DNA repair impairment protein-derived polypeptide and use thereof

Abstract

The application belongs to the technical field of molecular biology, and relates to a DNA repair damage protein-derived polypeptide and application thereof. An amino acid sequence of the polypeptide comprises a sequence shown in SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3. The polypeptide has a significant photoprotective effect, can effectively prevent and repair skin damage caused by light, has high safety and stability, and is suitable for various skin care products, thereby filling a blank in the prior art.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology and relates to a polypeptide derived from DNA repair damage protein and its applications. Background Technology

[0002] As the largest organ in the human body, the skin not only undertakes core physiological functions such as physical barrier, immune defense, and body temperature regulation, but its health and appearance also directly affect people's quality of life and psychological state. Sun damage has a significant impact on people, causing not only skin aging and darkening, but also potentially skin cancer.

[0003] There are many photoprotective ingredients, such as plant extracts, physical sunscreens, and chemical sunscreens. These ingredients have their own advantages, but they also have their own disadvantages, such as unclear ingredients and environmental unfriendliness. Peptides, as small molecule compounds formed by amino acids linked by peptide bonds, have become a research hotspot for repair-type active ingredients due to their advantages such as small molecular weight, strong skin penetration, good biocompatibility, and clear target of action.

[0004] Some existing active ingredients only indirectly assist in repair through antioxidant or anti-inflammatory pathways, lacking direct and efficient activation of specific repair pathways, resulting in imprecise and ineffective efficacy. How to obtain highly active and safe ingredients that protect against photo-induced skin damage is a problem that researchers in this field have been working to solve.

[0005] Therefore, it is of great significance to develop a novel peptide with high activity that targets specific pathways for repair. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a polypeptide derived from DNA repair damage proteins and its applications.

[0007] To achieve this objective, the present invention employs the following technical solution:

[0008] In a first aspect, the present invention provides a polypeptide derived from DNA repair damage proteins, wherein the amino acid sequence of the polypeptide includes the sequence shown in SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3.

[0009] SEQ ID No. 1: KDSST.

[0010] SEQ ID No.2: ASSKG.

[0011] SEQ ID No.3: ASSSST.

[0012] The polypeptide described in this invention has a significant photoprotective effect, effectively preventing and repairing skin damage caused by sunlight. It is highly safe and has excellent anti-aging, soothing, and anti-inflammatory effects.

[0013] Preferably, the DNA repair damage protein-derived polypeptide is obtained by a method comprising the following steps: virtual enzymatic digestion of the DNA repair damage protein to obtain polypeptide fragments; and screening for polypeptides with repair efficacy by virtual docking method based on their affinity ranking with target proteins.

[0014] Preferably, the target proteins include PARP1 and CDK1.

[0015] Preferably, the enzyme includes chymotrypsin, trypsin, pepsin, papain, and bromelain.

[0016] Preferably, the amino acid sequence of the DNA repair damage protein includes the sequence shown in SEQ ID No. 4.

[0017] SEQ ID No.4:

[0018] MASTHQSSTEPSSTGKSEETKKDASQGSGQDSKNVTVTKGTGSSATSAAIVKTGGSQGKDSSTTAGSSSTQGQKFSTTPTTDPKTFSSDQKEKSKSPAKEVPSGGDSKSQGD TKSQSDAKSSGQSQGQSKDSGKSSSDSSKSHSVIGAVKDVVAGAKDVAGKAVEDAPSIMHTAVDAVKNAATTVKDVASSAASTVAEKVVDAYHSVVGDKTDDKKEGEHSGDK KDDSKAGSGSGQGGDNKKSEGETSGQAESSSGNEGAAPAKGRGRGRPPAAAKGVAKGAAKGAAASKGAKSGAESSKGGEQSSGDIEMADASSKGGSDQRDSAATVGEGGAS GSEGGAKKGRGRGAGKKADAGDTSAEPPRRSSRLTSSGTGAGSAPAAAKGGAKRAASSSSTPSNAKKQATGGAGKAAATKATAAKSAASKAPQNGAGAKKKGGKAGGRKRK.

[0019] In a second aspect, the present invention provides a composition with repair efficacy, the composition comprising a DNA repair damage protein-derived polypeptide and an extract of *Elaeocarpus yunnanensis* from Yunnan and Tibet, wherein the amino acid sequence of the polypeptide comprises the sequence shown in SEQ ID No. 1.

[0020] This invention creatively discovers that when peptides are combined with extracts of *Elaeocarpus yunnanensis* from Yunnan and Tibet, the two exhibit an unexpected synergistic effect, and the combined effect is better than that of using them alone.

[0021] Preferably, the composition comprises, by weight, 3-10 parts of a DNA repair damage protein-derived polypeptide and 1-5 parts of *Elaeocarpus decipiens* extract from Yunnan and Tibet.

[0022] The mass fractions of DNA repair damage protein-derived peptides can be selected from 3, 4, 5, 6, 7, 8, 9, 10, etc., and the mass fractions of Yunnan-Tibet Elaeocarpus extract can be selected from 1, 2, 3, 4, 5, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0023] Preferably, the Yunnan-Tibet Elaeocarpus extract is prepared by a method comprising the following steps:

[0024] (1) Mix the Yunnan and Tibetan Elaeocarpus yunnanensis with an ethanol-water solution, reflux and extract, combine the extracts and concentrate to obtain a concentrated solution;

[0025] (2) Load the concentrated solution onto the macroporous resin for adsorption, wash with water and an ethanol aqueous solution with a mass percentage of 10-30% (e.g., 10%, 12%, 15%, 18%, 20%, 22%, 25%, 28%, 30%, etc.), elute with an ethanol aqueous solution with a mass percentage of 40-60% (e.g., 40%, 42%, 45%, 48%, 50%, 52%, 55%, 58%, 60%, etc.), collect the eluent, concentrate under reduced pressure, and dry to obtain the final product.

[0026] Preferably, the ethanol content in the aqueous ethanol solution in step (1) is 40-60% by mass, such as 40%, 42%, 45%, 48%, 50%, 52%, 55%, 58%, 60%, etc. Other specific values ​​within the above range can be selected, and will not be described in detail here.

[0027] Preferably, the mass ratio of the Yunnan Tibetan Elaeocarpus to the ethanol aqueous solution is 1:(10-15). The specific point values ​​in (10-15) can be selected from 10, 11, 12, 13, 14, 15, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0028] Thirdly, the present invention provides the application of the DNA repair damage protein-derived polypeptide according to the first aspect in the preparation of products with repair, anti-aging, and soothing effects.

[0029] Fourthly, the present invention provides the use of the composition with repairing effects according to the second aspect in the preparation of products with repairing effects.

[0030] Preferably, the product includes cosmetics or skincare products.

[0031] Fifthly, the present invention provides the use of the DNA repair damage protein-derived polypeptide according to the first aspect in the preparation of CDK1 activator, CDK2 activator, CDC6 activator, CDC25 activator or inflammatory factor inhibitor.

[0032] In a sixth aspect, the present invention provides the use of the composition according to the second aspect in the preparation of a CDC6 activator.

[0033] According to the research results of this invention, the polypeptides prepared by this invention have a significant inhibitory effect on the secretion of inflammatory factors and have an activating effect on CDK1, CDK2, CDC6, and CDC25. The polypeptide composition has an activating effect on CDC6. Therefore, the prepared polypeptides or compositions can be used as in vitro preparations for basic scientific research, such as exploring the mechanism of action of physiological activities related to inflammatory responses.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The polypeptide described in this invention has a significant photoprotective effect, effectively preventing and repairing skin damage caused by sunlight. It is highly safe and has excellent anti-aging, soothing, and anti-inflammatory effects. Attached Figure Description

[0036] Figure 1 This is a test result image of the light-based healing effect.

[0037] Figure 2 This is a graph showing the test results of the light protection effect.

[0038] Figure 3 This is a graph showing the results of the peptide stability test.

[0039] Figure 4A This is a graph showing the CDK1 expression levels.

[0040] Figure 4B This is a graph showing the CDK2 expression levels.

[0041] Figure 5A This is a graph showing the expression levels of CCND1.

[0042] Figure 5B This is a graph showing the expression levels of CCNE1.

[0043] Figure 5C This is a graph showing the CCNA2 expression level.

[0044] Figure 6A This is a graph showing the CDC6 expression level.

[0045] Figure 6BThis is a graph showing the CDC25 expression level.

[0046] Figure 7 This is a graph showing the PARP expression levels.

[0047] Figure 8 This is a graph showing the PCNA expression levels.

[0048] Figure 9 This is a graph showing the relative proportion of SA-β-gal positive cells.

[0049] Figure 10A This is a graph showing the relative expression levels of NO.

[0050] Figure 10B This is a graph showing the relative expression levels of TNF-α.

[0051] Figure 11A It is RFI CD86 Graph showing expression levels.

[0052] Figure 11B It is RFI CD54 Graph showing expression levels.

[0053] Figure 12 This is a graph showing the relative expression levels of CDC6. Detailed Implementation

[0054] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0055] Preparation method of Elaeocarpus decipiens extract from Yunnan and Tibet:

[0056] (1) Mix the Yunnan and Tibetan Elaeocarpus yunnanensis with an ethanol aqueous solution with a mass content of 50% at a mass ratio of 1:10, reflux extract twice, with a single extraction time of 1.5 h, and combine the extracts and concentrate to 1 / 11 of the extract mass.

[0057] (2) Load the concentrated solution onto AB-8 macroporous resin and allow it to adsorb overnight. Wash with water for 2 column volumes, wash with 20% ethanol aqueous solution for 5 column volumes, and elute with 50% ethanol aqueous solution for 10 column volumes. Collect the eluent, concentrate under reduced pressure to remove ethanol, and dry under negative pressure to obtain the final product.

[0058] Example 1

[0059] Acquisition and screening of peptides

[0060] 1. Peptide discovery:

[0061] (1) A polypeptide fragment was obtained by virtual enzymatic digestion of a DNA protective protein in tardigrades, and then a polypeptide with potential for safety, stability and activity was obtained by virtual docking technology.

[0062] (2) Virtual enzyme digestion:

[0063] Peptide fragments were obtained by virtual enzymatic digestion using five types of proteases (chymotrypsin, trypsin, pepsin, papain, and bromelain).

[0064] Virtual enzyme digestion was performed using the enzyme action program in the BIOPEP UWM online tool. The DNA repair damage protein of tardigrades was digested with chymotrypsin, trypsin, pepsin, papain and bromelain respectively. After removing repetitive peptides, 274 amino acid sequences were collected.

[0065] (3) Peptide screening:

[0066] After removing sequences containing more than 10 amino acids, 2 peptides, and sequences containing unstable amino acids, toxicity, sensitization, and novelty predictions were performed, resulting in 23 innovative monomeric peptides with no toxicity or sensitization risk.

[0067] Toxicity prediction URL: https: / / webs.iiitd.edu.in / raghava / toxinpred / multi_submit.php

[0068] The website for sensitization prediction is: https: / / www.ddg-pharmfac.net / AllerTOP / , with a sensitization threshold of 0.35.

[0069] Novelty prediction URL: https: / / biochemia.uwm.edu.pl / biopep / peptide_data.php.

[0070] (4) Virtual docking:

[0071] The 23 selected monomeric peptides were molecularly docked with the DNA repair target proteins PARP1 (PDB: 7KK4) and CDK1 (PDB: 5HQ0), respectively. Based on the ranking of the peptides' binding affinity to the two target proteins, four amino acid sequences from the DNA repair protein source were selected for synthesis.

[0072] Table 1

[0073]

[0074] 2. Peptide synthesis

[0075] The four peptides screened in (4) were synthesized by solid-phase synthesis, and this synthesis work was entrusted to Dangang Biotechnology Co., Ltd. The specific process of solid-phase synthesis of peptides is as follows: Based on the amino acid residue composition of the peptides, various amino acids containing a 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group at the amino terminus were used as raw materials. Their carboxyl terminus was covalently linked to 2-chlorotriphenylmethyl chloro resin, and dimethylformamide containing 20% ​​(V / V) piperidine was added. The reaction was carried out for 0.5 h to remove the amino protecting Fmoc. At the same time, acetic anhydride was added to the resin to acetylate the N-terminal amino acid. An excess of Fmoc-S-triphenylmethyl-L-cysteine ​​was added, and hydroxyphenyltriazole was used as a condensing agent. The reaction was carried out at 30°C for 2 h, so that the carboxyl group on Fmoc-S-triphenylmethyl-L-cysteine ​​condensed with the active amino group of glycine on the resin. The deprotection and condensation reactions were repeated, and the remaining amino acids were sequentially linked. The peptide was then cleaved from the resin, separated and purified by a C18 column, and freeze-dried to obtain a peptide with a purity greater than 95%.

[0076] Test Example 1

[0077] Photorepair effect: HaCaT cells in logarithmic growth phase with good morphology were seeded into 96-well plates and incubated for 24 h. A blank control group, an irradiation group, and a sample group were set up. The cells were incubated at 37℃ in a 5% CO2 incubator for 24 h. Except for the blank control group, the irradiation group and the sample group were given a dose of 30 mJ / cm². 2 Cells were irradiated with UVB, and then the irradiated group was replaced with complete culture medium. The sample group was treated with the drug and incubated in a 37°C 5% CO2 incubator for 24 h. The test concentration of the test substance was 200 μg / mL. After removing the original supernatant, 100 μL CCK-8 test solution was added to each well, and the cells were incubated for 2 h. The absorbance at 450 nm was measured using a microplate reader, and the cell viability was calculated.

[0078] Cell viability (%) = (A2 - A1) / (A3 - A1) × 100%

[0079] Wherein, A1: absorbance of uninoculated cells, A2: absorbance of inoculated cells + UV irradiation + sample incubation, and A3: absorbance of inoculated cells.

[0080] Photoprotective effect: HaCaT cells in logarithmic growth phase with good morphology were seeded into 96-well plates and incubated for 24 h. A blank control group, an irradiated group, and a sample group were set up. The plates were incubated at 37℃ in a 5% CO2 incubator for 24 h. The blank and irradiated groups were replaced with complete culture medium, while the sample group was treated with the drug and incubated at 37℃ in a 5% CO2 incubator for 2 h. The test concentration of the test substance was 200 μg / mL. Then, except for the blank control group, the irradiated group and the sample group were each given a dose of 30 mJ / cm³. 2 The cells were irradiated with UVB. They were then incubated at 37°C in 5% CO2 for 24 h. The original supernatant was removed, and CCK-8 test solution was added to each well. The cells were incubated for 2 h, and the absorbance at 450 nm was measured using a microplate reader to calculate the cell viability.

[0081] Cell viability (%) = (A2 - A1) / (A3 - A1) × 100%

[0082] Where A1: Absorbance of uninoculated cells, A2: Absorbance of inoculated cells + sample incubation + UV irradiation, and A3: Absorbance of inoculated cells.

[0083] The results are as follows Figure 1 and Figure 2 As shown, * indicates p < 0.05 compared to the UVB group, ** indicates p < 0.01 compared to the UVB group, and *** indicates p < 0.001 compared to the UVB group. Based on photoprotection and photorepair efficacy, T-7, T-8, and T-9 were preferred for further testing of gene expression related to DNA repair damage.

[0084] Test Example 2

[0085] Stability test

[0086] Stability of the peptide was investigated using an Agilent 1290 high-performance liquid chromatograph (HPLC). The column was a ZORBAX SB-Aq (100 mm × 3.0 mm, 3.5 μm), the column temperature was 30 ℃, and the flow rate was 0.4 mL / min. The mobile phase was 0.1% trifluoroacetic acid acetonitrile solution, and the aqueous phase was 0.1% trifluoroacetic acid aqueous solution. The detection wavelength was 220 nm, and the injection volume was 1 μL. The lyophilized peptide powder was dissolved in an aqueous solution at a concentration of 1 mg / mL and placed in 5 mL centrifuge tubes. The structural and pH changes were measured after 0 and 28 days of storage under cycling conditions at -15℃, 4℃, 25℃, 30℃ + light, and 49℃. After obtaining the HPLC chromatograms and retention time data, the peak area change rate was calculated by comparing the peak areas at 0 and 28 days to assess the peptide stability. The results are as follows: Figure 3As shown, the peak area of ​​the four peptides changed by less than 10% after 28 days of storage under various temperature conditions, and the pH change was not significant, indicating that the four monomeric peptides all have a certain degree of stability.

[0087] Test Example 3

[0088] Cell culture: Cells were rapidly revived in a 37°C water bath and seeded in DMEM medium containing 10% fetal bovine serum (FBS). The cells were then cultured in a 5% CO2 incubator at 37°C. When the cells reached 70-80% confluence, they were digested with trypsin to prepare a single-cell suspension, which was then passaged.

[0089] Grouping: A blank control group, a UVB-induced stimulation group, and a sample group were set up. The blank control group received no treatment. The UVB irradiation group received UVB irradiation treatment, and the sample treatment group received UVB irradiation treatment plus the test sample. The test concentration of the test substance was 200 μg / mL, and the UVB irradiation dose was 30 mJ / cm². 2 .

[0090] HaCaT cells, after three passages of resuscitation, were seeded in 6-well plates to achieve a cell concentration of 3.5 × 10⁻⁶ cells / well. 5 Cells were cultured overnight for 24 h. The test substance was then added, and the cells were pre-cultured for 2 h. The culture medium was removed, and the cells were irradiated with UVB, followed by incubation with normal culture medium for 24 h. Cells were then collected for subsequent RT-PCR analysis.

[0091] RT-PCR assay:

[0092] RNA was extracted using the Thermo Scientific™ GeneJET RNA Purification Kit to obtain purified RNA samples, which were then reverse transcribed into cDNA. The reaction system was as follows: 1 μg RNA; 5×Hiscript Buffer; ddH2O (RNase free) added to 20 μL. Reaction conditions: 37℃ for 15 min in a water bath, 85℃ for 5 s to inactivate the enzyme, and stored at -20℃.

[0093] Real-time quantitative PCR detection:

[0094] Reaction conditions: Pre-denaturation 95℃ 300 s; denaturation 95℃ 10 s; 60℃ 10 s; 72℃ 10 s; annealing 95℃ 10 s; 65℃ 60 s; 97℃ 1 s; extension 37℃ 30 s; a total of 40 cycles.

[0095] (1) Promotes CDK1 / 2 expression

[0096] The results are as follows Figure 4A and Figure 4BAs shown, * indicates p < 0.05 compared to the UVB group, ** indicates p < 0.01 compared to the UVB group, *** indicates p < 0.001 compared to the UVB group, and ### indicates p < 0.001 compared to the control group (BC). UVB induces a DNA damage response, leading to cell cycle arrest, reduced CDK1 / 2 expression, and ultimately inhibiting cell cycle progression. The addition of peptides T-7 / T-8 / T-9 can reverse this process and promote normal cell cycle progression.

[0097] (2) CCND1 / CCNE1 / CCNA2

[0098] Cyclins bind to CDKs to form cyclin-CDK complexes, which regulate the cell cycle. For example, cyclin D1-CDK4 / 6 initiates cell cycle progression, cyclin E1-CDK2 regulates entry into S phase, cyclin A2-CDK2 regulates DNA replication in S phase, and cyclin A2 / B2-CDK1 triggers mitosis.

[0099] The results are shown in Figure 5. In the figure, ns indicates no significant difference compared to the UVB group; * indicates p < 0.05 compared to the UVB group; ** indicates p < 0.01 compared to the UVB group; *** indicates p < 0.001 compared to the UVB group; ## indicates p < 0.01 compared to the control group (BC); and ### indicates p < 0.001 compared to the control group (BC). UVB blocks the activation of the Cyclin-CDK complex through the DNA damage response pathway, further inhibiting G1 / S phase checkpoint activation, S phase arrest, and G2 / M phase checkpoint activation, ultimately leading to cell cycle dysregulation. The addition of T-7 and T-9 reverses this process, promoting normal cyclin expression and thus advancing the cell cycle normally.

[0100] (3) CDC6 / 25

[0101] The results are shown in Figure 6. Here, ns indicates no significant difference compared to the UVB group, ** indicates p < 0.01 compared to the UVB group, and # indicates p < 0.05 compared to the control group (BC). In the S phase, cyclin A-CDK2 can translocate from its nuclear location in the G1 phase to the cytoplasm via CDC6, thereby regulating the role of CDC6 in initiating DNA replication and preventing DNA replication in the S and G2 phases. CDC25 can activate cyclin D-CDK4 / CDK6 by removing phosphorylation at specific sites, promoting the G1-S phase transition. The addition of T-7 reverses this process, advancing the cell cycle normally.

[0102] (4) PARP

[0103] The results are as follows Figure 7 As shown, ** indicates a p-value < 0.01 compared to the UVB group, *** indicates a p-value < 0.001 compared to the UVB group, and ### indicates a p-value < 0.001 compared to the control group (BC). PARP is a key substrate of Caspase-3 during apoptosis. When Caspase-3 is activated, it cleaves specific sites on PARP, ultimately leading to apoptosis. UVB induction can inhibit NF-κB / Caspase-3 activation, leading to PARP cleavage and increased apoptosis. The addition of T-7, T-8, and T-9 can reverse this process, promoting normal PARP expression and thus reducing apoptosis.

[0104] (5) PCNA

[0105] The results are as follows Figure 8 As shown, ns indicates no significant difference compared to the UVB group, * indicates p < 0.05 compared to the UVB group, ** indicates p < 0.01 compared to the UVB group, and ## indicates p < 0.01 compared to the blank group (BC). After UVB induces DNA damage, the Caspase cascade is activated, leading to the cleavage of PCNA at the Asp145 site, resulting in the inactivation of the functional fragment and a decrease in expression level. The addition of T-7 and T-9 significantly enhances PCNA expression, indicating that T-7 and T-9 have the ability to enhance DNA replication and damage repair.

[0106] Test Example 4

[0107] Anti-aging efficacy test

[0108] The system included a blank control group (untreated group), an induced aging group (D-gal induced aging group), and a sample group (D-gal induced aging + peptide treatment group).

[0109] HFF-1 cells were stored at a rate of 2 × 10⁶ cells per well. 4 Cells were seeded at a density of [number] cells per well in 48-well plates and cultured for 24 hours. The growth medium was then discarded and replaced with a test sample solution containing a final concentration of 20 mg / mL D-galactose (D-gal), resulting in a sample concentration of 200 μg / mL. The cells were cultured for another 72 hours. SA-β-galase activity was detected using an X-gal staining kit (Beyotime). The number of β-galactosidase-positive (β-gal positive) cells reflects the degree of cellular senescence. Figure 9As shown, * indicates a p-value < 0.05 compared to the D-gal group, ** indicates a p-value < 0.01 compared to the D-gal group, and ## indicates a p-value < 0.01 compared to the control group (NC). After D-galactose treatment, the number of blue senescent cells increased compared to the NC group. After T-7 pretreatment, the number of blue senescent cells significantly decreased, indicating that the peptide has a certain anti-aging effect.

[0110] Test Example 5

[0111] Soothing efficacy test

[0112] The experiment consisted of four groups: a blank control group (NC group) with complete culture medium; an induction stimulation group (LPS group); a dexamethasone (DEX) positive control group with 5 μg / mL DEX diluent; and a sample group with 200 μg / mL peptide diluent. Each group had three replicates. After pre-incubation for 2 h, all groups except the NC group were incubated with 1 μg / mL LPS for 24 h. The levels of NO and TNF-α were measured according to the instructions of the NO and ELISA kits. Absorbance was measured at the specified wavelength using a microplate reader, and the inhibition rate of inflammatory factors was calculated.

[0113] Inflammatory factor inhibition rate (%) = (A LPS -A S ) / (A LPS -A NC ) × 100%

[0114] In the formula, A LPS The absorbance of the LPS group; A S A represents the absorbance of the sample group. BC This represents the absorbance value of group NC.

[0115] As shown in Figure 10, ** indicates p < 0.01 compared to the LPS group, *** indicates p < 0.001 compared to the LPS group, and ### indicates p < 0.001 compared to the blank group (NC). T-7 pretreatment can significantly inhibit the secretion of NO and TNF-α inflammatory factors, thus T-7 has a soothing effect.

[0116] Test Example 6

[0117] Security test

[0118] (1) Safety evaluation of h-CLAT sensitization

[0119] The h-CLAT method is an in vitro assay that quantifies the expression of cell surface markers (i.e., CD4+) in the human monocytic leukemia cell line THP-1 cells 24 hours after exposure to the test substance. 86 and CD 54Changes in surface marker expression were observed. These surface molecules are typical markers of THP-1 activation in monocytes and can mimic DC activation, which plays a crucial role in T cell initiation. After cell staining with fluorescently labeled antibodies, changes in surface marker expression were measured by flow cytometry. The relative fluorescence intensity of the surface markers was calculated and used in a predictive model to support the differentiation between sensitizers and non-sensitizers. 86 and CD 54 Expression was analyzed by flow cytometry using the FL1 acquisition channel. Based on geometric mean fluorescence intensity (MFI), CD4+ expression was analyzed in positive control (ctrl) cells and chemically treated cells. 86 and CD 54 The relative fluorescence intensity (RFI) is calculated according to the following equation:

[0120] CD 86 An RFI of 150% or greater at at least one test concentration (with cell viability ≥ 50%) is considered a positive sensitizer; CD 54 An RFI (reactive protein index) of a monomeric peptide is defined as equal to or greater than 200% at at least one test concentration (cell viability ≥ 50%). As shown in Figure 11, the RFI of the monomeric peptide... CD86 Expression level <150%, RFI CD54 The expression level was <200%, therefore the peptide was not sensitizing.

[0121] (2) Neutral Red Absorption (NRU) Measurement

[0122] Add 100 μL of culture medium to the outer wells of a 96-well tissue culture plate (blank control), and add 100 μL of medium at a density of 1×10⁻⁶ to the remaining wells. 5 / mL of cell suspension (i.e., 1×10⁶) 4Cells were cultured for 24 h (5% CO2, 37℃) in two plates for each experiment, including the same series of test substance concentrations, solvent control, blank control, and positive control (chlorpromazine hydrochloride). After 24 h of culture, the culture medium was removed, and the cells were washed with PBS. 100 μL of the test substance culture medium (200 μg / mL) was added to each well at once. After 1 h of culture, one plate was exposed to light to determine phototoxicity (+Irr), and the other plate was placed in the dark to determine cytotoxicity (-Irr). Cells were examined under a phase-contrast microscope, and changes in cell morphology caused by the cytotoxicity of the test substance were recorded to eliminate experimental errors. Cells absorb neutral red into lysosomes and vacuoles within viable cells, which can be used as a quantitative indicator of cell number and viability. Cells were washed twice with 150 μL of pre-warmed EBSS or PBS, and 100 μL of culture medium containing 50 μg / mL neutral red was added. Cells were cultured for 3 h. Remove the neutral red culture medium, wash the cells once or twice with 150 μL EBSS or PBS, add 150 μL of neutral red desorption solution, and shake the 96-well plate thoroughly for 10 minutes until the neutral red is extracted from the cells and a homogeneous solution is formed. Detect the absorbance at 540 nm using a microplate reader. Use blank wells as a reference control.

[0123] Table 2

[0124]

[0125] Note: PIF = IC50(+Irr) / IC50(-Irr), PIF≤2, MPE≤0.1, then the prediction is no phototoxicity.

[0126] As shown in Table 2, the PIF value of the three peptides is <2 and the MPE value is <0.1. Therefore, according to the evaluation criteria, the three peptides showed negative results in the in vitro 3T3 neutral red uptake phototoxicity test and had no phototoxicity.

[0127] Test Example 7

[0128] Repair efficacy test

[0129] Refer to Test Example 2 for the testing method.

[0130] Test samples: T-7 peptide, Elaeocarpus decipiens extract from Yunnan and Tibet, and a combination of T-7 and Elaeocarpus decipiens extract from Yunnan and Tibet.

[0131] The test method is the same as in Test Example 2. The concentration of T-7 sample is 200 μg / mL; the concentration of E. coli extract from Yunnan and Tibet is 60 μg / mL; in the combination of T-7 and E. coli extract from Yunnan and Tibet, the concentration of T-7 is 120 μg / mL and the concentration of E. coli extract from Yunnan and Tibet is 30 μg / mL.

[0132] The results are as follows Figure 12As shown, ** indicates a p-value < 0.01 compared to the UVB group, * indicates a p-value < 0.05 compared to the UVB group, *** indicates a p-value < 0.001 compared to the UVB group, and # indicates a p-value < 0.05 compared to the control group (NC). In the S phase, cyclin A-CDK2 can translocate from its nuclear location in the G1 phase to the cytoplasm via CDC6, thereby regulating the role of CDC6 in initiating DNA replication and preventing DNA replication in the S and G2 phases. The combination of T-7 and *Elaeocarpus yunnanensis* significantly enhances CDC6 expression, exhibiting a synergistic effect.

[0133] The above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A polypeptide derived from DNA repair damage proteins, characterized in that, The amino acid sequence of the polypeptide is the sequence shown in SEQ ID No.

1.

2. A composition with restorative effects, characterized in that, The composition comprises a DNA repair damage protein-derived polypeptide and an extract of *Elaeocarpus yunnanensis* from Yunnan and Tibet, wherein the amino acid sequence of the polypeptide is the sequence shown in SEQ ID No.

1.

3. The composition with repairing effects according to claim 2, characterized in that, The composition comprises, by weight, 3-10 parts of a DNA repair damage protein-derived polypeptide and 1-5 parts of *Elaeocarpus decipiens* extract from Yunnan and Tibet.

4. The composition with repairing effects according to claim 2, characterized in that, The Yunnan-Tibet Elaeocarpus extract was prepared by a method comprising the following steps: (1) Mix the Yunnan and Tibetan Elaeocarpus yunnanensis with an ethanol-water solution, reflux and extract, combine the extracts and concentrate to obtain a concentrated solution; (2) Load the concentrated solution onto the macroporous resin for adsorption, wash with water and 10-30% ethanol aqueous solution, elute with 40-60% ethanol aqueous solution, collect the eluent, concentrate under reduced pressure, and dry to obtain the final product.

5. The application of the DNA repair damage protein-derived polypeptide according to claim 1 in the preparation of products with at least one of the effects of repair, anti-aging, and soothing.

6. The use of the composition with restorative effects according to any one of claims 2-4 in the preparation of products with restorative effects.

Images