123]

[0124] Cell seeding: Human dermal fibroblasts were seeded into 96-well plates (5 × 10⁻⁶ cells per well). 3 (each well) was incubated at 37°C and 5% CO2 for 24 hours.

[0125] Administration: After incubation, remove the culture medium, wash 1-2 times with D-Hanks, and then expose the sample group, negative control group, and positive control group to 9J / cm². 2Irradiation with UVA lamp. After modeling, the blank control group and negative control group were replaced with fresh culture medium, the sample group was added with fresh culture medium containing the sample, and the positive control group was added with fresh culture medium containing TGF-β. Incubation was continued at 37℃, 5% CO2 for 24 hours. The supernatant was discarded, and the process was repeated: washing, fixation, permeabilization, and antibody incubation. Immunofluorescence images were taken under a fluorescence microscope, and the results were quantitatively analyzed using ImageJ software.

[0126] Upregulation rate (%) = (S sample group - S negative control group) × 100% / S negative control group;

[0127] In the formula: S: average fluorescence intensity.

[0128] (2) Detection scheme for MMP-1 and MMP-3 expression levels:

[0129] Table 4-2. Experimental grouping of MMP-1 and MMP-3 expression levels

[0130]

[0131] Cell seeding: Human dermal fibroblasts were seeded into 96-well plates (5 × 10⁻⁶ cells per well). 3 (each well) was incubated at 37°C and 5% CO2 for 24 hours.

[0132] Administration: After incubation, remove the culture medium, wash 1-2 times with D-Hanks, and then expose the sample group, negative control group, and positive control group to 9J / cm². 2 Irradiate with UVA ultraviolet light. After modeling, replace the blank control group and negative control group with fresh culture medium, add fresh culture medium containing the sample to the sample group, and add fresh culture medium containing vitamin C to the positive control group. Continue incubation at 37℃ and 5% CO2 for 24 hours. Discard the supernatant, and perform washing-fixation-permeabilization-antibody incubation. Take pictures under a fluorescence microscope, and use ImageJ software to quantify the immunofluorescence results.

[0133] Downregulation rate (%) = (S negative control group - S sample group) × 100% / S negative control group;

[0134] In the formula: S: average fluorescence intensity.

[0135] 4-2. Firming efficacy test results:

[0136] (1) Results of elastin (ELN) expression detection:

[0137] Table 4-3. Experimental results of elastin (ELN) expression.

[0138]

[0139] Note: Compared with the blank control group, " ### "" indicates p < 0.001; compared with the negative control group, " * " indicates p < 0.05", *** "" indicates p < 0.001; p < 0.05 indicates a significant difference.

[0140] In this experiment, after UVA modeling, the average fluorescence intensity of ELN decreased, proving that UVA leads to ELN degradation. The addition of the positive control group TGF-β significantly upregulated the average fluorescence intensity of ELN, confirming the validity of the model. The addition of the sample from Example 2 significantly upregulated the average fluorescence intensity of ELN, demonstrating that the sample from Example 2 effectively reduced UVA-induced degradation of human fibroblast elastin (ELN), and its upregulation ability was dose-dependent. This demonstrates that Example 2 has a good firming effect.

[0141] (2) Results of CollagenⅠ (COL1) expression detection:

[0142] Table 4-4. Experimental results of Collagen I (COL1) expression.

[0143]

[0144] Note: Compared with the blank control group, " ## "" indicates p < 0.01; compared with the negative control group, " * " indicates p < 0.05", ** "" indicates p < 0.01; p < 0.05 indicates a significant difference.

[0145] In this experiment, after UVA modeling, the average fluorescence intensity of COL1 decreased, proving that UVA can lead to the degradation of COL1. However, the addition of the positive control group TGF-β significantly upregulated the average fluorescence intensity of COL1, confirming the validity of the model. The addition of the sample from Example 2 significantly upregulated the average fluorescence intensity of COL1, demonstrating that the sample from Example 2 effectively reduced UVA-induced degradation of human fibroblast Collagen I (COL1), and that its upregulation was dose-dependent. This demonstrates that the sample from Example 2 has a good firming effect.

[0146] (3) Results of Collagen III (COL3) expression detection:

[0147] Table 4-5. Experimental results of Collagen III (COL3) expression.

[0148]

[0149] Note: Compared with the blank control group, " ### "" indicates p < 0.001; compared with the negative control group, " * " indicates p < 0.05", ** " indicates p < 0.01", *** "" indicates p < 0.001; p < 0.05 indicates a significant difference.

[0150] In this experiment, after UVA modeling, the average fluorescence intensity of COL3 decreased, proving that UVA can lead to COL3 degradation. However, the addition of the positive control group TGF-β significantly upregulated the average fluorescence intensity of COL3, confirming the validity of the model. The addition of the sample from Example 2 significantly upregulated the average fluorescence intensity of COL3, demonstrating that the sample from Example 2 effectively reduced UVA-induced degradation of human fibroblast Collagen III (COL3), and that its upregulation was dose-dependent. This demonstrates that the sample from Example 2 has a good firming effect.

[0151] (4) Results of matrix metalloproteinase-1 (MMP-1) expression detection:

[0152] Table 4-6. Experimental results of matrix metalloproteinase-1 (MMP-1) expression.

[0153]

[0154] Note: Compared with the blank control group, " ### "" indicates p < 0.001; compared with the negative control group, " ** " indicates p < 0.01", *** "" indicates p < 0.001; p < 0.05 indicates a significant difference.

[0155] In this experiment, after UVA modeling, the average fluorescence intensity of MMP-1 increased, proving that UVA can induce MMP-1 expression. The addition of vitamin C (positive control group) significantly reduced the average fluorescence intensity of MMP-1, confirming the validity of the model. The addition of the sample from Example 2 significantly reduced the average fluorescence intensity of MMP-1, demonstrating that the sample from Example 2 effectively reduced the decline in skin mechanical properties caused by UVA and reduced the degradation of elastin and collagen caused by high MMP-1 expression, with the downregulation ability being dose-dependent. This demonstrates that Example 2 has a good firming effect.

[0156] (5) Results of matrix metalloproteinase-3 (MMP-3) expression detection:

[0157] Table 4-7. Experimental results of matrix metalloproteinase-3 (MMP-3) expression.

[0158]

[0159] Note: Compared with the blank control group, " ## "" indicates p < 0.01; compared with the negative control group, " ** " indicates p < 0.01", *** "" indicates p < 0.001; p < 0.05 indicates a significant difference.

[0160] In this experiment, after UVA modeling, the average fluorescence intensity of MMP-3 increased, proving that UVA can induce MMP-3 expression. The addition of vitamin C (positive control group) significantly reduced the average fluorescence intensity of MMP-3, confirming the validity of the model. The addition of the sample from Example 2 significantly reduced the average fluorescence intensity of MMP-3, demonstrating that the sample from Example 2 effectively reduced the decline in skin mechanical properties caused by UVA and reduced the degradation of elastin and collagen caused by high MMP-3 expression, with the downregulation ability being dose-dependent. This demonstrates that Example 2 has a good firming effect.

[0161] The parts of this invention not described in detail are prior art and therefore will not be specifically described here.

Claims

1. The application of a walnut bark extract in the preparation of cosmetics with at least one of the following effects: firming, anti-wrinkle, and anti-photoaging.

2. The application of the walnut bark extract according to claim 1, characterized in that: The polyphenol content in the walnut bark extract is greater than 50%.

3. The application of the walnut bark extract according to claim 1, characterized in that: The walnut bark extract can improve the decrease in the expression levels of elastin, type I collagen, and type III collagen, and the increase in the expression level of matrix metalloproteinases caused by ultraviolet radiation.

4. The application of the walnut bark extract according to claim 1, characterized in that: The preparation method of the walnut bark extract includes the following steps: A. Raw material processing: Preparation of walnut bark powder; B. Compound enzymatic hydrolysis: Add deionized water to the walnut bark powder, and add 0.7-1.0% (by weight of raw material) of cellulase and 0.2-0.4% (by weight of raw material) of hemicellulase, and perform enzymatic hydrolysis to obtain walnut enzymatic hydrolysate. C. Component extraction: Add 90-95% ethanol (v / v) to the walnut enzymatic hydrolysate, extract and filter at room temperature; add 50-80% ethanol (v / v) to the residue, extract and filter at room temperature; add 50-80% ethanol (v / v) to the residue, heat and extract and filter; combine all extracts to obtain walnut extract containing polyphenols. D. Concentration under reduced pressure: The polyphenol-containing walnut extract is concentrated under reduced pressure to obtain walnut concentrate. E. Macroporous resin purification: The concentrated walnut extract is loaded onto macroporous resin for adsorption, and then washed with water; after washing with water, it is desorbed and eluted with 40-70% ethanol by volume, and the eluent is collected to obtain the walnut eluent. F. Concentration and Drying: The walnut eluent is distilled and concentrated to obtain walnut extract, which is then vacuum dried and pulverized to obtain a brown powdery walnut bark extract.

5. The application of the walnut bark extract according to claim 4, characterized in that: In step B, during enzymatic hydrolysis, the pH of the hydrolysate is adjusted to 4.8±0.5, and the hydrolysis is carried out in a water bath with stirring at 40-55℃ for 2.5-3.5 hours.

6. The application of the walnut bark extract according to claim 4, characterized in that: In step C, the volume ratio of 90-95% ethanol to walnut enzymatic hydrolysate is 1:1, the material-to-liquid ratio of ethanol extracted at room temperature is 1:10 (w:v), and the material-to-liquid ratio of ethanol extracted by heating is 1:20 (w:v).

7. The application of the walnut bark extract according to claim 4, characterized in that: In step D, the conditions for vacuum concentration are as follows: vacuum concentration is carried out at 40-55℃ until the mass is 6-8 times that of the walnut bark powder, the ethanol content is 10-20%, and the solid content is 1-3%.

8. The application of the walnut bark extract according to claim 4, characterized in that: In step E, the loading flow rate of the walnut concentrate is 2-4 BV / h, and the flow rate of deionized water during washing is 2-4 BV / h.

9. The application of the walnut bark extract according to claim 4, characterized in that: Step F specifically involves: distilling and concentrating the walnut eluent at 40-55℃ until its mass is 1 / 10 of the walnut bark powder mass to obtain walnut extract; vacuum drying the walnut extract at 35-45℃ and 15-25 Pa for 40-70 hours; and then pulverizing and grinding the dried solid to obtain a brown powdery walnut bark extract.

10. A cosmetic product that firms, reduces wrinkles, or combats photoaging, characterized in that: It contains extract from walnut bark.

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