A white peony bark peptide, its preparation method and application

Abstract

This invention relates to the field of natural plant peptide technology, specifically to a Dictamnus dasycarpus root bark peptide, its preparation method, and its applications. The amino acid sequence of the Dictamnus dasycarpus root bark peptide is as follows: A, a peptide with the amino acid sequence SEQ ID NO1: Tyr-Gln-Tyr-Asn-Glu-Arg-Arg; or B, a peptide with the amino acid sequence SEQ ID NO2: Trp-Gln-Gln-Thr-Asn-Arg; or C, a peptide with the amino acid sequence SEQ ID NO3: Gly-Ser-Ala-Asn-Pro-Tyr. These peptides possess antioxidant activity, effectively scavenging free radicals caused by ultraviolet radiation, reducing oxidative stress, and preventing and repairing photoaging of the skin. In vitro experimental results show that the Dictamnus dasycarpus root bark peptide can significantly improve the survival rate of skin cells and reduce cell membrane damage, oxidative lipidation, DNA damage, and apoptosis caused by ultraviolet radiation. The Dictamnus dasycarpus root bark peptide of this invention can be used in combination with existing sunscreens, anti-aging cosmetics, and after-sun repair agents to enhance ultraviolet protection and anti-aging effects, and is suitable for sensitive skin.

Description

Technical Field

[0001] This invention relates to the field of natural plant peptides, particularly Dictamnus dasycarpus root bark peptides, their preparation methods, and their applications in skin care. Specifically, this invention provides a method for preparing Dictamnus dasycarpus root bark peptides with antioxidant and anti-photoaging functions, which can be widely used in sunscreens and anti-aging cosmetics. Background Technology

[0002] Photoaging is a skin aging process caused by prolonged exposure to ultraviolet (UV) radiation. UV radiation can be divided into UVA, UVB, and UVC. UVA has the longest wavelength, penetrating the skin's surface to reach the dermis, causing deep skin damage. UVB primarily affects the skin's surface, leading to acute inflammatory reactions and DNA damage. Long-term exposure to UV radiation causes oxidative stress in the skin, producing a large number of free radicals, such as superoxide anions, hydrogen peroxide, and hydroxyl radicals. These free radicals damage proteins, lipids, and DNA within skin cells, leading to the destruction of skin structure, degradation of collagen and elastin, and ultimately manifesting as sagging skin, wrinkles, pigmentation, and decreased elasticity—a series of signs of aging. This type of damage is one of the root causes of photoaging.

[0003] Currently, products on the market designed to prevent photoaging of the skin are mainly sunscreens and anti-aging skincare products. These products typically rely on chemical ingredients such as UV filters and antioxidants to block harmful UV rays or to slow down skin aging by inhibiting the generation of free radicals. However, existing sunscreen and anti-aging ingredients have certain limitations in terms of effectiveness and safety. While UV filters can effectively absorb UV rays, they are usually only effective against UVB, and prolonged use may cause adverse skin reactions. Antioxidants such as vitamins C and E, although capable of scavenging free radicals to some extent, have poor stability and require high concentrations to achieve significant effects; long-term use may cause skin irritation.

[0004] Among numerous plants, Dictamnus dasycarpus root bark, a traditional Chinese medicine, possesses various pharmacological effects, including anti-rheumatism, strengthening of muscles and bones, and immune regulation. In recent years, scientists have also gradually recognized its potential anti-aging and antioxidant functions. Dictamnus dasycarpus root bark is rich in various bioactive components, such as polysaccharides, flavonoids, phenolic compounds, and amino acids. Its peptide components, due to their small molecule characteristics, are easily absorbed by the skin and rapidly exert biological effects. Studies have shown that certain peptides in Dictamnus dasycarpus root bark have strong antioxidant activity, effectively scavenging free radicals and slowing down skin oxidative damage caused by ultraviolet radiation. Dictamnus dasycarpus root bark peptides can react with free radicals, preventing free radicals from attacking skin cells, thereby reducing cell damage caused by oxidative stress. Furthermore, Dictamnus dasycarpus root bark peptides have also been found to have significant effects in inhibiting ultraviolet-induced skin inflammation, inhibiting the overexpression of matrix metalloproteinases (MMPs), and enhancing skin barrier function. These functions make it an ideal anti-photoaging ingredient.

[0005] In summary, Dictamnus dasycarpus peptide, as a natural antioxidant and anti-photoaging active ingredient, possesses enormous application potential in the prevention and treatment of skin photoaging due to its excellent biological activity and good skin permeability. Therefore, there is an urgent need to invent a Dictamnus dasycarpus peptide, its preparation method, and its applications to solve the aforementioned technical problems. Summary of the Invention

[0006] This invention relates to a Dictamnus dasycarpus root bark peptide with antioxidant or anti-photoaging functions, its preparation method, and its applications. By extracting and purifying peptide components from Dictamnus dasycarpus root bark, a class of peptides with significant antioxidant and anti-photoaging effects were screened, and their applications in skin care products were explored. The purpose of this invention is to provide a natural plant peptide that can effectively prevent and repair skin aging caused by ultraviolet radiation and protect the skin from free radical damage, with wide applications in sunscreens, anti-aging cosmetics, and after-sun repair agents.

[0007] A Dictamnus dasycarpus root bark peptide, the amino acid sequence of which is shown below:

[0008] A. A peptide with the amino acid sequence SEQ ID NO1:Tyr-Gln-Tyr-Asn-Glu-Arg-Arg; or B. A peptide with the amino acid sequence SEQ ID NO2:Trp-Gln-Gln-Thr-Asn-Arg; or C. A peptide with the amino acid sequence SEQ ID NO3: Gly-Ser-Ala-Asn-Pro-Tyr.

[0009] Preferably, a method for preparing Dictamnus dasycarpus peptides is provided, comprising the following steps: S1. Take the root bark of Dictamnus dasycarpus, defatt it and remove the tannins, then extract it with PBS buffer at a liquid-to-solid ratio of 40-60, collect the supernatant by centrifugation, precipitate with ammonium sulfate, dialyze, and freeze dry to obtain crude protein. S2. Take the crude protein, add protease for enzymatic hydrolysis, and obtain the hydrolysate; S3. The enzymatic hydrolysate is separated by gel column chromatography, and the components corresponding to the target absorption peak are collected.

[0010] Preferably, in step S1, the extraction temperature is 45-65℃, the ammonium sulfate precipitate is 80% saturated ammonium sulfate, and the PBS buffer solution has a liquid-to-solid ratio of 50:1.

[0011] Preferably, the protease in S2 is a neutral protease, and the preferred hydrolysis conditions for the neutral protease are: pH 7.0, hydrolysis time 1 hour, hydrolysis temperature 50°C, liquid-to-solid ratio 50:1, and enzyme addition amount 3000 U / g.

[0012] Preferably, in step S3, the target absorption peak is the absorption peak at 280 nm.

[0013] Preferably, the application of Dictamnus dasycarpus peptides in the preparation of cosmetic products or pharmaceuticals for anti-oxidation or anti-photoaging of the skin is proposed.

[0014] Preferably, the product is a sunscreen, an anti-aging cosmetic, or an after-sun repair agent.

[0015] Preferably, the Dictamnus dasycarpus peptide is used in the preparation of a sunscreen agent, wherein the Dictamnus dasycarpus peptide prevents damage to epidermal cells caused by ultraviolet radiation, and the epidermal cell damage is selected from one or more of the following: decreased cell vitality, cell membrane damage, oxidative lipidation, DNA damage, or apoptosis.

[0016] Preferably, the Dictamnus dasycarpus peptide is used to prepare an after-sun repair agent, wherein the Dictamnus dasycarpus peptide repairs the skin epidermal cell damage caused by ultraviolet radiation, and the skin epidermal cell damage is selected from one or more of the following: decreased cell vitality, cell membrane damage, oxidative lipidation, DNA damage, or apoptosis.

[0017] Preferably, the Dictamnus dasycarpus peptide is used in the preparation of anti-aging cosmetics that prevent or repair skin aging, wherein the skin aging is caused by ultraviolet radiation and is specifically manifested as one or more of the following: thickening of the skin epidermis, increased level of skin oxidative stress, aggravated skin oxidative lipidation, or loss of skin collagen.

[0018] The above amino acid sequences are SEQ ID NO1: Tyr-Gln-Tyr-Asn-Glu-Arg-Arg (Y-7-R) peptide, SEQ ID NO2: Trp-Gln-Gln-Thr-Asn-Arg (W-6-R) peptide, and SEQ ID NO3: Gly-Ser-Ala-Asn-Pro-Tyr (G-6-Y) peptide.

[0019] This invention uses Dictamnus dasycarpus peptides (Y-7-R, W-6-R, or G-6-Y) as the target, and has the following beneficial effects:

[0020] The Dictamnus dasycarpus peptides (especially Y-7-R) in this invention can significantly increase the survival rate of damaged cells from approximately 46% to 85%, while effectively inhibiting the release rate of lactate dehydrogenase, a marker of cell membrane damage, from 218% to 118%, far exceeding the levels of existing antioxidants. Simultaneously, the peptides of this invention can efficiently scavenge free radicals, significantly reduce the content of malondialdehyde (MDA), the end product of lipid peroxidation, and reduce the rate of UVB-induced cellular DNA damage and apoptosis by more than 50% and 65%, respectively. By blocking the photoaging damage chain from source to end, it can effectively address the cascade reaction of free radical bursts caused by ultraviolet radiation, which in turn damages cell membranes, lipids, and DNA, triggering apoptosis. The Dictamnus dasycarpus peptides in this invention exhibit comprehensive protective and repairing effects, fundamentally combating skin photoaging.

[0021] Experiments in this invention demonstrate that topical application of Dictamnus dasycarpus peptides possesses excellent and comprehensive in vivo protective and repairing effects. When used preventively with the Y-7-R peptides of this invention, it almost completely eliminates tissue lesions such as epidermal thickening and dermal inflammatory infiltration caused by ultraviolet radiation, restoring skin thickness from an abnormally thickened approximately 153 μm to a near-normal 75 μm, maintaining a clear and intact structure. The peptides of this invention can significantly prevent and reverse collagen loss caused by ultraviolet radiation. In the repair group using Y-7-R, skin collagen density effectively increased from approximately 59% in the model group to 86%, fundamentally maintaining skin elasticity and firmness, preventing and improving wrinkles, and effectively addressing the overall tissue lesions caused by epidermal thickening, collagen degradation, and decreased elasticity.

[0022] The Dictamnus dasycarpus peptide provided by this invention is derived from natural plants, has a well-defined chemical structure, and a small molecular weight (607-1028 Da). It is easy to synthesize on a large scale and its quality is controllable. It overcomes the shortcomings of chemically synthesized antioxidants, such as poor stability and high skin irritation, as well as the unclear composition of traditional plant crude extracts. Its low toxicity and high biocompatibility make it flexible and stable to be added to sunscreens, after-sun repair agents, and various anti-aging cosmetics. Attached Figure Description

[0023] Figure 1This is a purification diagram of the Dictamnus dasycarpus peptides (Y-7-R, W-6-R, or G-6-Y) used in this invention;

[0024] Figure 2 This is a time-of-flight mass spectrometry (TOF-MS) spectrum of the Dictamnus dasycarpus peptides (Y-7-R, W-6-R, or G-6-Y) used in this invention;

[0025] Figure 3 This is a liquid chromatography-tandem mass spectrometry (LC-MS / MS) secondary mass spectrum of the Dictamnus dasycarpus peptide (Y-7-R, W-6-R, or G-6-Y) used in this invention;

[0026] Figure 4 This is a diagram showing the cell survival rate of skin epidermal cells in the prevention of ultraviolet radiation caused by the white oyster shell peptide (Y-7-R, W-6-R, or G-6-Y) used in this invention.

[0027] Figure 5 This is a graph showing the lactate dehydrogenase release rate of the Dictamnus dasycarpus peptide (Y-7-R, W-6-R, or G-6-Y) of the present invention in preventing ultraviolet-induced damage to the cell membrane of skin epidermal cells.

[0028] Figure 6 This diagram shows the malondialdehyde content of the Dictamnus dasycarpus peptide (Y-7-R, W-6-R, or G-6-Y) in the present invention for preventing ultraviolet-induced oxidative lipidation of skin epidermal cells.

[0029] Figure 7 This is a diagram showing the ratio of DNA-damaged cells in skin epidermal cells to prevent ultraviolet-induced DNA damage by the white oyster shell peptide (Y-7-R, W-6-R, or G-6-Y) of the present invention.

[0030] Figure 8 This is a cell apoptosis rate diagram showing the prevention of ultraviolet-induced epidermal cell apoptosis by the Dictamnus dasycarpus peptide (Y-7-R, W-6-R, or G-6-Y) of the present invention.

[0031] Figure 9 This is a cell survival rate diagram showing the repair of skin epidermal cell damage caused by ultraviolet radiation using the Dictamnus dasycarpus peptide (Y-7-R, W-6-R, or G-6-Y) of the present invention.

[0032] Figure 10 This diagram illustrates the lactate dehydrogenase release rate of the Dictamnus dasycarpus peptide (Y-7-R, W-6-R, or G-6-Y) in repairing UV-induced epidermal cell membrane damage in the skin.

[0033] Figure 11 The diagram shows the malondialdehyde content of the white oyster shell peptide (Y-7-R, W-6-R, or G-6-Y) of this invention in repairing ultraviolet-induced oxidative lipidation of skin epidermal cells.

[0034] Figure 12 This is a diagram showing the percentage of DNA-damaged cells in skin epidermal cells repaired by the white oyster shell peptide (Y-7-R, W-6-R, or G-6-Y) of the present invention to repair DNA damage to skin epidermal cells caused by ultraviolet radiation.

[0035] Figure 13 This is a graph showing the apoptosis rate of the white oyster shell peptide (Y-7-R, W-6-R, or G-6-Y) of the present invention in repairing ultraviolet-induced apoptosis of skin epidermal cells.

[0036] Figure 14 H&E staining pathological image of the prevention of skin damage caused by ultraviolet radiation by the Dictamnus dasycarpus peptide (Y-7-R, W-6-R or G-6-Y) of the present invention;

[0037] Figure 15 This is a data graph showing how the Dictamnus dasycarpus peptide (Y-7-R, W-6-R, or G-6-Y) of the present invention prevents epidermal thickening caused by ultraviolet radiation.

[0038] Figure 16 This is a graph showing the hydrogen peroxide content of the Dictamnus dasycarpus peptide (Y-7-R, W-6-R, or G-6-Y) of the present invention in preventing skin oxidative stress caused by ultraviolet radiation.

[0039] Figure 17 This is a diagram showing the malondialdehyde content of the Dictamnus dasycarpus peptide (Y-7-R, W-6-R, or G-6-Y) in the present invention for preventing skin oxidation and lipidation caused by ultraviolet radiation.

[0040] Figure 18 This is a collagen density ratio diagram showing how the Dictamnus dasycarpus peptides (Y-7-R, W-6-R, or G-6-Y) of the present invention prevent skin collagen loss caused by ultraviolet radiation.

[0041] Figure 19 H&E staining pathological image of the repair of skin damage caused by ultraviolet radiation by the Dictamnus dasycarpus peptide (Y-7-R, W-6-R or G-6-Y) of the present invention;

[0042] Figure 20 This is a data graph showing how the Dictamnus dasycarpus peptide (Y-7-R, W-6-R, or G-6-Y) of the present invention repairs epidermal thickening caused by ultraviolet radiation.

[0043] Figure 21 The graph shows the hydrogen peroxide content of the Dictamnus dasycarpus peptide (Y-7-R, W-6-R, or G-6-Y) of this invention in repairing skin oxidative stress caused by ultraviolet radiation.

[0044] Figure 22 The malondialdehyde content diagram shows the malondialdehyde content of the white oyster shell peptide (Y-7-R, W-6-R or G-6-Y) of the present invention in repairing skin oxidative lipidation caused by ultraviolet radiation.

[0045] Figure 23 This is a collagen density ratio diagram showing how the dictamnus dasycarpus peptides (Y-7-R, W-6-R, or G-6-Y) of this invention repair collagen loss in the skin caused by ultraviolet radiation.

[0046] (The above appendix) Figure 4-13 In the ranges of 15-18 and 20-23, compared with the control group, ### P<0.001; compared with the UVB model group or the photoaging model group, * P<0.05; ** P<0.01; *** P<0.001; n=6; and attached Figure 14 , 19 (In the case of n=3.) Detailed Implementation

[0047] like Figures 1-23 As shown, this invention proposes a Dictamnus dasycarpus root bark peptide, its preparation method, and its application. In this invention, the amino acid sequences of the Dictamnus dasycarpus root bark peptides (such as Y-7-R, W-6-R, or G-6-Y) are determined by secondary mass spectrometry (e.g., the amino acid sequence of Y-7-R is SEQ ID NO1: tyrosine-glutamine-tyrosine-asparagine-glutamic acid-arginine-arginine; the amino acid sequence of W-6-R is SEQ ID NO2: tryptophan-glutamine-glutamine-threonine-asparagine-arginine; the amino acid sequence of G-6-Y is SEQ ID NO3: glycine-serine-alanine-asparagine-proline-tyrosine). These are active peptides with significant anti-photoaging properties extracted from the traditional Chinese medicinal herb Dictamnus dasycarpus root bark using an alkaline extraction and acid precipitation method combined with protease hydrolysis. To screen for peptides with the best antioxidant capacity, this invention verifies the strong in vitro antioxidant activity of Y-7-R, W-6-R, and G-6-Y through DPPH, ABTS, and PTIO free radical scavenging experiments.

[0048] The UVB mentioned in this invention refers to ultraviolet B, and the ultraviolet lamp used in the experiment was a Philips PL-S9W, with a wavelength range of 300-320 nm and a peak wavelength of 311 nm. Furthermore, the HaCaT cells used in this study represent human immortalized epidermal cells (this cell line was provided by Haixing Biotechnology Co., Ltd., product number: TCH-C388). The animal experiments used male BALB / c mice (weighing 20-25 g), which were obtained from Yisi Laboratory Animal Technology Co., Ltd. Prior to the experiments, this study was approved by the Laboratory Animal Ethics Committee of Beihua University, with ethics protocol number 2026030607.

[0049] Example 1:

[0050] This invention relates to the preparation of crude protein from Dictamnus dasycarpus root bark.

[0051] Fresh *Dictamnus dasycarpus* root bark was selected, and the mud, coarse stems, and moldy or mildewed parts were carefully removed, retaining only the root bark. The cleaned root bark was placed in an oven and dried at a constant temperature of 50℃ for 8 hours until the sample weight was constant and the moisture content was below 5%. The dried root bark was then pulverized using a high-speed pulverizer to obtain a relatively uniform coarse powder. After pulverization, the powder was sieved through a 60-mesh sieve to remove unpulverized particles, yielding fine *Dictamnus dasycarpus* root bark powder. The fine powder was weighed and petroleum ether was added at a material-to-liquid ratio of 1:5 (g / mL). The mixture was magnetically stirred at room temperature for 30 minutes to fully dissolve the fat-soluble components. The mixture was filtered under reduced pressure, and the filter residue was collected. Degreasing was repeated twice to ensure thorough removal of pectin and fat-soluble impurities. The filter residue was placed in a fume hood to evaporate any remaining petroleum ether, yielding dried, degreased *Dictamnus dasycarpus* root bark powder. The degreased powder was then added to deionized water at a material-to-liquid ratio of 1:10 and magnetically stirred at room temperature for 30 minutes to fully dissolve the tannins. After stirring, the dissolved tannins were removed by filtration. The filter residue was dried in a fume hood to obtain detanned Dictamnus dasycarpus root bark powder. The detanned powder was weighed and added to 0.05 mol / L PBS buffer (pH 7.0) at a material-to-liquid ratio of 1:20. The mixture was then magnetically stirred in an ice bath for 2 hours to ensure the protein was fully dissolved in the buffer. The extract was centrifuged at 10,000 rpm for 20 minutes at 4°C to remove insoluble substances. The supernatant was collected as the crude protein solution. Ammonium sulfate was slowly added to the crude protein solution until it reached 80% saturation. After stirring for 30 minutes, the mixture was allowed to stand at 4°C for 4 hours to allow the protein to precipitate completely. The mixture was then centrifuged at 10,000 rpm for 20 minutes, and the supernatant was discarded. The precipitate was the crude Dictamnus dasycarpus root bark protein. The protein precipitate was then reconstituted with a small amount of PBS buffer and transferred to a 3.5 kDa dialysis bag. Dialysis was performed using deionized water for 24 hours, with the dialysis water changed every 6 hours to remove salts and small molecule impurities. After dialysis, the protein solution was freeze-dried to obtain stable Dictamnus dasycarpus protein powder.

[0052] Example 2:

[0053] This invention optimizes the processing conditions for crude protein extraction from Dictamnus dasycarpus root bark.

[0054] Table 1: Summary of the effect of pH on extraction rate 7.0 3.82±0.11 6.84±2.1 ★Best 8.0 3.55±0.09 6.31±1.6 Slightly lower 9.0 3.29±0.14 5.86±2.4 — 10.0 3.10±0.12 5.52±2.0 — 11.0 2.85±0.10 5.07±1.8 Protein denaturation intensifies 12.0 1.96±0.08 3.51±1.5 Significant decline

[0055] Experimental results showed that the optimal pH for protein extraction from Dictamnus dasycarpus root bark was 7.0, under which the extraction rate was the highest, reaching 6.84±2.1%, and the protein content was 3.82±0.11 mg / mL.

[0056] Table 2: Summary of the effect of liquid-solid ratio on extraction rate 10:1 2.15±0.07 3.84±1.3 Insufficient solvent 20:1 3.01±0.10 5.36±1.8 — 30:1 3.45±0.12 6.14±2.0 — 40:1 3.87±0.11 6.22±2.2 ★The effect is average. 50:1 3.87±0.11 6.92±2.2 ★Best 60:1 3.90±0.15 6.97±2.8 Basically stable, with no significant increase.

[0057] According to the experimental results, the protein extraction rate reached its optimal level (6.92±2.2%) and the protein content was 3.87±0.11 mg / mL when the liquid-to-solid ratio was 50:1 (mL / g).

[0058] Table 3: Summary of the effect of extraction time on protein extraction 0 0.25±0.02 0.46±0.3 Almost no extraction 10 1.82±0.06 3.29±1.4 — 20 2.89±0.10 5.23±1.9 — 40 3.51±0.08 6.36±1.7 — 60 3.92±0.11 7.01±2.3 ★Best 120 3.90±0.15 6.95±2.5 Plateau period

[0059] The optimal extraction time was 60 minutes, under which the protein content was 3.92±0.11 mg / mL and the extraction rate reached its maximum (7.01±2.3%).

[0060] Table 4: Summary of the effect of extraction temperature on protein extraction 45 2.92±0.09 5.21±1.6 Low temperature, insufficient dissolution 50 3.90±0.12 6.99±2.1 ★Best 55 3.72±0.13 6.68±2.0 — 65 3.10±0.14 5.57±2.4 Partial denaturation of protein 70 2.58±0.10 4.64±1.9 obvious degeneration

[0061] Studies have shown that 50℃ is the optimal temperature for protein extraction, with a protein content of 3.90±0.12mg / mL and an extraction rate of 6.99±2.1%.

[0062] Table 5: Summary Table of Enzymatic Hydrolysis Optimization B-1 Alcalase (Bacillus) 8.5–9.5 29.1±1.2 145.8±6.7 73.0±3.1 76.2±3.3 High DH, strong antioxidant B-2 Subtilisin-like (basic; commercial formulation) 8.0–9.0 24.5±1.0 121.2±5.4 65.8±2.7 69.4±2.9 good N-1 Neutrase (Bacillus) 6.5–7.5 22.8±1.0 112.6±4.9 60.5±2.6 64.8±2.8 Gentle cutting, preferred candidate (selected) N-2 Neutralprotease-2 (neutral) 7.0 18.7±0.9 93.4±4.1 48.9±2.2 53.7±2.4 mild A-1 Pepsin (source: pig stomach) 1.5–2.5 21.3±1.2 105.7±5.2 58.2±2.9 61.4±3.0 Performs well in acidic environments A-2 Trypsin (alkaline protease; Bovine) 7.5–9.0 27.8±1.3 130.4±5.9 69.4±3.2 72.6±3.1 Strong hydrolytic properties and antioxidant activity C-1 Papain (Carica) 5.0–6.5 23.6±1.0 115.3±5.3 60.2±2.7 63.0±3.0 Good performance in neutral environment C-2 Ficin (fig proteinase) 5.0–6.0 25.2±1.1 120.5±5.4 62.7±2.9 65.1±2.8 It exhibits a high degree of hydrolysis under neutral conditions and good antioxidant properties.

[0063] By screening proteases to optimize the enzymatic hydrolysis process of Dictamnus dasycarpus root bark, neutral protease (Neutrase) was selected as the best choice, showing a high degree of hydrolysis (22.8±1.0%) and significant antioxidant activity.

[0064] In summary, to optimize protein extraction conditions, the optimal enzymatic hydrolysis conditions were screened based on single-factor experiments: using neutral protease, adjusting the pH to 7.0, hydrolysis time to 1 hour, hydrolysis temperature to 50℃, liquid-to-solid ratio to 50:1, and enzyme dosage to 3000 U / g. Under these conditions, the crude protein was successfully hydrolyzed, yielding the optimal Dictamnus dasycarpus root bark hydrolysate.

[0065] Example 3:

[0066] The present invention relates to the isolation, purification, and identification of Dictamnus dasycarpus peptides.

[0067] First, the protein hydrolysate from Dictamnus dasycarpus was separated and purified. The hydrolysate was initially separated using Sephadex-G50 gel column chromatography, and then further purified using a nucleic acid and protein purification instrument to obtain Dictamnus dasycarpus peptides. The experimental results are as follows: Figure 1As shown, within the time range of 0 to 160 minutes, the protein hydrolysate of *Dictamnus dasycarpus* exhibited three main absorption peaks at a wavelength of 280 nm, indicating that the liquid contains multiple bioactive peptides. Next, we used time-of-flight mass spectrometry (TOF-MS) to determine the precise molecular weights of these three main peptides. The experimental results showed that the molecular weights of the three peptides, Y-7-R, W-6-R, and G-6-Y, were 607.62 Da, 831.89 Da, and 1028.09 Da, respectively. Figure 2 As shown in the figure, this result demonstrates that we successfully obtained several small peptides with specific molecular weights through enzymatic hydrolysis. Furthermore, to conduct detailed identification of the bioactivity of these peptides, we employed liquid chromatography-tandem mass spectrometry (LC-MS / MS) for precise analysis of peptides containing CD activity. The peptides were separated using liquid chromatography, and fragment ion spectra were obtained using mass spectrometry, allowing for precise determination of their molecular weights. Secondary mass spectrometry analysis provided more detailed peptide information, and by comparing with entries in known databases, the amino acid sequences of the peptides were accurately deduced.

[0068] like Figure 3 As shown, the amino acid sequences of the three main peptides were identified by LC-MS / MS as follows: SEQ ID NO1: amino acid sequence is tyrosine (Y)-glutamine (Q)-tyrosine (Y)-asparagine (N)-glutamic acid (E)-arginine (R)-arginine (R) (Y-7-R); SEQ ID NO2: amino acid sequence is tryptophan (W)-glutamine (Q)-glutamine (Q)-threonine (T)-asparagine (N)-arginine (R) (W-6-R); SEQ ID NO3: amino acid sequence is glycine (G)-serine (S)-alanine (A)-asparagine (N)-proline (P)-tyrosine (Y) (G-6-Y). These peptide sequence information provides important basis for further functional studies and applications. After identification, to ensure the high purity and bioactivity of the Dictamnus dasycarpus peptides, all peptides were synthesized using solid-phase peptide synthesis (SPPS) to ensure their high purity and bioactivity.

[0069] In this invention, the recommended dosage of sunscreen agent is 2 mg / cm³. 2 The effective dose of Dictamnus dasycarpus peptides for anti-oxidation and anti-aging is 0.2 mg / cm³. 2Therefore, this invention proposes that dictamnus dasycarpus peptides (such as Y-7-R, W-6-R, or G-6-Y) can be added to existing sunscreens or after-sun repair products at a ratio of 1:10 to enhance their antioxidant and anti-aging effects, thereby preparing a compound antioxidant and anti-aging sunscreen or after-sun repair agent. Furthermore, dictamnus dasycarpus peptides (such as Y-7-R, W-6-R, or G-6-Y) can also be combined with various carriers or excipients to develop novel antioxidant and anti-aging sunscreen products or after-sun repair agents. This peptide component can also be combined with other ingredients in cosmetics (such as moisturizers, thickeners, fragrances, etc.) to form anti-aging cosmetics.

[0070] Example 4:

[0071] This invention presents an experimental study on the preventive effect of Dictamnus dasycarpus peptides (Y-7-R, W-6-R, or G-6-Y) on UVB-induced HaCaT cell damage.

[0072] HaCaT cells were used at a rate of 1×10 5 Cells were seeded at a density of 100 mL / mL in Dulbecco's Modified Eagle Medium (DMEM) containing 100 mL / L fetal bovine serum into 96-well plates and cultured in a cell culture incubator. When cells reached 85% confluence, they were divided into five groups: control group, UVB model group, G-6-Y prevention group, W-6-R prevention group, and Y-7-R prevention group. The control group received physiological saline, while the UVB model group was exposed to ultraviolet B (UVB) radiation. The specific steps were as follows: A UVB lamp (PL-S9W / 01, Royal Philips Electronics Ltd., wavelength range: 300-320 nm, peak: 311 nm) was used. The 96-well plate was placed vertically at a distance of 10 cm from the light source, with an irradiation power of 500 μW / cm². 2 The irradiation time was 1 minute, and the final radiation energy was 30 mJ / cm². 2Before irradiation, DMEM medium was removed from the wells and the cells were rinsed with physiological saline. After irradiation, DMEM medium was added back in, and the cells were cultured for another 24 hours. In the prevention group, G-6-Y, W-6-R, and Y-7-R cells were pretreated with 2 μg / mL for 1 hour before UVB irradiation. During irradiation, after removing DMEM medium from the wells, the cells were treated with physiological saline containing the corresponding concentration of Dictamnus dasycarpus peptide for 1 minute. After irradiation, the cells were treated with culture medium containing the same concentration of peptide for another 24 hours. After treatment, 20 μL of 5 mg / mL MTT solution was added to each well, and the cells were cultured for another 4 hours. After culture, the culture supernatant in the wells was discarded, and 150 μL of dimethyl sulfoxide (DMSO) was added. The cells were gently shaken for 10 minutes until the blue-purple formazan crystals were completely dissolved. The absorbance (OD) was then measured at 490 nm using a microplate reader, and the cell viability was calculated using the following formula: Cell viability (%) = (OD value of experimental group / OD value of control group) × 100%.

[0073] like Figure 4 As shown, under UVB radiation (30mJ / cm²), 2 In a HaCaT cell oxidative stress model induced by UVB radiation, the experimental results showed that the cell survival rate in the control group was 99.86±3.49%, while the cell survival rate in the UVB model group was significantly decreased to only 46.75±5.77%. In contrast, the cell survival rate in the prevention group containing 2 μg / mL LG-6-Y increased to 70.09±6.08%; the cell survival rate in the prevention group containing 2 μg / mL W-6-R was 78.03±5.34%; and the cell survival rate in the prevention group containing 2 μg / mL LY-7-R was 85.23±6.11%. These data indicate that G-6-Y, W-6-R, and Y-7-R can effectively alleviate UV-induced cell damage, with Y-7-R exhibiting the most significant protective effect.

[0074] Example 5:

[0075] This experiment investigated the preventive effect of Dictamnus dasycarpus peptides (Y-7-R, W-6-R, or G-6-Y) on UVB-induced cell membrane damage in HaCaT cells.

[0076] HaCaT cells at 1×10 5Cells were seeded at 100 mL / mL in 96-well plates containing Dulbecco's modified Eagle's medium (DMEM) with 100 mL / L fetal bovine serum and cultured in a cell culture incubator. When the cells reached 85% confluence, they were divided into five groups: control group, UVB model group, G-6-Y prevention group, W-6-R prevention group, and Y-7-R prevention group. The control group received physiological saline. The UVB model group was irradiated using a UVB lamp (PL-S9W / 01; Royal Philips Electronics Ltd., wavelength range: 300-320 nm; peak: 311 nm) at a vertical distance of 10 cm from the UV light source, with an irradiation power of 500 μW / cm². 2 The irradiation time was 1 minute, ultimately producing 30 mJ / cm³. 2 For the G-6-Y, W-6-R, and Y-7-R prevention groups, cells were pretreated with 2 μg / mL G-6-Y, W-6-R, and Y-7-R for 1 hour before UVB irradiation. During irradiation, the DMEM medium in the 96-well plates was discarded, and cells were treated with 2 μg / mL G-6-Y, W-6-R, and Y-7-R for 1 minute. After irradiation, 2 μg / mL G-6-Y, W-6-R, and Y-7-R were added again, and cells were treated for another 24 hours. Finally, the lactate dehydrogenase index in the cell supernatant was measured using the Nanjing Jiancheng lactate dehydrogenase kit (A020-2-2) according to the instructions, and expressed as the lactate dehydrogenase release rate.

[0077] like Figure 5 As shown, at 30 mJ / cm 2 In the UVB-induced HaCaT cell oxidative stress model, the release rate of lactate dehydrogenase (LDH) in the control group was 100.26±8.65%; the release rate in the UVB model group was 218.88±10.66%; while the release rate in the G-6-Y prevention group (containing 2 μg / mL LG-6-Y) was 138.03±13.33%; the release rate in the W-6-R prevention group was 130.56±10.22%; and the release rate in the Y-7-R prevention group was 118.78±9.49%. Compared with the UVB model group, the release rates of LDH in the G-6-Y, W-6-R, and Y-7-R prevention groups were significantly reduced, indicating that Dictamnus dasycarpus peptides can effectively prevent UV-induced damage to the epidermal cell membrane, with Y-7-R showing particularly significant protective effects.

[0078] Example 6:

[0079] This experiment investigated the preventive effect of Dictamnus dasycarpus peptides (Y-7-R, W-6-R, or G-6-Y) on UVB-induced oxidative lipidation in HaCaT cells.

[0080] HaCaT cells at 1×10 5 Cells were seeded at 100 mL / mL in 96-well plates containing Dulbecco's modified Eagle's medium (DMEM) with 100 mL / L fetal bovine serum and cultured in a cell culture incubator. When the cells reached 85% confluence, they were divided into five groups: control group, UVB model group, G-6-Y prevention group, W-6-R prevention group, and Y-7-R prevention group. The control group received physiological saline. The UVB model group was irradiated using a UVB lamp (PL-S9W / 01; Royal Philips Electronics Ltd., wavelength range: 300-320 nm; peak: 311 nm) at a vertical distance of 10 cm from the UV light source, with an irradiation power of 500 μW / cm². 2 The irradiation time was 1 minute, ultimately producing 30 mJ / cm³. 2 For the G-6-Y, W-6-R, and Y-7-R prevention groups, cells were pretreated with 2 μg / mL G-6-Y, W-6-R, and Y-7-R for 1 hour before UVB irradiation. During irradiation, the DMEM medium in the 96-well plates was discarded, and cells were treated with 2 μg / mL G-6-Y, W-6-R, and Y-7-R for 1 minute. After irradiation, 2 μg / mL G-6-Y, W-6-R, and Y-7-R were added again for 24 hours. Finally, the malondialdehyde (MDA) content in the cells was measured using the Nanjing Jiancheng Malondialdehyde Assay Kit (A003-4-1) according to the instructions.

[0081] like Figure 6 As shown, at 30 mJ / cm 2In the UVB-induced HaCaT cell oxidative stress model, the malondialdehyde (MDA) content in the control group was 1.92 ± 0.21 nmol / mg; the MDA content in the UVB model group was significantly increased, reaching 8.12 ± 0.52 nmol / mg; while the MDA content in the G-6-Y prevention group containing 2 μg / mL LG-6-Y was 5.36 ± 0.46 nmol / mg; the MDA content in the W-6-R prevention group containing 2 μg / mL W-6-R was 4.12 ± 0.42 nmol / mg; and the MDA content in the Y-7-R prevention group containing 2 μg / mL LY-7-R was 3.25 ± 0.39 nmol / mg. Compared to the UVB model group, the malondialdehyde content in cells of the G-6-Y, W-6-R, and Y-7-R prevention groups was significantly reduced, indicating that Dictamnus dasycarpus peptides effectively reduced the oxidative lipidation of skin epidermal cells caused by ultraviolet radiation, with Y-7-R showing the most significant protective effect.

[0082] Example 7:

[0083] This invention presents an experimental study on the preventive effect of Dictamnus dasycarpus peptides (Y-7-R, W-6-R, or G-6-Y) on UVB-induced DNA damage in HaCaT cells.

[0084] HaCaT cells at 1×10 5 Cells were seeded at 100 mL / mL in 96-well plates containing Dulbecco's modified Eagle's medium (DMEM) with 100 mL / L fetal bovine serum and cultured in a cell culture incubator. When the cells reached 85% confluence, they were divided into five groups: control group, UVB model group, G-6-Y prevention group, W-6-R prevention group, and Y-7-R prevention group. The control group received physiological saline. The UVB model group was irradiated using a UVB lamp (PL-S9W / 01; Royal Philips Electronics Ltd., wavelength range: 300-320 nm; peak: 311 nm) at a vertical distance of 10 cm from the UV light source, with an irradiation power of 500 μW / cm². 2 The irradiation time was 1 minute, ultimately producing 30 mJ / cm³. 2For the G-6-Y, W-6-R, and Y-7-R prevention groups, cells were pretreated with 2 μg / mL G-6-Y, W-6-R, and Y-7-R respectively for 1 hour before UVB irradiation. During irradiation, the DMEM medium in the 96-well plates was discarded, and physiological saline was added. After irradiation, DMEM medium was added back into the plates, and the cells were cultured in a cell incubator for another 24 hours. Cells were treated for 1 minute, then irradiated and treated again with 2 μg / mL of G-6-Y, W-6-R, and Y-7-R for 24 hours. After that, single-cell suspensions of each group of cells were prepared and low-melting-point agarose was added to prepare sample slides. The sample slides were placed in cell lysis buffer for 1 hour, dried, and electrophoresed for 30 minutes. Then, the sample slides were removed and stained with 25 μg / mL ethidium bromide (EB) in the dark for 10 minutes. 100 cells from each group of sample slides were randomly observed under a fluorescence microscope at 200x magnification. The tailed cells were counted and the tailing rate (cell DNA damage ratio) was calculated.

[0085] like Figure 7 As shown, at 30 mJ / cm 2 In the UVB-induced HaCaT cell oxidative stress model, the DNA damage rate in the control group was 5.03±0.19%; the DNA damage rate in the UVB model group was significantly increased, reaching 44.32±0.91%; while the DNA damage rate in the G-6-Y prevention group (containing 2 μg / mL LG-6-Y) was 24.56±0.48%; the DNA damage rate in the W-6-R prevention group (containing 2 μg / mL W-6-R) was 23.84±0.41%; and the DNA damage rate in the Y-7-R prevention group (containing 2 μg / mL LY-7-R) was 18.73±0.39%. Compared with the UVB model group, the DNA damage rates in the G-6-Y, W-6-R, and Y-7-R prevention groups were significantly reduced, indicating that Dictamnus dasycarpus peptides can significantly reduce UV-induced DNA damage in skin epidermal cells, with Y-7-R showing the most prominent protective effect.

[0086] Example 8:

[0087] This invention presents an experimental study on the preventive effect of Dictamnus dasycarpus peptides (Y-7-R, W-6-R, or G-6-Y) on UVB-induced apoptosis in HaCaT cells.

[0088] HaCaT cells at 1×10 5Cells were seeded at 100 mL / mL in 96-well plates containing Dulbecco's modified Eagle's medium (DMEM) with 100 mL / L fetal bovine serum and cultured in a cell culture incubator. When the cells reached 85% confluence, they were divided into five groups: control group, UVB model group, G-6-Y prevention group, W-6-R prevention group, and Y-7-R prevention group. The control group received physiological saline. The UVB model group was irradiated using a UVB lamp (PL-S9W / 01; Royal Philips Electronics Ltd., wavelength range: 300-320 nm; peak: 311 nm) at a vertical distance of 10 cm from the UV light source, with an irradiation power of 500 μW / cm². 2 The irradiation time was 1 minute, ultimately producing 30 mJ / cm³. 2 For the G-6-Y, W-6-R, and Y-7-R prevention groups, cells were pretreated with 2 μg / mL G-6-Y, W-6-R, and Y-7-R for 1 hour before UVB irradiation. During irradiation, the DMEM medium in the 96-well plates was discarded, and cells were treated with 2 μg / mL G-6-Y, W-6-R, and Y-7-R in saline for 1 minute. After irradiation, 2 μg / mL G-6-Y, W-6-R, and Y-7-R were added again for 24 hours. Following this, the Annexin V / PI apoptosis detection kit (556547) from BD was used, following the instructions. Cells were collected and washed with binding buffer. The cells were then stained with 5 μL Annexin V-FITC and 10 μL LPI staining solution and incubated at room temperature in the dark for 15 minutes. Finally, apoptotic cells were analyzed using flow cytometry. Apoptosis was expressed as the apoptosis rate.

[0089] like Figure 8 As shown, at 30 mJ / cm 2In the UVB-induced HaCaT cell oxidative stress model, the apoptosis rate in the control group was 5.13±0.91%; the apoptosis rate in the UVB model group was significantly increased, reaching 47.25±4.78%; while the apoptosis rate in the G-6-Y prevention group containing 2 μg / mL LG-6-Y was 30.15±2.12%; the apoptosis rate in the W-6-R prevention group containing 2 μg / mL W-6-R was 18.94±2.06%; and the apoptosis rate in the Y-7-R prevention group containing 2 μg / mL LY-7-R was 15.52±1.98%. Compared with the UVB model group, the apoptosis rates in the G-6-Y, W-6-R, and Y-7-R prevention groups were significantly reduced, indicating that Dictamnus dasycarpus peptides can effectively reduce UV-induced epidermal cell apoptosis, with Y-7-R showing the most significant protective effect.

[0090] In conjunction with the above embodiments 4-8, the preventive effect of the Dictamnus dasycarpus peptide of the present invention on HaCaT cells, at 30 mJ / cm 2 Based on a UVB-induced HaCaT cell oxidative stress model, this study verified the preventive and protective effects of Dictamnus dasycarpus peptides (Y-7-R, W-6-R, and G-6-Y). The experiment included a control group, a UVB model group, and three peptide pretreatment groups (2 μg / mL). The effects were assessed using five indicators: MTT assay, LDH release rate, malondialdehyde (MDA) content, DNA damage rate, and apoptosis rate. The results showed that in the UVB model group, cell survival rate plummeted to 46.75±5.77%, LDH release rate increased to 218.88±10.66%, MDA content reached 8.12±0.52 nmol / mg, DNA damage rate was 44.32±0.91%, and apoptosis rate was 47.25±4.78%. Pretreatment with the three peptides significantly improved the above-mentioned damage, with Y-7-R showing the best effect, increasing cell survival rate to 85.23±6.11%, reducing LDH release rate to 118.78±9.49%, MDA content to 3.25±0.39 nmol / mg, DNA damage rate to 18.73±0.39%, and apoptosis rate to 15.52±1.98%, all significantly better than the W-6-R and G-6-Y groups. This confirms that Dictamnus dasycarpus peptides can effectively prevent UVB-induced epidermal cell damage by inhibiting oxidative damage, membrane rupture, DNA breakage, and apoptosis.

[0091] Example 9:

[0092] This invention includes experiments on the repair effect of Dictamnus dasycarpus peptides (Y-7-R, W-6-R, or G-6-Y) on UVB-induced HaCaT cell damage.

[0093] HaCaT cells at 1×10 5Cells were seeded at 100 mL / mL in 96-well plates of Dulbecco's modified Eagle's medium (DMEM) containing 100 mL / L fetal bovine serum and cultured in a cell culture incubator. When the cells reached 85% confluence, they were divided into five groups: control group, UVB model group, G-6-Y repair group, W-6-R repair group, and Y-7-R repair group. The control group received physiological saline. The UVB model group was irradiated using a UVB lamp (PL-S9W / 01; Royal Philips Electronics Ltd., wavelength range: 300-320 nm; peak: 311 nm) at a vertical distance of 10 cm from the UV light source, with an irradiation power of 500 μW / cm². 2 The irradiation time was 1 minute, ultimately producing 30 mJ / cm³. 2 For the 96-well plate irradiation, the DMEM medium was discarded and physiological saline was added. After irradiation, DMEM medium was added back and the plate was placed in a cell incubator for another 24 hours. For the G-6-Y, W-6-R, and Y-7-R repair groups, 2 μg / mL of G-6-Y, W-6-R, and Y-7-R solutions were added to each well 1 hour after UVB irradiation for 24 hours. Then, 20 μL of 5 mg / mL MTT solution was added to each well, and the cells were cultured for another 4 hours. After terminating the culture, the supernatant was discarded, and 150 μL of dimethyl sulfoxide (DMSO) was added to each well to terminate the reaction. The cells were shaken for 10 minutes to dissolve the water-insoluble blue-purple formazan crystals. The absorbance of each well was measured at 490 nm using a microplate reader, and the cell viability was calculated: Cell viability (%) = (OD value of experimental group / OD value of control group) × 100%.

[0094] like Figure 9 As shown, at 30 mJ / cm 2 In the UVB-induced HaCaT cell oxidative stress model, the cell survival rate in the control group was 100.14±4.12%; the cell survival rate in the G-6-Y group was 98.73±4.23%; the cell survival rate in the UVB model group was significantly decreased, only 49.87±5.89%; while the cell survival rate in the G-6-Y repair group containing 2 μg / mL LG-6-Y was 70.53±6.49%; the cell survival rate in the W-6-R repair group containing 2 μg / mL W-6-R was 74.28±6.08%; and the cell survival rate in the Y-7-R repair group containing 2 μg / mL LY-7-R was 78.92±6.16%. These results indicate that G-6-Y, W-6-R, and Y-7-R can effectively improve cell survival rate induced by ultraviolet radiation, demonstrating their potential in repairing UV-induced epidermal cell damage.

[0095] Example 10:

[0096] This invention presents an experimental study on the repair effect of Dictamnus dasycarpus peptides (Y-7-R, W-6-R, or G-6-Y) on UVB-induced cell membrane damage in HaCaT cells.

[0097] HaCaT cells at 1×10 5 Cells were seeded at 100 mL / mL in 96-well plates of Dulbecco's modified Eagle's medium (DMEM) containing 100 mL / L fetal bovine serum and cultured in a cell culture incubator. When the cells reached 85% confluence, they were divided into five groups: control group, UVB model group, G-6-Y repair group, W-6-R repair group, and Y-7-R repair group. The control group received physiological saline. The UVB model group was irradiated using a UVB lamp (PL-S9W / 01; Royal Philips Electronics Ltd., wavelength range: 300-320 nm; peak: 311 nm) at a vertical distance of 10 cm from the UV light source, with an irradiation power of 500 μW / cm². 2 The irradiation time was 1 minute, ultimately producing 30 mJ / cm³. 2 For the energy, the DMEM medium in the 96-well plate was discarded and physiological saline was added before irradiation. After irradiation, DMEM medium was added back and the cells were placed in a cell incubator for another 24 hours. For the G-6-Y repair group, W-6-R repair group, and Y-7-R repair group, 2 μg / mL of G-6-Y, W-6-R, and Y-7-R were added to the cells 1 hour after UVB irradiation and the cells were treated for 24 hours. Then, the lactate dehydrogenase index in the cell supernatant was measured using the Nanjing Jiancheng lactate dehydrogenase kit (A020-2-2) according to the steps described in the instructions, and expressed as lactate dehydrogenase release rate.

[0098] like Figure 10 As shown, at 30 mJ / cm 2In the UVB-induced HaCaT cell oxidative stress model, the release rate of lactate dehydrogenase (LDH) in the control group was 98.73±6.98%; the release rate of LDH in the UVB model group was significantly increased, reaching 235.62±14.85%; while the release rate of LDH in the G-6-Y repair group containing 2 μg / mL LG-6-Y was 167.39±16.02%; the release rate of LDH in the W-6-R repair group containing 2 μg / mL W-6-R was 160.21±15.93%; and the release rate of LDH in the Y-7-R repair group containing 2 μg / mL LY-7-R was 143.58±14.92%. Compared to the UVB model group, the release rate of lactate dehydrogenase in the G-6-Y, W-6-R, and Y-7-R repair groups was significantly reduced, indicating that Dictamnus dasycarpus peptides can effectively reduce UV-induced damage to the epidermal cell membrane, with Y-7-R showing the most significant effect.

[0099] Example 11:

[0100] Experimental study on the repair effect of Dictamnus dasycarpus peptide (Y-7-R, W-6-R or G-6-Y) on UVB-induced oxidative lipidation in HaCaT cells.

[0101] HaCaT cells at 1×10 5 Cells were seeded at 100 mL / mL in 96-well plates of Dulbecco's modified Eagle's medium (DMEM) containing 100 mL / L fetal bovine serum and cultured in a cell culture incubator. When the cells reached 85% confluence, they were divided into five groups: control group, UVB model group, G-6-Y repair group, W-6-R repair group, and Y-7-R repair group. The control group received physiological saline. The UVB model group was irradiated using a UVB lamp (PL-S9W / 01; Royal Philips Electronics Ltd., wavelength range: 300-320 nm; peak: 311 nm) at a vertical distance of 10 cm from the UV light source, with an irradiation power of 500 μW / cm². 2 The irradiation time was 1 minute, ultimately producing 30 mJ / cm³. 2 For the energy, the DMEM medium in the 96-well plate was discarded and physiological saline was added before irradiation. After irradiation, DMEM medium was added back and the cells were placed in a cell incubator for 24 hours. For the G-6-Y repair group, W-6-R repair group, and Y-7-R repair group, 2 μg / mL of G-6-Y, W-6-R, and Y-7-R were added to the cells 1 hour after UVB lamp irradiation and the cells were treated for 24 hours. Then, the malondialdehyde (MDA) content in the cells was measured using the Nanjing Jiancheng Malondialdehyde Assay Kit (A003-4-1) according to the instructions.

[0102] like Figure 11 As shown, at 30 mJ / cm 2 In the UVB-induced HaCaT cell oxidative stress model, the malondialdehyde (MDA) content in the control group was 2.96 ± 0.29 nmol / mg; the MDA content in the UVB model group was significantly increased, reaching 7.24 ± 0.42 nmol / mg; while the MDA content in the G-6-Y repair group containing 2 μg / mLG-6-Y was 4.11 ± 0.47 nmol / mg; the MDA content in the W-6-R repair group containing 2 μg / mLW-6-R was 3.05 ± 0.45 nmol / mg; and the MDA content in the Y-7-R repair group containing 2 μg / mLY-7-R was 2.82 ± 0.40 nmol / mg. Compared to the UVB model group, the malondialdehyde content in cells of the G-6-Y, W-6-R, and Y-7-R repair groups was significantly reduced, indicating that Dictamnus dasycarpus peptides can effectively reduce the oxidative lipidation of skin epidermal cells caused by ultraviolet radiation, with Y-7-R showing the most significant effect.

[0103] Example 12:

[0104] Experimental study on the repair effect of Dictamnus dasycarpus peptide (Y-7-R, W-6-R or G-6-Y) on UVB-induced DNA damage in HaCaT cells.

[0105] HaCaT cells at 1×10 5 Cells were seeded at 100 mL / mL in 96-well plates of Dulbecco's modified Eagle's medium (DMEM) containing 100 mL / L fetal bovine serum and cultured in a cell culture incubator. When the cells reached 85% confluence, they were divided into five groups: control group, UVB model group, G-6-Y repair group, W-6-R repair group, and Y-7-R repair group. The control group received physiological saline. The UVB model group was irradiated using a UVB lamp (PL-S9W / 01; Royal Philips Electronics Ltd., wavelength range: 300-320 nm; peak: 311 nm) at a vertical distance of 10 cm from the UV light source, with an irradiation power of 500 μW / cm². 2 The irradiation time was 1 minute, ultimately producing 30 mJ / cm³. 2For the energy, the DMEM medium in the 96-well plate was discarded and physiological saline was added before irradiation. After irradiation, DMEM medium was added again and the plate was placed in a cell incubator for 24 hours. For the G-6-Y repair group, W-6-R repair group, and Y-7-R repair group, 2 μg / mL of G-6-Y, W-6-R, and Y-7-R were added to the cells 1 hour after UVB irradiation and the cells were treated for 24 hours. Then, the cells in each group were made into single-cell suspensions and low-melting-point agarose was added to prepare sample slides. The sample slides were placed in cell lysis buffer for 1 hour, dried, and electrophoresed for 30 minutes. After that, the sample slides were removed and stained with 25 μg / mL ethidium bromide (EB) in the dark for 10 minutes. 100 cells from each sample slide were randomly observed under a fluorescence microscope at 200x magnification. The tailed cells were counted and the tailing rate (cell DNA damage ratio) was calculated.

[0106] like Figure 12 As shown, at 30 mJ / cm 2 In the UVB-induced HaCaT cell oxidative stress model, the DNA damage rate in the control group was 5.42±0.38%; the DNA damage rate in the UVB model group was significantly increased, reaching 52.28±6.14%; while the DNA damage rate in the G-6-Y repair group containing 2 μg / mLG-6-Y was 42.12±1.02%; the DNA damage rate in the W-6-R repair group containing 2 μg / mLW-6-R was 29.76±0.95%; and the DNA damage rate in the Y-7-R repair group containing 2 μg / mLY-7-R was 23.88±0.83%. Compared with the UVB model group, the DNA damage rates in the G-6-Y, W-6-R, and Y-7-R repair groups were significantly reduced, indicating that Dictamnus dasycarpus peptides can effectively reduce UV-induced DNA damage in skin epidermal cells, with Y-7-R showing the most significant effect.

[0107] Example 13:

[0108] Experimental study on the repair effect of Dictamnus dasycarpus peptide (Y-7-R, W-6-R or G-6-Y) on UVB-induced apoptosis in HaCaT cells.

[0109] HaCaT cells at 1×10 5Cells were seeded at 100 mL / mL in 96-well plates of Dulbecco's modified Eagle's medium (DMEM) containing 100 mL / L fetal bovine serum and cultured in a cell culture incubator. When the cells reached 85% confluence, they were divided into five groups: control group, UVB model group, G-6-Y repair group, W-6-R repair group, and Y-7-R repair group. The control group received physiological saline. The UVB model group was irradiated using a UVB lamp (PL-S9W / 01; Royal Philips Electronics Ltd., wavelength range: 300-320 nm; peak: 311 nm) at a vertical distance of 10 cm from the UV light source, with an irradiation power of 500 μW / cm². 2 The irradiation time was 1 minute, ultimately producing 30 mJ / cm³. 2 For the first group, DMEM medium in 96-well plates was discarded and physiological saline was added before irradiation. After irradiation, DMEM medium was added back and the cells were cultured in a cell incubator for 24 hours. For the G-6-Y, W-6-R, and Y-7-R repair groups, 2 μg / mL of G-6-Y, W-6-R, and Y-7-R solutions were added 1 hour after UVB irradiation and the cells were treated for 24 hours. Then, the Annexin V / PI apoptosis detection kit (556547) from BD was used, following the manufacturer's instructions. Cells were collected and washed with binding buffer. They were then stained with 5 μL Annexin V-FITC and 10 μL LPI staining solution and incubated at room temperature in the dark for 15 minutes. Finally, flow cytometry was used to analyze apoptotic cells. Apoptosis was expressed as the apoptosis rate.

[0110] like Figure 13 As shown, at 30 mJ / cm 2 In the UVB-induced HaCaT cell oxidative stress model, the apoptosis rate in the control group was 6.18±0.87%; the apoptosis rate in the UVB model group was significantly increased, reaching 47.23±3.42%; while the apoptosis rate in the G-6-Y repair group containing 2 μg / mL LG-6-Y was 32.19±2.48%; the apoptosis rate in the W-6-R repair group containing 2 μg / mL W-6-R was 21.88±2.33%; and the apoptosis rate in the Y-7-R repair group containing 2 μg / mL LY-7-R was 18.47±2.17%. Compared with the UVB model group, the apoptosis rates in the G-6-Y, W-6-R, and Y-7-R repair groups were significantly reduced, indicating that Dictamnus dasycarpus peptides can effectively reduce UV-induced epidermal cell apoptosis, with Y-7-R showing the most significant effect.

[0111] In conjunction with the above embodiments 9-13, the present invention focuses on the repair effect of Dictamnus dasycarpus peptide on HaCaT cells after UVB irradiation. The model and cell treatment are the same as before, except that 2 μg / mL peptide is administered for 24 hours after irradiation 1 hour later. The results showed that the cell survival rate in the UVB model group was only 49.87±5.89%, the LDH release rate was 235.62±14.85%, the MDA content was 7.24±0.42 nmol / mg, the DNA damage rate was 52.28±6.14%, and the apoptosis rate was 47.23±3.42%. After repair by the three peptides, the cell status was significantly improved. Y-7-R still performed the best, with the survival rate recovering to 78.92±6.16%, the LDH release rate decreasing to 143.58±14.92%, the MDA content decreasing to 2.82±0.40 nmol / mg, the DNA damage rate decreasing to 23.88±0.83%, and the apoptosis rate decreasing to 18.47±2.17%. Moreover, the repair effects of W-6-R and G-6-Y were also significantly better than those of the model group, indicating that Dictamnus dasycarpus peptides can effectively reverse the UVB cell damage that has occurred and have the potential for post-sun exposure repair.

[0112] Example 14:

[0113] Experimental study on the preventive effect of Dictamnus dasycarpus peptide (Y-7-R, W-6-R or G-6-Y) on UVB-induced skin damage in mice.

[0114] SPF-grade BALB / c mice weighing 20-25g were randomly assigned to groups and housed in a suitable temperature environment with a 12-hour light / dark cycle. The mice had free access to food and water for 7 days to acclimatize. Afterward, the mice were divided into 5 groups: a control group, a UVB model group, a G-6-Y prevention group, a W-6-R prevention group, and a Y-7-R prevention group. Hair was removed from the backs of all mice until the skin was completely exposed, with a removal area of ​​6-8 cm². 2 The control group received no UVB irradiation during each session; instead, physiological saline (20 μL / cm²) was applied directly. 2 The photoaging model group used a UVB lamp (PL-S9W / 01; Royal Philips Electronics Ltd., wavelength range: 300-320nm; peak: 311nm) to irradiate the backs of hairless mice at a vertical distance of 5cm from the UV light source, with an irradiation power of 1000μW / cm. 2 The irradiation time was 2 minutes, ultimately producing 120 mJ / cm². 2 The energy was applied three times a week for four weeks. The G-6-Y prevention group, W-6-R prevention group, and Y-7-R prevention group applied 0.2 mg / cm² of UVB solution one hour before each UVB irradiation session. 2Y-7-R, W-6-R, or G-6-Y (total volume 20 μL); after the experiment, take approximately 1 cm from the back of the mouse. 2 After skin tissue was extracted, it was first fixed with 4% paraformaldehyde to maintain the integrity of the tissue structure. Subsequently, the tissue was dehydrated with graded ethanol and cleared with xylene before being embedded in paraffin for subsequent sectioning. Using a Leica micro-sectioner (Germany), the paraffin-embedded tissue was cut into sections approximately 5 μm thick. To observe epidermal proliferation and morphological changes, the sections were stained with hematoxylin-eosin (H&E). Hematoxylin stains the nuclei blue-purple, while eosin stains the cytoplasm and matrix pink. This staining method clearly shows the arrangement of epidermal cells, the thickness of the stratum corneum, and the overall morphology of the dermal structure, allowing for microscopic observation of the degree of skin damage in different groups of mice.

[0115] like Figure 14 As shown, at 120 mJ / cm 2 In a UVB-induced mouse model of skin photoaging, the skin of mice in the UVB model group, as observed by H&E staining, showed epidermal thickening, hyperkeratosis, and significant damage to the skin tissue structure. The boundary between the epidermis and dermis was unclear, with inflammatory cell infiltration, significantly dry and rough skin, pronounced wrinkles, and a lack of elasticity. Compared to the UVB model group, mice with 0.2 mg / cm³ of UVB showed significantly different results. 2 H&E staining results of the skin of mice in the G-6-Y prevention group showed that the epidermal structure was relatively intact, the inflammatory cell infiltration in the dermis was significantly reduced, skin damage was significantly improved, skin wrinkles and roughness were significantly reduced, and good elasticity was restored; 0.2 mg / cm 2 The W-6-R prevention group mice also showed less skin damage, more regular epidermal tissue, moderate keratinization, smoother skin, and effective suppression of dermal inflammatory response with good elasticity recovery; (containing 0.2 mg / cm³) 2 The H&E staining results of the skin of mice in the Y-7-R prevention group were the most significant. The epidermal structure was clear, the infiltration of inflammatory cells in the dermis almost completely disappeared, the skin wrinkles were significantly reduced, and the skin elasticity was optimally restored.

[0116] Example 15:

[0117] Experimental study on the preventive effect of Dictamnus dasycarpus peptide (Y-7-R, W-6-R or G-6-Y) on UVB-induced epidermal thickening in mice.

[0118] SPF-grade BALB / c mice weighing 20-25g were randomly assigned to groups and housed in a suitable temperature environment with a 12-hour light / dark cycle. The mice had free access to food and water for 7 days to acclimatize. Afterward, the mice were divided into 5 groups: a control group, a UVB model group, a G-6-Y prevention group, a W-6-R prevention group, and a Y-7-R prevention group. Hair was removed from the backs of all mice until the skin was completely exposed, with a removal area of ​​6-8 cm². 2 The control group received no UVB irradiation during each session; instead, physiological saline (20 μL / cm²) was applied directly. 2 The photoaging model group used a UVB lamp (PL-S9W / 01; Royal Philips Electronics Ltd., wavelength range: 300-320nm; peak: 311nm) to irradiate the backs of hairless mice at a vertical distance of 5cm from the UV light source, with an irradiation power of 1000μW / cm. 2 The irradiation time was 2 minutes, ultimately producing 120 mJ / cm². 2 The energy was applied three times a week for four weeks. The G-6-Y prevention group, W-6-R prevention group, and Y-7-R prevention group applied 0.2 mg / cm² of UVB solution one hour before each UVB irradiation session. 2 Y-7-R, W-6-R, or G-6-Y (total volume 20 μL) were used. After the experiment, mice were euthanized by cervical dislocation, and mouse skin tissue was obtained and immersed in 4% paraformaldehyde overnight, then embedded in paraffin to form tissue blocks. The tissue blocks were then cut into 5 μm sections using a paraffin microtome. Finally, the tissue sections were stained with skin hematoxylin-eosin (H&E), and the skin epidermis was observed under an optical microscope. Skin epidermal thickness was measured using AxioVisionRel.4.8 software. Three images were selected for each group, and six non-overlapping epidermal thickness measurements were taken consecutively at points where there was no significant change in epidermal thickness within each group. The average value was used as the quantitative indicator of epidermal thickness.

[0119] like Figure 15 As shown, at 120 mJ / cm 2 In a UVB-induced mouse model of skin photoaging, the epidermal thickness of the control group mice was 18.62±2.12 μm; the epidermal thickness of the UVB model group mice was significantly increased, reaching 153.47±17.9 μm; while the thickness of the skin containing 0.2 mg / cm³ was significantly increased. 2 The epidermal thickness of mice in the G-6-Y prevention group was 95.43±6.12 μm; containing 0.2 mg / cm³. 2 The epidermal thickness of mice in the W-6-R prevention group was 84.21±5.62μm; containing 0.2mg / cm³. 2The epidermal thickness of mice in the Y-7-R prevention group was 75.72±6.45 μm. Compared with the photoaging model group, the epidermal thickness of mice in the G-6-Y, W-6-R, and Y-7-R prevention groups was significantly reduced, indicating that these Dictamnus dasycarpus peptides can effectively prevent epidermal thickening caused by ultraviolet radiation, with Y-7-R showing the most significant effect.

[0120] Example 16:

[0121] Experimental study on the preventive effect of Dictamnus dasycarpus peptide (Y-7-R, W-6-R or G-6-Y) on UVB-induced oxidative stress in mouse skin.

[0122] SPF-grade BALB / c mice weighing 20-25g were randomly assigned to groups and housed in a suitable temperature environment with a 12-hour light / dark cycle. The mice had free access to food and water for 7 days to acclimatize. Afterward, the mice were divided into 5 groups: a control group, a UVB model group, a G-6-Y prevention group, a W-6-R prevention group, and a Y-7-R prevention group. Hair was removed from the backs of all mice until the skin was completely exposed, with a removal area of ​​6-8 cm². 2 The control group received no UVB irradiation during each session; instead, physiological saline (20 μL / cm²) was applied directly. 2 The photoaging model group used a UVB lamp (PL-S9W / 01; Royal Philips Electronics Ltd., wavelength range: 300-320nm; peak: 311nm) to irradiate the backs of hairless mice at a vertical distance of 5cm from the UV light source, with an irradiation power of 1000μW / cm. 2 The irradiation time was 2 minutes, ultimately producing 120 mJ / cm². 2 The energy was applied three times a week for four weeks. The G-6-Y prevention group, W-6-R prevention group, and Y-7-R prevention group applied 0.2 mg / cm² of UVB solution one hour before each UVB irradiation session. 2 Y-7-R, W-6-R, or G-6-Y (total volume 20 μL); after the experiment, mice were euthanized by cervical dislocation, and mouse skin tissue was obtained and prepared into a tissue homogenate. Hydrogen peroxide in the mouse skin tissue was measured using the Solarbio Biotechnology Hydrogen Peroxide Assay Kit (BC3590) according to the instructions.

[0123] like Figure 16 As shown, at 120 mJ / cm 2In a UVB-induced mouse skin photoaging model, the hydrogen peroxide content in the skin of the control group mice was 10.45±1.04 (μmol / mg); the hydrogen peroxide content in the skin of the UVB model group mice was significantly increased, reaching 39.86±2.34 (μmol / mg); while the content of 0.2 mg / cm³ was significantly higher. 2 The hydrogen peroxide content in the skin of mice in the G-6-Y prevention group was 29.32 ± 3.12 (μmol / mg); it contained 0.2 mg / cm³. 2 The hydrogen peroxide content in the skin of mice in the W-6-R prevention group was 18.87 ± 2.98 (μmol / mg); containing 0.2 mg / cm³. 2 The hydrogen peroxide content in the skin of mice in the Y-7-R prevention group was 15.65±2.57 (μmol / mg). Compared with the UVB model group, the hydrogen peroxide content in the skin of mice in the G-6-Y, W-6-R and Y-7-R prevention groups was significantly reduced, indicating that Dictamnus dasycarpus peptides can effectively prevent skin oxidative stress caused by ultraviolet radiation, with Y-7-R showing the most significant effect.

[0124] Example 17:

[0125] Experimental study on the preventive effect of Dictamnus dasycarpus peptide (Y-7-R, W-6-R or G-6-Y) on UVB-induced oxidative lipidation of mouse skin.

[0126] SPF-grade BALB / c mice weighing 20-25g were randomly assigned to groups and housed in a suitable temperature environment with a 12-hour light / dark cycle. The mice had free access to food and water for 7 days to acclimatize. Afterward, the mice were divided into 5 groups: a control group, a UVB model group, a G-6-Y prevention group, a W-6-R prevention group, and a Y-7-R prevention group. Hair was removed from the backs of all mice until the skin was completely exposed, with a removal area of ​​6-8 cm². 2 The control group received no UVB irradiation during each session; instead, physiological saline (20 μL / cm²) was applied directly. 2 The photoaging model group used a UVB lamp (PL-S9W / 01; Royal Philips Electronics Ltd., wavelength range: 300-320nm; peak: 311nm) to irradiate the backs of hairless mice at a vertical distance of 5cm from the UV light source, with an irradiation power of 1000μW / cm. 2 The irradiation time was 2 minutes, ultimately producing 120 mJ / cm². 2 The energy was applied three times a week for four weeks. The G-6-Y prevention group, W-6-R prevention group, and Y-7-R prevention group applied 0.2 mg / cm² of UVB solution one hour before each UVB irradiation session. 2Y-7-R, W-6-R, or G-6-Y (total volume 20 μL); after the experiment, mice were euthanized by cervical dislocation, and mouse skin tissue was obtained and prepared into a tissue homogenate. Malondialdehyde (MDA) in the skin tissue was determined using the Nanjing Jiancheng Company Malondialdehyde Assay Kit (A003-4-1) according to the instructions, and the MDA content was expressed as MDA content.

[0127] like Figure 17 As shown, at 120 mJ / cm 2 In a UVB-induced mouse skin photoaging model, the malondialdehyde (MDA) content in the skin of the control group mice was 1.42 ± 0.73 (nmol / mg); the MDA content in the skin of the UVB model group mice was significantly increased, reaching 8.56 ± 1.21 (nmol / mg); while the content of 0.2 mg / cm³ was significantly lower. 2 The malondialdehyde (MDA) content in the skin of mice in the G-6-Y prevention group was 5.12 ± 1.02 (nmol / mg); and the content was 0.2 mg / cm³. 2 The malondialdehyde (MDA) content in the skin of mice in the W-6-R prevention group was 4.04 ± 0.96 (nmol / mg); and the content was 0.2 mg / cm³. 2 The malondialdehyde (MDA) content in the skin of mice in the Y-7-R prevention group was 3.81 ± 1.04 (nmol / mg). Compared with the UVB model group, the MDA content in the skin of mice in the G-6-Y, W-6-R, and Y-7-R prevention groups was significantly reduced, indicating that Dictamnus dasycarpus peptides can effectively prevent skin oxidative lipidation caused by ultraviolet radiation, with Y-7-R showing the most significant effect.

[0128] Example 18:

[0129] Experimental study on the preventive effect of Dictamnus dasycarpus peptide (Y-7-R, W-6-R or G-6-Y) on UVB-induced collagen loss in mouse skin.

[0130] SPF-grade BALB / c mice weighing 20-25g were randomly assigned to groups and housed in a suitable temperature environment with a 12-hour light / dark cycle. The mice had free access to food and water for 7 days to acclimatize. Afterward, the mice were divided into 5 groups: a control group, a UVB model group, a G-6-Y prevention group, a W-6-R prevention group, and a Y-7-R prevention group. Hair was removed from the backs of all mice until the skin was completely exposed, with a removal area of ​​6-8 cm². 2 The control group received no UVB irradiation during each session; instead, physiological saline (20 μL / cm²) was applied directly. 2The photoaging model group used a UVB lamp (PL-S9W / 01; Royal Philips Electronics Ltd., wavelength range: 300-320nm; peak: 311nm) to irradiate the backs of hairless mice at a vertical distance of 5cm from the UV light source. The irradiation power was 1000μW / cm2, and the irradiation time was 2 minutes, ultimately producing 120mJ / cm2. 2 The energy was applied three times a week for four weeks. The G-6-Y prevention group, W-6-R prevention group, and Y-7-R prevention group applied 0.2 mg / cm² of UVB solution one hour before each UVB irradiation session. 2 Y-7-R, W-6-R, or G-6-Y (total volume 20 μL) were used. After the experiment, mice were euthanized by cervical dislocation, and mouse skin tissue was obtained and immersed in 4% paraformaldehyde overnight, then embedded in paraffin to form tissue blocks. The tissue blocks were then cut into 5 μm sections using a paraffin microtome. Finally, the tissue sections were stained with Sirius red staining solution, and collagen fibers in the skin tissue were observed under an optical microscope. The collagen density ratio of each group of images was measured using ImageJ 1.52 software.

[0131] like Figure 18 As shown, at 120 mJ / cm 2 In a UVB-induced photoaging model of mouse skin, the collagen density ratio in the skin of the control group mice was 102.1±5.76%; the collagen density ratio in the skin of the UVB model group mice was significantly decreased to 59.93±5.88%; while the collagen density ratio in the skin of mice containing 0.2 mg / cm³ was significantly lower. 2 The collagen density ratio in the skin of mice in the G-6-Y prevention group was 77.68±6.19%; containing 0.2 mg / cm³. 2 The collagen density ratio in the skin of mice in the W-6-R prevention group was 75.43±5.94%; containing 0.2 mg / cm³. 2 The collagen density ratio in the skin of mice in the Y-7-R prevention group was 83.12±6.31%. Compared with the UVB model group, the collagen density in the skin of mice in the G-6-Y, W-6-R and Y-7-R prevention groups was significantly increased, indicating that dictamnus dasycarpus peptides can effectively prevent collagen loss caused by ultraviolet radiation, with Y-7-R showing the most significant effect.

[0132] In conjunction with the above embodiments 14-18, the Dictamnus dasycarpus peptide of the present invention, in its preventive effect at the animal level, via 120 mJ / cm 2 A UVB-induced photoaging model of BALB / c mouse skin (3 times a week for 4 weeks) was established to validate the use of 0.2 mg / cm² UVB. 2The preventive effect of Dictamnus dasycarpus peptides. The experiment included a control group, a UVB model group, and three peptide pretreatment groups (applied 1 hour before irradiation). Results showed that in the UVB model group, the epidermis of mice thickened to 153.47±17.9 μm, and H&E staining revealed hyperkeratosis, inflammatory infiltration, and structural disorder; the skin hydrogen peroxide (H2O2) content increased to 39.86±2.34 μmol / mg, the MDA content reached 8.56±1.21 nmol / mg, and the collagen density ratio decreased to 59.93±5.88%. Pretreatment with all three peptides significantly improved the above phenotypes, with Y-7-R showing the best effect—epidermal thickness decreased to 75.72±6.45μm, hydrogen peroxide content was 15.65±2.57μmol / mg, MDA content was 3.81±1.04nmol / mg, collagen density ratio was restored to 83.12±6.31%, dermal inflammation almost disappeared, and skin elasticity was significantly restored, confirming that Dictamnus dasycarpus peptides can systematically prevent UVB-induced skin structural damage, oxidative stress, and collagen loss.

[0133] Example 19:

[0134] Experimental study on the repair effect of Dictamnus dasycarpus peptide (Y-7-R, W-6-R or G-6-Y) on UVB-induced skin damage in mice.

[0135] SPF-grade BALB / c mice weighing 20-25g were randomly divided into five groups and housed in a suitable environment. The laboratory light / dark cycle was 12 hours, and the mice had free access to food and water. The mice were allowed to acclimatize for 7 days. After the acclimatization period, experiments were conducted according to the following groups: control group, UVB model group, G-6-Y repair group, W-6-R repair group, and Y-7-R repair group. Before the experiment, the backs of the mice were shaved to ensure complete skin exposure; the shaved area was controlled to be 6-8 cm². 2 The control group received only physiological saline (20 μL / cm²) during UVB irradiation. 2 The mice were not exposed to UVB light. The UVB model group was treated with a UVB lamp (PL-S9W / 01; Royal Philips Electronics Ltd., wavelength range: 300-320nm, peak: 311nm), with the mice's backs placed at a vertical distance of 5cm from the UV light source, and the irradiation power was 1000μW / cm². 2 Each irradiation lasted 2 minutes, with a final irradiation energy of 120 mJ / cm². 2 The irradiation frequency was three times a week for four weeks. In the repair groups, the G-6-Y, W-6-R, and Y-7-R repair groups applied 0.2 mg / cm³ of ozone solution one hour after each UVB irradiation session. 2 G-6-Y, W-6-R, or Y-7-R solutions (total volume 20 μL). After the experiment, approximately 1 cm of tissue was taken from the back of the mouse.2 Skin tissue was first fixed with 4% paraformaldehyde to ensure structural integrity. Next, the tissue was dehydrated using a gradient of ethanol and cleared with xylene before being embedded in paraffin for subsequent sectioning. Using a Leica micro-sectioner (Germany), the paraffin-embedded tissue was cut into sections approximately 5 μm thick. To observe epidermal proliferation and morphological changes, the sections were stained with hematoxylin and eosin (H&E). Hematoxylin stains the cell nuclei, appearing blue-purple, while eosin stains the cytoplasm and matrix, appearing pink. This staining method clearly shows the arrangement of epidermal cells, the thickness of the stratum corneum, and the overall morphology of the dermal structure. After staining, the degree of skin damage in different groups of mice was observed under a microscope to assess the skin damage and repair effects caused by UVB irradiation.

[0136] like Figure 19 As shown, at 120 mJ / cm 2 In a UVB-induced mouse model of skin photoaging, the skin of mice in the UVB model group, as observed by H&E staining, showed epidermal thickening, hyperkeratosis, significantly damaged skin tissue structure, blurred boundaries between the epidermis and dermis, obvious infiltration of inflammatory cells, dry, rough skin, pronounced wrinkles, and lack of elasticity. Compared to the UVB model group, mice with 0.2 mg / cm³ of UVB showed... 2 H&E staining results of the skin of mice in the G-6-Y repair group showed that the epidermal structure was relatively intact, the inflammatory cell infiltration in the dermis was significantly reduced, skin damage was significantly improved, wrinkles and roughness were significantly reduced, and skin elasticity was well restored; containing 0.2 mg / cm 2 The W-6-R repair group mice also showed fewer skin lesions in their skin H&E staining results, with more regular epidermal tissue, moderate keratinization, smoother skin surface, significantly reduced dermal inflammation, and restored skin elasticity; containing 0.2 mg / cm³ 2 The Y-7-R repair group of mice showed the most significant H&E staining results on their skin. The epidermal structure was clear, the inflammatory cell infiltration in the dermis almost completely disappeared, skin wrinkles were significantly reduced, and the skin elasticity recovery effect was the best.

[0137] Example 20:

[0138] Experimental study on the repair effect of Dictamnus dasycarpus peptide (Y-7-R, W-6-R or G-6-Y) on UVB-induced epidermal thickening in mouse skin.

[0139] SPF-grade BALB / c mice weighing 20-25g were randomly divided into five groups and housed in a suitable environment. The laboratory light / dark cycle was 12 hours, and the mice had free access to food and water. The mice were allowed to acclimatize for 7 days. After the acclimatization period, experiments were conducted according to the following groups: control group, UVB model group, G-6-Y repair group, W-6-R repair group, and Y-7-R repair group. Before the experiment, the backs of the mice were shaved to ensure complete skin exposure; the shaved area was controlled to be 6-8 cm². 2 The control group received only physiological saline (20 μL / cm²) during UVB irradiation. 2 The mice were not exposed to UVB light. The UVB model group was treated with a UVB lamp (PL-S9W / 01; Royal Philips Electronics Ltd., wavelength range: 300-320nm, peak: 311nm), with the mice's backs placed at a vertical distance of 5cm from the UV light source, and the irradiation power was 1000μW / cm². 2 Each irradiation lasted 2 minutes, with a final irradiation energy of 120 mJ / cm². 2 The irradiation frequency was three times a week for four weeks. In the repair groups, the G-6-Y, W-6-R, and Y-7-R repair groups applied 0.2 mg / cm³ of ozone solution one hour after each UVB irradiation session. 2 G-6-Y, W-6-R, or Y-7-R solutions (total volume 20 μL) were used. After the experiment, mice were euthanized by cervical dislocation, and their skin tissue was then removed and fixed overnight in 4% paraformaldehyde. The fixed tissue was then embedded in paraffin to form tissue blocks. Next, the tissue blocks were sectioned into 5 μm thick tissue sections using a paraffin microtome.

[0140] To observe morphological changes in the skin epidermis, tissue sections were stained with skin hematoxylin and eosin (H&E). The stained sections were observed under an optical microscope. The thickness of the skin epidermis was measured using AxioVisionRel.4.8 software. Three representative photographs were selected for each group, and in each photograph, within an area where there was no significant change in epidermal thickness, six non-overlapping epidermal thickness measurements were taken consecutively, and their average value was calculated as a quantitative indicator of skin epidermal thickness.

[0141] like Figure 20 As shown, at 120 mJ / cm 2 In a UVB-induced photoaging model of mouse skin, the epidermal thickness of the control group mice was 16.66±3.06 μm. The epidermal thickness of the photoaging model group mice significantly increased, reaching 168.05±16.19 μm; while the thickness of the epidermal layer containing 0.2 mg / cm³ was significantly increased. 2 The epidermal thickness of mice in the G-6-Y repair group was 106.49±6.79 μm, containing 0.2 mg / cm³ of G-6-Y.2 The epidermal thickness of the W-6-R repair group was 98.72±7.13μm, containing 0.2mg / cm³. 2 The epidermal thickness of the Y-7-R repair group was 82.18±8.01 μm. Compared with the UVB model group, the epidermal thickness of mice in the G-6-Y, W-6-R, and Y-7-R repair groups was significantly reduced, indicating that dictamnus dasycarpus peptides can effectively alleviate epidermal thickening caused by ultraviolet radiation, with Y-7-R showing the most significant effect.

[0142] Example 21:

[0143] Experimental study on the repair effect of Dictamnus dasycarpus peptide (Y-7-R, W-6-R or G-6-Y) on UVB-induced oxidative stress in mouse skin.

[0144] SPF-grade BALB / c mice weighing 20-25g were randomly divided into five groups and housed in a suitable environment. The laboratory light / dark cycle was 12 hours, and the mice had free access to food and water. The mice were allowed to acclimatize for 7 days. After the acclimatization period, experiments were conducted according to the following groups: control group, UVB model group, G-6-Y repair group, W-6-R repair group, and Y-7-R repair group. Before the experiment, the backs of the mice were shaved to ensure complete skin exposure; the shaved area was controlled to be 6-8 cm². 2 The control group received only physiological saline (20 μL / cm²) during UVB irradiation. 2 The mice were not exposed to UVB light. The UVB model group was treated with a UVB lamp (PL-S9W / 01; Royal Philips Electronics Ltd., wavelength range: 300-320nm, peak: 311nm), with the mice's backs placed at a vertical distance of 5cm from the UV light source, and the irradiation power was 1000μW / cm². 2 Each irradiation lasted 2 minutes, with a final irradiation energy of 120 mJ / cm². 2 The irradiation frequency was three times a week for four weeks. In the repair groups, the G-6-Y, W-6-R, and Y-7-R repair groups applied 0.2 mg / cm³ of ozone solution one hour after each UVB irradiation session. 2 G-6-Y, W-6-R, or Y-7-R solutions (total volume 20 μL) were prepared. After the experiment, mice were euthanized by cervical dislocation, and mouse skin tissue was obtained and prepared into a tissue homogenate. Hydrogen peroxide in the mouse skin tissue was measured using the Solarbio Biotechnology Hydrogen Peroxide Assay Kit (BC3590) according to the instructions.

[0145] like Figure 21 As shown, at 120 mJ / cm 2In a UVB-induced photoaging model of mouse skin, the hydrogen peroxide content in the skin of the control group mice was 9.46±1.34 (μmol / mg); the hydrogen peroxide content in the skin of the UVB model group mice was significantly increased, reaching 31.08±3.09 (μmol / mg); while the content of 0.2 mg / cm³ was significantly lower. 2 The hydrogen peroxide content in the skin of mice in the G-6-Y repair group was 20.49±3.47 (μmol / mg), containing 0.2 mg / cm³. 2 The hydrogen peroxide content in the skin of mice in the W-6-R repair group was 19.34±3.22 (μmol / mg), containing 0.2 mg / cm³. 2 The hydrogen peroxide content in the skin of mice in the Y-7-R repair group was 18.72±2.98 (μmol / mg). Compared with the UVB model group, the hydrogen peroxide content in the skin of mice in the G-6-Y, W-6-R and Y-7-R repair groups was significantly reduced, indicating that Dictamnus dasycarpus peptides can effectively alleviate skin oxidative stress caused by ultraviolet radiation, with Y-7-R showing the most significant effect.

[0146] Example 22:

[0147] Experimental study on the repair effect of Dictamnus dasycarpus peptide (Y-7-R, W-6-R or G-6-Y) on UVB-induced oxidative lipidation in mouse skin.

[0148] SPF-grade BALB / c mice weighing 20-25g were randomly divided into five groups and housed in a suitable environment. The laboratory light / dark cycle was 12 hours, and the mice had free access to food and water. The mice were allowed to acclimatize for 7 days. After the acclimatization period, experiments were conducted according to the following groups: control group, UVB model group, G-6-Y repair group, W-6-R repair group, and Y-7-R repair group. Before the experiment, the backs of the mice were shaved to ensure complete skin exposure; the shaved area was controlled to be 6-8 cm². 2 The control group received only physiological saline (20 μL / cm²) during UVB irradiation. 2 The mice were not exposed to UVB light. The UVB model group was treated with a UVB lamp (PL-S9W / 01; Royal Philips Electronics Ltd., wavelength range: 300-320nm, peak: 311nm), with the mice's backs placed at a vertical distance of 5cm from the UV light source, and the irradiation power was 1000μW / cm². 2 Each irradiation lasted 2 minutes, with a final irradiation energy of 120 mJ / cm². 2 The irradiation frequency was three times a week for four weeks. In the repair groups, the G-6-Y, W-6-R, and Y-7-R repair groups applied 0.2 mg / cm³ of ozone solution one hour after each UVB irradiation session. 2G-6-Y, W-6-R, or Y-7-R solutions (total volume 20 μL) were used. After the experiment, mice were euthanized by cervical dislocation, and mouse skin tissue was obtained and prepared into a tissue homogenate. Malondialdehyde (MDA) in the skin tissue was determined using the Nanjing Jiancheng Company's Malondialdehyde Assay Kit (A003-4-1) according to the instructions, and the result was expressed as MDA content.

[0149] like Figure 22 As shown, at 120 mJ / cm 2 In a UVB-induced mouse skin photoaging model, the malondialdehyde (MDA) content in the skin of the control group mice was 1.89 ± 0.81 (nmol / mg); the MDA content in the skin of the UVB model group mice was significantly increased, reaching 6.73 ± 1.21 (nmol / mg); while the content of 0.2 mg / cm³ was significantly lower. 2 The malondialdehyde (MDA) content in the skin of mice in the G-6-Y repair group was 4.32 ± 0.99 (nmol / mg), containing 0.2 mg / cm³. 2 The malondialdehyde (MDA) content in the skin of mice in the W-6-R repair group was 4.12 ± 1.03 (nmol / mg), containing 0.2 mg / cm³. 2 The malondialdehyde (MDA) content in the skin of mice in the Y-7-R repair group was 3.65 ± 0.95 (nmol / mg). Compared with the UVB model group, the MDA content in the skin of mice in the G-6-Y, W-6-R, and Y-7-R repair groups was significantly reduced, indicating that Dictamnus dasycarpus peptides can effectively repair skin oxidative lipidation caused by ultraviolet radiation, with Y-7-R showing the most significant effect.

[0150] Example 23:

[0151] Experimental study on the repair effect of Dictamnus dasycarpus peptide (Y-7-R, W-6-R or G-6-Y) on UVB-induced collagen loss in mouse skin.

[0152] SPF-grade BALB / c mice weighing 20-25g were randomly divided into five groups and housed in a suitable environment. The laboratory light / dark cycle was 12 hours, and the mice had free access to food and water. The mice were allowed to acclimatize for 7 days. After the acclimatization period, experiments were conducted according to the following groups: control group, UVB model group, G-6-Y repair group, W-6-R repair group, and Y-7-R repair group. Before the experiment, the backs of the mice were shaved to ensure complete skin exposure; the shaved area was controlled to be 6-8 cm². 2 The control group received only physiological saline (20 μL / cm²) during UVB irradiation. 2The mice were not exposed to UVB light. The UVB model group was treated with a UVB lamp (PL-S9W / 01; Royal Philips Electronics Ltd., wavelength range: 300-320nm, peak: 311nm), with the mice's backs placed at a vertical distance of 5cm from the UV light source, and the irradiation power was 1000μW / cm². 2 Each irradiation lasted 2 minutes, with a final irradiation energy of 120 mJ / cm². 2 The irradiation frequency was three times a week for four weeks. In the repair groups, the G-6-Y, W-6-R, and Y-7-R repair groups applied 0.2 mg / cm³ of ozone solution one hour after each UVB irradiation session. 2 G-6-Y, W-6-R, or Y-7-R solutions (total volume 20 μL) were used. After the experiment, mice were euthanized by cervical dislocation, and their skin tissue was removed and fixed overnight in 4% paraformaldehyde. The fixed tissue was then embedded in paraffin to form tissue blocks. The tissue blocks were cut into 5 μm thin sections using a paraffin microtome. The tissue sections were then stained with Sirius red, which specifically stains collagen fibers. The distribution and morphology of collagen fibers in the skin tissue were observed under an optical microscope. To quantify the collagen fiber content, ImageJ 1.52 software was used to analyze the images of each group, measuring the collagen density ratio to assess changes in collagen fibers in the skin of different treatment groups.

[0153] like Figure 23 As shown, at 120 mJ / cm 2 In a UVB-induced photoaging model of mouse skin, the collagen density ratio in the skin of the control group mice was 100.02±7.99%; the collagen density ratio in the skin of the UVB model group mice was significantly reduced, to only 59.79±6.08%; while the collagen density ratio in the skin of mice containing 0.2 mg / cm³ was significantly lower. 2 The collagen density ratio in the skin of mice in the G-6-Y repair group was 72.94±6.13%, containing 0.2 mg / cm³. 2 The collagen density ratio in the skin of mice in the W-6-R repair group was 79.15±6.30%, containing 0.2 mg / cm³. 2 The collagen density ratio in the skin of mice in the Y-7-R repair group was 86.23±5.88%. Compared with the UVB model group, the collagen density in the skin of mice in the G-6-Y, W-6-R and Y-7-R repair groups was significantly increased, indicating that dictamnus dasycarpus peptides can effectively repair the loss of skin collagen caused by ultraviolet radiation, with Y-7-R showing the most significant effect.

[0154] In conjunction with the above Examples 19-23, the animal-level repair effect of the Dictamnus dasycarpus peptide of the present invention uses the same model as in Examples 14-18, but the method is adjusted to apply 0.2 mg / cm² of the peptide 1 hour after irradiation.2 Dictamnus dasycarpus peptides. Results showed that the epidermis in the UVB model group further thickened to 168.05±16.19 μm, with hydrogen peroxide content of 31.08±3.09 μmol / mg, MDA content of 6.73±1.21 nmol / mg, and collagen density ratio of only 59.79±6.08%. All three peptides significantly alleviated damage after repair, with Y-7-R showing outstanding performance—epidermal thickness decreased to 82.18±8.01 μm, hydrogen peroxide content to 18.72±2.98 μmol / mg, MDA content to 3.65±0.95 nmol / mg, and collagen density ratio restored to 86.23±5.88%. H&E staining showed clear epidermal structure and near-complete disappearance of inflammatory infiltration, confirming that Dictamnus dasycarpus peptides can effectively repair existing photoaging of the skin, reverse collagen loss and structural damage, and possess application value for post-sun exposure repair and anti-aging.

[0155] The above description is a further detailed explanation of the present invention in conjunction with specific preferred embodiments. For those skilled in the art, several simple deductions or substitutions can be made without departing from the present invention, and all such deductions or substitutions should be considered as falling within the scope of patent protection determined by the submitted claims.

Claims

1. A peptide derived from Dictamnus dasycarpus root bark, characterized in that: The amino acid sequence of the Dictamnus dasycarpus peptide is shown below: A. A peptide with the amino acid sequence SEQ ID NO1:Tyr-Gln-Tyr-Asn-Glu-Arg-Arg; or B. A peptide with the amino acid sequence SEQ ID NO2:Trp-Gln-Gln-Thr-Asn-Arg; or C. A peptide with the amino acid sequence SEQ ID NO3:Gly-Ser-Ala-Asn-Pro-Tyr.

2. The method for preparing Dictamnus dasycarpus peptide according to claim 1, characterized in that: Includes the following steps: S1. Take the root bark of Dictamnus dasycarpus, defatt it and remove the tannins, then extract it with PBS buffer at a liquid-to-solid ratio of 40-60, collect the supernatant by centrifugation, precipitate with ammonium sulfate, dialyze, and freeze dry to obtain crude protein. S2. Take the crude protein, add protease for enzymatic hydrolysis, and obtain the hydrolysate; S3. The enzymatic hydrolysate is separated by gel column chromatography, and the components corresponding to the target absorption peak are collected.

3. The method for preparing Dictamnus dasycarpus peptide according to claim 2, characterized in that: In step S1, the extraction temperature is 45-65℃, the ammonium sulfate precipitate is 80% saturated ammonium sulfate, and the PBS buffer solution has a liquid-to-solid ratio of 50:

1.

4. The method for preparing Dictamnus dasycarpus peptide according to claim 2, characterized in that: The protease in S2 is a neutral protease, and the hydrolysis conditions of the neutral protease are: pH 7.0, hydrolysis time 1 hour, hydrolysis temperature 50℃, liquid-to-solid ratio 50:1, and enzyme addition amount 3000U / g.

5. The method for preparing Dictamnus dasycarpus peptide according to claim 2, characterized in that: In step S3, the target absorption peak is the absorption peak at 280 nm.

6. The use of the Dictamnus dasycarpus peptide according to claim 1 in the preparation of cosmetic products or pharmaceuticals for anti-oxidation or anti-photoaging of the skin.

7. The application of the Dictamnus dasycarpus peptide according to claim 6 in the preparation of cosmetic products or pharmaceuticals for anti-oxidation or anti-photoaging of the skin, characterized in that: The cosmetic products or medicines mentioned are sunscreens, anti-aging cosmetics, or after-sun repair agents.

8. The application of the Dictamnus dasycarpus peptide according to claim 7 in the preparation of cosmetic products or pharmaceuticals for anti-oxidation or anti-photoaging of the skin, characterized in that: The Dictamnus dasycarpus peptide is used in the preparation of sunscreens to prevent skin epidermal cell damage caused by ultraviolet radiation. The skin epidermal cell damage includes one or more of the following: decreased cell viability, cell membrane damage, oxidative lipidation, DNA damage, or apoptosis.

9. The application of the Dictamnus dasycarpus peptide according to claim 7 in the preparation of cosmetic products or pharmaceuticals for anti-oxidation or anti-photoaging of the skin, characterized in that: The Dictamnus dasycarpus peptide is used to prepare an after-sun repair agent. The Dictamnus dasycarpus peptide repairs skin epidermal cell damage caused by ultraviolet radiation. The skin epidermal cell damage includes one or more of the following: decreased cell vitality, cell membrane damage, oxidative lipidation, DNA damage, or apoptosis.

10. The application of the Dictamnus dasycarpus peptide according to claim 7 in the preparation of cosmetic products or pharmaceuticals for anti-oxidation or anti-photoaging of the skin, characterized in that: The Dictamnus dasycarpus peptide is used to prepare anti-aging cosmetic products or medicines that prevent or repair skin aging. The skin aging is caused by ultraviolet radiation and is specifically manifested as one or more of the following: thickening of the epidermis, increased level of skin oxidative stress, aggravated skin oxidative lipidation, or loss of skin collagen.

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