Fish type 1 collagen-derived peptide, external composition including the same, and use thereof in preparation of external composition

Abstract

collagen-derived peptide serves as an active ingredient and can be applied in use in preparation of an external composition involving antioxidation, whitening, promotion of extracellular matrix production, and prevention of photoaging.

Description

Technical Field

[0001] This invention relates to a fish collagen and its applications, particularly to a type I collagen-derived peptide from fish scales, topical components containing the peptide, and its use in preparing components for anti-oxidation, promoting extracellular matrix formation, whitening, and preventing photoaging. Prior Technology

[0002] Type I collagen is the most abundant collagen in the human body, playing a crucial role in skin elasticity, joint health, and overall connective tissue strength. Over the past decade, fish collagen has received widespread attention due to its excellent bioavailability and potential health benefits.

[0003] Fish collagen can be obtained from fish skin, scales, and bones. Bioactive compounds derived from the hydrolysis of fish collagen are called fish type I collagen-derived peptides or simply fish collagen peptides. Generally, fish collagen is broken down into smaller peptides by enzymes, making it easier for the human body to absorb. Compared to mammalian collagen, fish contain a higher proportion of collagen, and after hydrolysis, fish collagen has a smaller molecular weight, making it easier for the digestive system to absorb. Therefore, it is considered a more sustainable and effective source.

[0004] Current research shows that oral administration of fish collagen peptides can improve skin hydration and elasticity and reduce wrinkles. Furthermore, fish collagen peptides can also protect joints and cartilage by stimulating the production of type II collagen.

[0005] Therefore, there is an urgent need to develop a new fragment and new efficacy of fish collagen peptides to increase added value and expand application areas. Summary of the Invention

[0006] Therefore, one aspect of the present invention is to provide a type I collagen-derived peptide from fish.

[0007] Secondly, another aspect of the present invention is to provide a topical composition comprising a type I collagen-derived peptide from fish as an active ingredient.

[0008] Furthermore, another aspect of the present invention is to provide the use of fish type I collagen-derived peptides in the preparation of antioxidant components, wherein the antioxidant components use fish type I collagen-derived peptides as the active ingredient.

[0009] Furthermore, another aspect of the present invention is to provide the use of fish type I collagen-derived peptides in the preparation of topical components that promote extracellular matrix generation, wherein the topical components use fish type I collagen-derived peptides as the active ingredient.

[0010] Furthermore, another aspect of the present invention provides the use of fish type I collagen-derived peptides in the preparation of a whitening topical composition, wherein the topical composition uses fish type I collagen-derived peptides as the active ingredient.

[0011] In addition, another aspect of the present invention provides the use of fish type I collagen-derived peptides in the preparation of topical components for preventing photoaging, wherein the topical components use fish type I collagen-derived peptides as the active ingredient.

[0012] According to the above description of the present invention, a type I collagen-derived peptide for fish is proposed, comprising an amino acid sequence as shown in sequence identification number SEQ ID NO:1.

[0013] According to another aspect of the present invention, a topical composition is provided comprising a fish type I collagen-derived peptide as an active ingredient, wherein the fish type I collagen-derived peptide comprises an amino acid sequence as shown in SEQ ID NO:1.

[0014] In the above embodiments, the dosage forms of the aforementioned external components include, for example, liquids, powders, emulsions, and aerosols.

[0015] According to another aspect of the present invention, a fish type I collagen-derived peptide is proposed for use in the preparation of an antioxidant composition, wherein the antioxidant composition uses the fish type I collagen-derived peptide as the active ingredient, and the fish type I collagen-derived peptide may contain an amino acid sequence as shown in SEQ ID NO:1.

[0016] In the above embodiments, the aforementioned fish type I collagen-derived peptides may have free radical scavenging activity. The free radicals may include 2,2-diphenyl-1-trinitrophenylhydrazine (DPPH) free radicals and reactive oxygen species (ROS), and the reactive oxygen species may include superoxide (O2-) free radicals, hydroxyl (·OH) free radicals and hydrogen peroxide (H2O2).

[0017] According to another embodiment of the present invention, a fish type I collagen-derived peptide is proposed for use in the preparation of an external composition that promotes extracellular matrix formation, wherein the external composition may use the fish type I collagen-derived peptide as an active ingredient, and the fish type I collagen-derived peptide contains an amino acid sequence as shown in SEQ ID NO:1.

[0018] In the above embodiments, the aforementioned extracellular matrix may contain collagen and elastin.

[0019] According to another aspect of the present invention, a fish type I collagen-derived peptide is proposed for use in the preparation of a whitening topical composition, wherein the topical composition uses the fish type I collagen-derived peptide as an active ingredient, and the fish type I collagen-derived peptide may contain an amino acid sequence as shown in SEQ ID NO:1.

[0020] In the above embodiments, the aforementioned fish type I collagen-derived peptides inhibit the activity of mushroom tyrosinase.

[0021] According to yet another aspect of the present invention, a fish type I collagen-derived peptide is proposed for use in the preparation of a topical composition for preventing photoaging, wherein the topical composition uses the fish type I collagen-derived peptide as the active ingredient, the fish type I collagen-derived peptide may contain an amino acid sequence as shown in SEQ ID NO:1, and the topical composition may be administered in vitro to fibroblasts treated with ultraviolet light.

[0022] The above-mentioned fish type I collagen-derived peptides and topical compositions containing them are used as active ingredients in the present invention. They can be used to prepare topical compositions that promote antioxidant activity, promote extracellular matrix formation, whiten skin, and prevent photoaging.

[0023] It is understood that the foregoing general description and the following detailed description are merely illustrative examples intended to provide further explanation of the claimed invention. Simple Explanation of the Diagram

[0024] To make the above and other objects, features, advantages and embodiments of the present invention more apparent and understandable, the detailed description of the accompanying drawings is as follows: Figure 1 shows a partial sequence alignment of fish and human type I collagen-derived peptides according to an embodiment of the present invention. Figure 2 is a dot pattern analysis of fish type I collagen-derived peptides (hereinafter referred to as fish scale collagen peptides) according to some embodiments of the present invention. Figure 3 is a bar graph showing the cell survival rate of human dermal fibroblasts after treatment with fish scale collagen peptides according to an embodiment of the present invention. Figure 4 is a bar graph showing the total collagen content of human dermal fibroblasts after treatment with fish scale collagen peptides according to an embodiment of the present invention (Figure number *** represents p<0.001). Figures 5A and 5B respectively show the Western ink dot analysis diagram (Figure 5A) and the relative intensity bar graph (Figure 5B, figure number * represents p<0.05) of elastin in human dermal fibroblasts after treatment with fish scale collagen peptides according to an embodiment of the present invention. Figure 6 is a bar graph showing the mushroom casein inhibition rate of fish scale collagen peptides according to an embodiment of the present invention (Figure number *** represents p<0.001). Figure 7 is a bar graph showing the DPPH free radical scavenging rate of fish scale collagen peptides according to an embodiment of the present invention (Figure number *** represents p<0.001). Figure 8 is a bar graph showing the hydroxyl radical scavenging rate of fish scale collagen peptides according to an embodiment of the present invention (Figure number *** represents p<0.001). Figure 9 is a bar graph showing the hydrogen peroxide scavenging rate of fish scale collagen peptides according to an embodiment of the present invention (Figure number *** represents p<0.001). Figures 10A to 10C respectively show fluorescence staining images (Figure 10A) of human dermal fibroblasts irradiated with UVA and treated with fish scale collagen peptides according to an embodiment of the present invention, bar graphs of ROS scavenging ability (Figure 10B, where *** represents p<0.001 compared with the UVA irradiated control group, and ### represents p<0.001 compared with the control group), and bar graphs of increased SOD activity (Figure 10C, where ** represents p<0.01 compared with the UVA irradiated control group, and ## represents p<0.01 compared with the control group). Figures 11A and 11B respectively show the Western ink dot analysis diagram (Figure 11A) and the relative intensity bar graph (Figure 11B) of type I procollagen in human dermal fibroblasts after UVA irradiation and treatment with fish scale collagen peptides according to an embodiment of the present invention. Figure number *** represents p<0.001 compared with the UVA irradiation control group, and ## represents p<0.01 compared with the control group. Figures 12A and 12B respectively show the staining photographs (Figure 12A) and the percentage bar chart of SA-β-gal positive cells (Figure 12B) of human dermal fibroblasts after UVA irradiation and treatment with fish scale collagen peptides according to an embodiment of the present invention. (Figure 12B, where *** represents p < 0.001 compared with the UVA irradiation control group, and ### represents p < 0.001 compared with the control group.) Implementation

[0025] If a definition or usage of a term in a cited reference is inconsistent with or contrary to the definition of that term herein, the definition herein shall apply, and not the definition in the cited reference. Secondly, unless the context otherwise defines, a singular term may include a plural term, and a plural term may include a singular term. The elements and arrangements described in the specific examples are for illustration and simplification of the disclosure and are not intended to limit the invention.

[0026] As mentioned above, the present invention provides a fish type I collagen-derived peptide and an external composition containing the same, wherein the fish type I collagen-derived peptide is used as the active ingredient.

[0027] The type I collagen-derived peptides described here are derived from fish. Therefore, type I collagen-derived peptides can be obtained by extraction from fish or by artificially synthesizing specific amino acid sequences.

[0028] In other words, there are no particular limitations on the aforementioned fish species. However, in some embodiments, the aforementioned fish species may include the genus Epinephelus, such as Epinephelus lanceolatus, Epinephelus coioides, or other species of the same genus.

[0029] There are no particular restrictions on the parts of the fish used, but in some embodiments, fish scales are preferred. Fish scales are generally considered to be worthless and difficult to process, and are often discarded as waste. However, fish scales are rich in collagen and hydroxyapatite. This invention uses grouper scales as a source of type I collagen, thereby increasing the added value and application areas of fish scales.

[0030] The type I collagen-derived peptides described herein are derived from fish type I collagen-derived peptides, and generally, their amino acid sequence length can be, for example, 27 to 50, 27 to 42, or 27 amino acid residues. In some specific examples, the sequence length of the type I collagen-derived peptides can be, for example, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, or 27 amino acid residues.

[0031] In some specific examples, fish type I collagen-derived peptides may contain amino acid sequences as shown in SEQ ID NO:1. Please refer to Figure 1, which shows a partial sequence alignment of fish and human type I collagen-derived peptides according to an embodiment of the present invention. In Figure 1, box 103 shows the sequence of amino acid residues 80 to 106 of the dotted type I collagen α1 chain (GenBank: AER42673.1), box 103 shows the sequence of amino acid residues 104 to 130 of the dotted type I collagen α2 chain (GenBank: ADG29147.1), box 103 shows the sequence of amino acid residues 30 to 56 of the dotted type I collagen α chain (GenBank: ADG29173.1), and box 103 shows the sequence of amino acid residues 206 to 232 of the human type I collagen α1 chain (PDB: 5K31_A). Box 103 represents the functional region predicted to promote collagen production. The symbols "*", ":", and "‧" below the sequences in Figure 1 are defined as follows: "*" indicates that all sequences have the same amino acid at that site. ":" indicates that three sequences have the same amino acid at that site, and another sequence has an amino acid of a different type but the same property (e.g., positively charged, negatively charged, polar, nonpolar, or containing a specific amino acid as a side chain); or, two sequences have the same amino acid at that site, and two other sequences have the same amino acid at that site, with the two sets of identical amino acids being different in type but the same in property. "‧" indicates that three sequences have the same amino acid at that site, and another sequence has an amino acid of a different type and property at that site; or, two sequences have the same amino acid at that site, and two other sequences have amino acids of a different type but the same property at that site. Blank numbers on the figure indicate that the sequences do not have the same amino acid at that site, or that two sequences have the same amino acid at that site, but two other sequences have amino acids of different types and properties at that site.

[0032] In some embodiments, the fish type I collagen-derived peptides of SEQ ID NOs:1 to 3 were confirmed by immunoassay to have the collagen-promoting functional region shown in Figure 1, obtained from the scales of the gentian grouper (Epinephelus lanceolatus).

[0033] The aforementioned fish scale collagen peptides can be added as active ingredients to topical formulations. In some embodiments, the dosage form of the aforementioned topical formulation may include, but is not limited to, liquids, powders, emulsions, and aerosols, but the present invention is not limited to these examples. In vitro experiments (e.g., cell experiments) have confirmed that this topical formulation has antioxidant, extracellular matrix-promoting, whitening, and photoaging-preventing effects, and can be used to prepare antioxidant formulations, topical formulations that promote extracellular matrix production, whitening topical formulations, and photoaging-preventing topical formulations, etc.

[0034] In other words, in the above embodiments, there are no particular limitations on the types of cells suitable for in vitro experiments. However, in some specific examples, the cell types may be, for example, skin cells, including but not limited to dermal fibroblasts and epidermal keratinocytes. The aforementioned antioxidant components refer to fish type I collagen-derived peptides, which have the effects of scavenging free radicals, reducing intracellular oxidative stress, and enhancing SOD activity. There are no particular limitations on the types of free radicals mentioned above, which may include, but are not limited to, 2,2-diphenyl-1-trinitrophenylhydrazine (DPPH) free radicals and reactive oxygen species (ROS), and reactive oxygen species may include, but are not limited to, superoxide (O2-) free radicals, hydroxyl (·OH) free radicals, and hydrogen peroxide (H2O2).

[0035] In the above embodiments, the aforementioned topical composition that promotes extracellular matrix generation refers to fish type I collagen-derived peptides that have the effect of promoting the generation of extracellular matrix such as collagen and elastin.

[0036] In the above embodiments, the aforementioned whitening topical composition refers to a fish type I collagen-derived peptide that inhibits the activity of mushroom tyrosinase.

[0037] In the above embodiments, the aforementioned topical composition for preventing photoaging refers to fibroblasts treated with ultraviolet light. After being administered in vitro with fish type I collagen-derived peptides, the proportion of aging-related biomarkers (such as senescence-associated β-galactosidase, SA-β-gal) positive cells can be significantly reduced.

[0038] It should be noted that, compared to existing fish type I collagen, the fish type I collagen-derived peptides of the present invention have a specific sequence, are not only of moderate molecular size (with 27 to 50, 27 to 42, or 27 amino acid residues), making them easy to absorb, but also have multiple functions, which can increase the added value of fish scales and expand their application areas.

[0039] The following examples illustrate the application of the present invention, but they are not intended to limit the invention. Those skilled in the art can make various modifications and refinements without departing from the spirit and scope of the invention.

[0040] Example 1: Preparation of Gentian Grouper Type I Collagen-Derived Peptides

[0041] First, a cleaning step is performed, which involves washing the gentian grouper scales with tap water that is 10 times the weight of the fish scales.

[0042] Next, the fish scales undergo a skin removal process, which involves soaking them in a 0.8% sodium hydroxide aqueous solution (10 times their weight) for 12 hours. Afterward, the scales are drained, and then rinsed 4-6 times with tap water (10 times their weight) (30 minutes of rinsing followed by 10 minutes of settling) until the pH of the rinse solution drops to 7.0-9.0.

[0043] Next, the first decalcification step is carried out, which involves stirring and soaking the fish scales in a 0.8% hydrochloric acid aqueous solution (i.e., decalcification solution) that is 10 times the weight of the fish scales for 2.5 hours, and then discarding the decalcification solution.

[0044] Then, a second decalcification step is carried out, which involves stirring and soaking the fish scales in a 0.8% hydrochloric acid aqueous solution (i.e., decalcification solution) that is 7 times the weight of the fish scales for 2.5 hours, and then discarding the decalcification solution.

[0045] Next, the pH value is adjusted by stirring and rinsing the fish scales 1-2 times (15 minutes each time) with reverse osmosis (RO) water that is 5 times the weight of the fish scales, so that the pH value of the rinsed solution is maintained between 3.0 and 4.0, in order to obtain decalcified gentian grouper scales.

[0046] Then, the enzyme-removed peptide step is carried out, which involves soaking the aforementioned decalcified gentian grouper scales in a 50mM hydrochloric acid aqueous solution (50 times the weight of the fish scales), adding 0.2% pepsin, and stirring (100 to 120 rpm) in a 10°C water bath for 48 hours to obtain gentian grouper scale hydrolysate.

[0047] Next, a salting-out step is performed, in which sodium chloride is added to the enzymatic hydrolysate of gentian grouper scales to a final concentration of 1M, and the mixture is allowed to stand for 30 minutes to obtain the gentian grouper scale salting-out solution. Then, the aforementioned gentian grouper scale salting-out solution is centrifuged at 4000 rpm for 30 minutes to obtain type I collagen-derived peptides from gentian grouper scales. Hydrolysis of gentian grouper scales with 0.2% pepsin yields 1% collagen.

[0048] Example 2: Sequence Verification of Type I Collagen-Derived Peptides from Gentian Grouper Scales

[0049] The literature [Katayama K. et al., J. Biol. Chem. 268(14):9941-4, May 15, 1993] reveals that human type I collagen has a collagen-promoting functional region (SEQ ID NO:4, CTSHTGAWGKTVI EYKTTKSSRLPIID), but currently there are no commercially available monoclonal antibodies for type I collagen that can identify an amino acid sequence that is the same as or similar to SEQ ID NO:4. Therefore, in Example 2, an antibody was developed to evaluate and confirm whether the fish scale type I collagen-derived peptide of Example 1 has an amino acid sequence that is the same as or similar to SEQ ID NO:4.

[0050] Based on protein sequences from the NCBI database, the fish scale type I collagen-derived peptides in Example 1 were determined to likely belong to fish type I collagen. Next, three sequences from spotted grouper type I collagen that promote collagen production—SEQ ID NO:1 (CTSHTGTWGKTVIDYKTTKTSRLPIID), SEQ ID NO:2 (CTRHTGEWSKTVIEYRTNKPSRLPILD), and SEQ ID NO:3 (CTTHTGSWGKTVIDYKTSKTSRL PIID)—were compared with a partial amino acid sequence of human type I collagen (SEQ ID NO:4, CTSHTGAWGKTVIEYKTTKSSRLPIID). The results are shown in Figure 1.

[0051] Please refer to Figure 1, which shows a partial sequence alignment of fish and human type I collagen-derived peptides according to an embodiment of the present invention. In Figure 1, SEQ ID NO:1 shows the sequence of amino acid residues 73 to 114 of the type I collagen α1 chain of spotted grouper (GenBank: AER42673.1), SEQ ID NO:2 shows the sequence of amino acid residues 97 to 138 of the type I collagen α2 chain of spotted grouper (GenBank: ADG29147.1), SEQ ID NO:3 shows the sequence of amino acid residues 23 to 64 of the type I collagen α chain of spotted grouper (GenBank: ADG29173.1), and SEQ ID NO:4 shows the sequence of amino acid residues 199 to 240 of the type I collagen α1 chain of human (PDB: 5K31_A). The thick boxes in Figure 1 predict functional regions that may promote collagen production. The symbols "*", ":", and "‧" below the sequences in Figure 1 are defined as follows: "*" indicates that all sequences have the same amino acid at that site. ":" indicates that three sequences have the same amino acid at that site, and another sequence has an amino acid of a different type but the same property (e.g., positively charged, negatively charged, polar, nonpolar, or containing a specific amino acid as a side chain); or, two sequences have the same amino acid at that site, and two other sequences have the same amino acid at that site, with the two sets of identical amino acids being different in type but the same in property. "‧" indicates that three sequences have the same amino acid at that site, and another sequence has an amino acid of a different type and property at that site; or, two sequences have the same amino acid at that site, and two other sequences have amino acids of a different type but the same property at that site. Blank numbers on the figure indicate that the sequences do not have the same amino acid at that site, or that two sequences have the same amino acid at that site, but two other sequences have amino acids of different types and properties at that site.

[0052] As shown in Figure 1, the amino acid sequences of SEQ ID NO: 1 to 3 are roughly similar to some of the amino acid sequences of human type I collagen. Although there are some differences in some amino acid sites, this belongs to the species difference.

[0053] Subsequently, the amino acid sequences of SEQ ID NOs:1 to 3 were artificially synthesized. HPLC and MASS mass spectrometry analyses confirmed that the purity of the synthesized sequences was greater than 95%, and the molecular weights of each peptide sequence were correct.

[0054] Generally, to generate an effective immune response and neutralizing antibody IgG, the molecular weight of the immunoantigen must be greater than 5 kDa. Therefore, the aforementioned artificially synthesized amino acid sequences of SEQ ID NOs:1 to 3 were covalently bound to Keyhole Limpet Hemocyanin (KLH) to obtain fusion antigens with a molecular weight sufficient to elicit an effective immune response. Then, mice were immunized with the aforementioned fusion antigens, and the serum from the immunized mice was collected as multiple antibody strains.

[0055] The multi-antibody strains from Example 2 were used to perform dot blot analysis on the fish scale type I collagen-derived peptides from Example 1. The analysis procedure is briefly described below.

[0056] First, 1 µL of fish scale type I collagen-derived peptide solutions of different concentrations (0.5, 1, 2, 5, 10 mg / mL) from Example 1 (protein amounts of 0.5, 1, 2, 5, 10 μg; positive control group: 0.01 μg mouse serum; negative control group: 1 μg bovine serum albumin (BSA)) were dropped onto commercially available protein transfer membranes [Immun-Blot PVDF Membrane / Precut for mini gels (Bio-Rad Laboratories Inc.)] and fixed at room temperature (generally 10˚C to 40˚C) or air-dried for 5 minutes. Next, the transfer membranes were blocked with 1 mL of blocking solution (PBS solution containing 5% skim milk powder) for 1 hour. Subsequently, the blocking solution was removed, and the transfected membrane was rinsed once with 1 mL of 1X PBS, allowing it to stand for 10 seconds each time. Then, 1 mL of the multi-antibody solution from Example 2 (diluted 1:4000) was added, and the membrane was reacted at 4˚C overnight (more than 16 hours). Afterward, the multi-antibody solution was removed, and the transfected membrane was rinsed three times with 1 mL of 1X wash buffer, allowing it to stand for at least 10 seconds each time. Next, 1 mL of secondary antibody solution [HRP-conjugated Goat anti-Mouse IgG, diluted 1:5000] was added, and the membrane was reacted at room temperature for 1 hour. Then, the secondary antibody solution was removed, and the transfected membrane was rinsed three times with 1 mL of 1X wash buffer, allowing it to stand for at least 10 seconds each time. Finally, the transfected membrane was rinsed once with 1 mL of 1X PBS. Next, 1.2 mL of ECL Plus reaction solution (0.1 mL / cm²) was added to each transfer membrane, and the reaction was carried out at room temperature for 5 minutes. Subsequently, the ECL Plus reaction solution was removed, and the transferred membrane was photographed using a cryoluminescence analyzer. The results are shown in Figure 2.

[0057] Please refer to Figure 2, which shows the dot patch analysis of fish type I collagen-derived peptides (hereinafter referred to as fish scale collagen peptides) from some embodiments of the present invention. The positive control group is mouse serum, and the negative control group is BSA. As shown in Figure 2, the multiple antibodies obtained from the artificially synthesized amino acid sequences of SEQ ID NOs: 1 to 3 in Example 2 can indeed identify the fish scale type I collagen-derived peptides from Example 1.

[0058] In addition, the serum (multi-strain antibody) obtained from 9 mice was reacted with the fish scale type I collagen-derived peptide of Example 1. The serum (multi-strain antibody) obtained by immunizing mice with SEQ ID NO:1 showed better immunorecognition effect, as shown in Figure 2(f), which shows that the fish scale type I collagen-derived peptide of Example 1 has the functional region that promotes collagen production as shown in SEQ ID NO:1.

[0059] Example 3: Formulation of fish type I collagen-derived peptides

[0060] The artificially synthesized fish type I collagen-derived peptide (such as the amino acid sequence shown in SEQ ID NO:1, with an average molecular weight of 3.02 kDa) of Example 2 was dissolved in sterile water to prepare a 50 mM preservation solution, which was stored at -20°C and thawed and diluted before use.

[0061] Example 4: Extracellular matrix formation assay of human dermal fibroblasts

[0062] In this example, human dermal fibroblasts (Hs68, BCRC number 60038 or ATCC number CRL-1635) were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antimicrobial agent (Penicillin-Streptomycin-Glutamine, PSG) in a 37°C, 5% CO2 incubator for various tests.

[0063] 4.1 Cell viability analysis

[0064] Human dermal fibroblasts (hereinafter referred to as Hs68 cells) were seeded into 24-well cell culture dishes at a cell density of 5 × 10⁴ cells / mL. After cell attachment, culture medium containing different concentrations of the artificially synthesized fish type I collagen-derived peptide (SEQ ID NO:1, also known as fish scale collagen peptide) from Example 2 was added to each well, and the cells were cultured for 24 hours. Then, MTT solution was added to each well, and the cells were cultured for another 3 hours to allow the cells to metabolize and formazan crystals (blue-purple MTT). Subsequently, the formazan crystals were dissolved with isopropanol, and their absorbance at 570 nm was measured to evaluate whether the addition of the fish scale collagen peptide (artificially synthesized SEQ ID NO:1) from Example 2 affected cell viability.

[0065] Please refer to Figure 3, which is a bar graph 301 showing the cell survival rate of human dermal fibroblasts after treatment with fish scale collagen peptide according to an embodiment of the present invention. The results in Figure 3 show that after treatment with fish scale collagen peptide (SEQ ID NO:1) of Example 2 at concentrations of 0.1, 0.2, 0.5, 1, and 2 µM, the cell survival rates of Hs68 cells were 96.38 ± 3.17%, 96.43 ± 5.05%, 94.70 ± 6.57%, 95.92 ± 5.42%, and 84.35 ± 2.68%, respectively. The cell survival rates of all of them were greater than 80%, and there was no significant difference, indicating that the fish scale collagen peptide (SEQ ID NO:1) of Example 2 did not have cytotoxicity to Hs68 cells.

[0066] 4.2 Collagen content determination

[0067] Human dermal fibroblasts (Hs68) were seeded into 6-well cell culture dishes at a density of 2 × 10⁵ cells / mL. After cell attachment, culture medium containing fish scale collagen peptide (SEQ ID NO: 1) from Example 2 was added to each well, and the cells were cultured for 24 hours. Then, the cell culture medium from each well was removed, and 250 μL of concentrated reagent (model 90626, Chondrex Inc., Redmond, WA, USA) was added. The mixture was mixed at 4°C and shaken overnight. The mixture was centrifuged the next day, and the supernatant was collected. Sirius Red Solution (model 90622, Chondrex Inc., Redmond, WA, USA) was added, and the mixture was incubated at room temperature for 20 minutes. Residual reagent was then washed away with Wash Solution (model 90623, Chondrex Inc., Redmond, WA, USA). Then, the extraction buffer (model 90624, Chondrex Inc., Redmond, WA, USA) was added and mixed evenly. The absorbance was measured at 540 nm, and the content was calculated using the calibration curve of the collagen standard.

[0068] Please refer to Figure 4, which is a bar graph 401 showing the total collagen content of human dermal fibroblasts after treatment with fish scale collagen peptides according to an embodiment of the present invention (Figure number *** represents p<0.001). The results in Figure 4 show that after treatment with fish scale collagen peptides (SEQ ID NO:1) of Example 2 at concentrations of 0.1, 0.2, 0.5, 1, and 2 µM, the collagen content of Hs68 cells was 20.70 ± 1.37, 20.61 ± 2.79, 22.19 ± 2.38, 17.87 ± 6.14, and 17.92 ± 3.27 µg / mL, respectively. Among them, the fish scale collagen peptides (SEQ ID NO:1) of Example 2 at concentrations of 0.1, 0.2, and 0.5 µM significantly increased the collagen content of Hs68 cells, demonstrating a clear effect in promoting collagen production.

[0069] 4.3 Elastin Performance Analysis

[0070] Human dermal fibroblasts (Hs68) were seeded into 10 cm diameter cell culture dishes. After cell attachment, culture medium containing fish scale collagen peptide (SEQ ID NO: 1) from Example 2 was added, and the cells were cultured for 24 hours. Lysis buffer (model R0278, Sigma-Aldrich Co., St Louis, MO, USA) was added to lyse the cell membrane, and intracellular proteins were collected and separated by SDS-PAGE electrophoresis. The proteins were then transferred onto a PVDF membrane, blocked with skim milk, and then primary and secondary antibodies were added sequentially. A chemiluminescence agent was then added, and the expression of elastin was observed and quantified using the commercially available software ImageJ.

[0071] Please refer to Figures 5A and 5B, which respectively show the Western ink dot analysis (Figure 501) and relative intensity bar graph (Figure 503) of elastin in human dermal fibroblasts treated with fish scale collagen peptide according to an embodiment of the present invention (Figure 5A, * indicates p<0.05). The results in Figures 5A and 5B show that after treatment with fish scale collagen peptide (SEQ ID NO:1) of Example 2 at concentrations of 0.1, 0.2, 0.5, and 1 µM, the elastin expression level in Hs68 cells increased to 1.76, 1.65, 1.57, and 1.3 times that of the control group (with a band concentration of 1.00), respectively. Furthermore, even low concentrations of fish scale collagen peptide (SEQ ID NO:1) of Example 2 significantly enhanced the elastin expression level, demonstrating a clear effect in promoting elastin production.

[0072] Example 5: Determination of the inhibitory capacity of mushroom tyrosinase

[0073] The aqueous solution of fish scale collagen peptide (SEQ ID NO:1) from Example 2 and the mushroom tyrosinase solution were sequentially added to a 96-well cell culture dish and reacted for 5 minutes. Then, 0.03% (w / v) tyrosine solution was added, and the mixture was reacted at 37˚C for 25 minutes. The absorbance was then measured at 492 nm, and the inhibition rate of the fish scale collagen peptide (SEQ ID NO:1) from Example 2 against mushroom tyrosinase was calculated.

[0074] Please refer to Figure 6, which is a bar graph 601 showing the mushroom casein inhibition rate of fish scale collagen peptides according to an embodiment of the present invention (Figure number *** represents p<0.001).

[0075] Figure 6 shows that the inhibition rates of the fish scale collagen peptide (SEQ ID NO:1) of Example 2 at concentrations of 62.5, 125, 250, 500, 1000, and 5000 μg / mL against mushroom tyrosinase activity were 14.73 ± 5.46%, 31.35 ± 6.10%, 52.18 ± 5.72%, 76.51 ± 7.80%, 99.58 ± 0.61%, and 99.29 ± 0.58%, respectively, with a half-maximal inhibitory concentration (IC50) of 371 ± 4.23 μg / mL. This indicates that the fish scale collagen peptide (SEQ ID NO:1) of Example 2 can effectively inhibit mushroom tyrosinase activity and does indeed have a whitening effect.

[0076] Example 6: In vitro antioxidant test

[0077] 6.1 Determination of DPPH free radical scavenging ability

[0078] An aqueous solution of fish scale collagen peptide (SEQ ID NO:1) from Example 2 was added to a 96-well cell culture dish. Next, 0.2 mM DPPH methanol solution was added to each well, and after thorough mixing, the mixture was allowed to stand and react in the dark for 30 minutes. Afterward, the absorbance of each well was measured at 540 nm, and the scavenging rate of DPPH free radicals by the fish scale collagen peptide (SEQ ID NO:1) from Example 2 was calculated.

[0079] Please refer to Figure 7, which is a bar graph 701 showing the DPPH free radical scavenging rate of the fish scale collagen peptide of an embodiment of the present invention (Figure number *** represents p<0.001). The results in Figure 7 show that the fish scale collagen peptide of Example 2 (SEQ ID NO:1) has good DPPH free radical scavenging ability, and the scavenging rate shows an upward trend with the increase of the concentration of the fish scale collagen peptide of Example 2. Its half-inhibitory concentration (IC50) is 92.37 ± 5.18 μg / mL, which does have antioxidant effect.

[0080] 6.2 Determination of hydroxyl radical (HO·) scavenging ability

[0081] An aqueous solution of fish scale collagen peptide (SEQ ID NO:1) from Example 2 was added to a 1.5 mL microcentrifuge tube, followed by the addition of 2-deoxyribose and thorough mixing. Next, 4 mM ferric chloride (FeCl3), 2 mM ascorbic acid, and 12 mM hydrogen peroxide (H2O2) were added sequentially to generate hydroxyl radicals, which attacked 2-deoxyribose to produce malondialdehyde (MDA). Subsequently, a 2-thiobarbituric acid (TBA) solution was added, and after thorough mixing, the mixture was heated at 100˚C for 10 minutes. After cooling, the mixture was centrifuged, and the supernatant was collected and added to a 96-well cell culture dish. The absorbance of each well was then measured at 532 nm. The scavenging rate of hydroxyl radicals by the fish scale collagen peptide (SEQ ID NO:1) from Example 2 was calculated based on the change in the concentration of the pink MDA-TBA product generated by the reaction of TBA and MDA.

[0082] Please refer to Figure 8, which is a bar graph 801 showing the hydroxyl radical scavenging rate of fish scale collagen peptides according to an embodiment of the present invention (Figure number *** represents p<0.001).

[0083] Figure 8 shows that the fish scale collagen peptide (SEQ ID NO:1) of Example 2 has good hydroxyl radical scavenging ability and is concentration-dependent, and does have antioxidant effects.

[0084] 6.3 Determination of hydrogen peroxide (H2O2) scavenging ability

[0085] An aqueous solution of fish scale collagen peptide (SEQ ID NO:1) from Example 2 was added to a 96-well UV transparent cell culture plate. 40 mM H2O2 solution was added, mixed thoroughly, and allowed to react in the dark for 10 minutes. The absorbance of each well was then measured at 230 nm, and the inhibition rate of H2O2 by the fish scale collagen peptide (SEQ ID NO:1) from Example 2 was calculated.

[0086] Please refer to Figure 9, which is a bar graph 901 showing the hydrogen peroxide scavenging rate of fish scale collagen peptide according to an embodiment of the present invention (Figure number *** represents p<0.001). The results in Figure 9 show that the fish scale collagen peptide (SEQ ID NO:1) of Example 2 exhibits significant hydrogen peroxide scavenging ability, which is concentration-dependent, and it does indeed have free radical scavenging activity.

[0087] Example 7: Anti-photoaging test of human dermal fibroblasts

[0088] 7.1 Measurement of intracellular oxidative stress in human dermal fibroblasts

[0089] 2',7'-dichlorofluorescin diacetate (DCFDA) is a membrane-permeable ROS fluorescent probe and is not itself a fluorescent substance. After entering the cell membrane, DCFDA is broken down by esterases in the cell, losing its carboxyl group and membrane permeability. When DCFDA interacts with intracellular ROS, it is oxidized by ROS to fluorescent DCF, which can be excited at 488 nm and emits green fluorescence at a wavelength of approximately 520 nm.

[0090] Hs68 cells were seeded at a density of 2 × 10⁵ cells / well in 60 mm diameter cell culture dishes and cultured for 24 hours to allow cell adhesion. After washing with PBS, the cells were irradiated with UVA at a dose of 20 J / cm². Then, DMEM culture medium containing fish scale collagen peptide (SEQ ID NO: 1) and 0.5% FBS from Example 2 was added and the cells were cultured for 24 hours to evaluate its effect on intracellular ROS scavenging. Subsequently, 10 μM DCFDA solution (10 mM DCFDA storage solution stored at -20°C protected from light; diluted with FluoroBrite™ DMEM to 10 μM DCFDA solution before use) was added and the cells were cultured at 37°C for 30 minutes. After rinsing with PBS to remove excess DCFDA, images were immediately captured at 20x magnification using a fluorescence microscope (EVOS M5000, Invitrogen™ Thermo Fisher Scientific Inc., USA), and the average fluorescence intensity was quantitatively analyzed using the commercially available software ImageJ.

[0091] Please refer to Figures 10A to 10C, which respectively show the fluorescence staining photograph 1001 (Figure 10A) of human dermal fibroblasts irradiated with UVA and treated with fish scale collagen peptides according to an embodiment of the present invention, and the bar graph 1003 (Figure 10B) of ROS scavenging ability. Figure number *** represents p<0.001 compared with the UVA irradiated control group, and figure number ### represents p<0.001 compared with the control group.

[0092] Figures 10A and 10B show that after UVA irradiation at a dose of 20 J / cm², the DCF fluorescence intensity of Hs68 cells increased by 7.82 times compared to the untreated control group, indicating a significant difference. This demonstrates that intracellular ROS was significantly induced by UVA irradiation. Treatment with 0.1 to 2 μM of the fish scale collagen peptide (SEQ ID NO:1) from Example 2 significantly reduced intracellular ROS levels to 5.58, 5.06, 2.71, and 1.23 times that of the UVA-induced control group, respectively. The fish scale collagen peptide (SEQ ID NO:1) from Example 2 exhibited a significant and concentration-dependent scavenging effect on UVA-induced ROS in cells, and at high concentrations, it effectively reduced cellular oxidative stress to near that of the UVA-induced control group, as shown in Figures 10A and 10B, demonstrating its efficacy in reducing intracellular oxidative stress.

[0093] 7.2 Assay of superoxide dismutase activity in human dermal fibroblasts

[0094] Superoxide dismutase (SOD) primarily catalyzes the dismutation of superoxide anions (O2-), converting them into oxygen (O2) and hydrogen peroxide (H2O2), thereby maintaining intracellular redox balance and preventing cell damage caused by oxidative stress. In this example, xanthine oxidase generates superoxide anions, which reduce WST-1 (Dojindo's highly water-soluble tetrazolium salt, a compound similar to MTT) to a water-soluble formazan compound, increasing the absorbance at 450 nm. After SOD dismutates the superoxide anions, the amount of formazan produced decreases, thus reducing the absorbance at 450 nm. Hs68 cells were seeded at a density of 2 × 10⁵ cells / well in 6-well cell culture dishes. When the cells reached approximately 80% confluence, they were irradiated with UVA at a dose of 20 J / cm², and culture medium containing fish scale collagen peptide (artificially synthesized SEQ ID NO: 1) from Example 2 was added. The cells were cultured for 24 hours. The cell culture medium was centrifuged at 14000g at 4°C for 5 minutes, and the supernatant was used as the SOD activity detection solution. SOD activity was detected according to the instruction manual of the commercially available detection kit [Superoxide Dismutase Activity Assay Kit (Abcam, Ltd., UK)]. In short, 20 μL of sample was added to the sample group and control group (i.e., blank group)-2, and 20 μL of sterile water was added to the control group (i.e., blank group)-1 and control group (i.e., blank group)-3. Add 200 μL of WST working solution (add 19 mL of SOD assay buffer to the WST test tube and store at 4°C protected from light) to each group. Then, add 20 μL of SOD dilution buffer to control group-2 and control group-3 respectively. Add 20 μL of enzyme working solution (add 110 μL of sterile water to the SOD tube, dispense into brown microcentrifuge tubes, store at -20°C, and dilute 20 times with SOD dilution buffer before use) to the test group and control group-1. After mixing thoroughly, react at 37°C for 20 minutes and then measure the absorbance at 440 nm.

[0095] To better understand whether the fish scale collagen peptide (artificially synthesized SEQ ID NO:1) of Example 2 can further enhance the activity of the antioxidant enzyme SOD by clearing the large amount of ROS generated in cells due to UVA irradiation, Hs68 cells were irradiated with 20 J / cm2 UVA and then treated with different concentrations of the fish scale collagen peptide (artificially synthesized SEQ ID NO:1) of Example 2, and the activity intensity of the antioxidant enzyme SOD was measured.

[0096] Please refer to Figure 10C, which is a bar graph 1005 showing the increase in SOD activity of human dermal fibroblasts after UVA irradiation and treatment with fish scale collagen peptides according to an embodiment of the present invention (Figure 10C, figure number ** represents p<0.01 compared with the UVA irradiation control group, figure number ## represents p<0.01 compared with the control group). As shown in Figure 10C, after UVA irradiation, the SOD activity of Hs68 cells increased to 9.02 times that before irradiation. Further treatment with 0.1, 0.5, 1, and 2 μM of the fish scale collagen peptide (artificially synthesized SEQ ID NO:1) from Example 2 further increased SOD activity, reaching 14.6, 15.62, 16.34, and 21.36 times the original levels, respectively. The 2 μM fish scale collagen peptide (artificially synthesized SEQ ID NO:1) from Example 2 showed the highest SOD activity with a significant difference, demonstrating a clear efficacy in enhancing SOD activity.

[0097] 7.3 Determination of Collagen Concentration in Human Dermal Fibroblasts

[0098] Hs68 cells were seeded into 6-well cell culture dishes at a density of 2 × 10⁵ cells / well. When the cells reached approximately 80% confluence, they were irradiated with UVA at a dose of 20 J / cm². Then, culture medium containing fish scale collagen peptides (synthetically synthesized SEQ ID NO: 1) from Example 2 was added, and the cells were cultured for 24 hours. After removing the culture medium, the cells were washed with ice-cold PBS, and lysis buffer was added to break the cells and collect the cell lysate. The supernatant was obtained by centrifugation at 12,000 rpm and 4°C for 10 minutes, and the supernatant was the intracellular protein. The obtained supernatant (containing intracellular proteins) was then subjected to electrophoresis using 10% SDS-PAGE. The protein film was then transferred to a PVDF membrane, and primary antibodies (specifically recognizing type I collagen) and secondary antibodies were added. Afterward, a cryogenic reaction was performed, and the protein expression of type I collagen was observed using the commercially available ImageQuant™ 800 software. Protein quantification was performed using the commercially available ImageJ software.

[0099] Please refer to Figures 11A and 11B, which respectively show the Western ink dot analysis of type I procollagen of human dermal fibroblasts after UVA irradiation and treatment with fish scale collagen peptides according to an embodiment of the present invention. Figure 1101 (Figure 11A) and relative intensity bar graph 1103 (Figure 11B, where *** represents p < 0.001 compared with the UVA irradiation control group, and ## represents p < 0.01 compared with the control group).

[0100] The results showed that after UVA irradiation at a dose of 20 J / cm², the protein expression of procollagen I in Hs68 cells significantly decreased to 0.63 times that of the control group, indicating that UVA irradiation-induced photodamage to cells inhibited collagen synthesis in fibroblasts. With increasing concentrations of the fish scale collagen peptide (synthetic SEQ ID NO: 1) from Example 2, the protein expression of procollagen I gradually recovered. Particularly at concentrations of 0.5, 1, and 2 μM of the fish scale collagen peptide (synthetic SEQ ID NO: 1) from Example 2, the protein expression of procollagen I significantly increased to 1.23, 1.28, and 1.17 times that of the control group, respectively. This demonstrates that the fish scale collagen peptide (synthetic SEQ ID NO: 1) from Example 2 has excellent efficacy in promoting collagen synthesis and protecting the integrity of the extracellular matrix under light irradiation.

[0101] 7.4 Staining assay for senescent cells in human dermal fibroblasts

[0102] When cells cease growth and enter a senescence state, their morphology and metabolic activities change, including increased cell volume and activation of senescence-associated β-galactosidase (SA-β-Gal). SA-β-Gal is a lysosomal hydrolase that is normally active at pH 4.0, but in senescent cells, it remains active even at pH 6.0. Using 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) as the receptor for the SA-β-Gal enzyme reaction, the resulting blue precipitate can be used as an indicator of cellular senescence. Hs68 cells were seeded at a density of 1 × 10⁵ cells / well in 6-well cell culture dishes. The following day, after the cells adhered, the culture medium was removed and the cells were rinsed with PBS. The cells were then irradiated with UVA at a dose of 20 J / cm², and culture medium containing fish scale collagen peptide (artificially synthesized SEQ ID NO: 1) from Example 2 was added. After culturing for 24 hours, staining was performed. The senescent cell staining assay was performed according to the instruction manual of the commercially available kit [Senescence Detection Kit (Abcam Ltd., UK)]. In short, after removing the cell culture medium, wash the cells with PBS, add 1 mL of fixative solution to each well, fix the cells at room temperature for 10 minutes, remove the fixative solution, wash the cells again with PBS, and then add 1 mL of staining solution mixture (weigh 20 mg X-gal powder, dissolve in 1 mL DMSO to make a 20 mg / mL X-gal stock solution, store at -20°C for later use; when using, prepare according to the instruction manual, take 6.11 mL of staining solution, add 65 μL of staining supplement and 325 μL of X-gal stock solution, mix well, and incubate overnight at 37°C, 0% CO2 (to avoid CO2 interference with pH). After staining, image the cells using a commercially available fluorescence imaging system (EVOS M5000 multi-functional fluorescence imaging system). The method for quantifying SA-β-Gal positive cells involves randomly selecting at least three images from different fields of view, counting 100 cells, and expressing the result as a percentage of cell count.

[0103] To evaluate the efficacy of fish scale collagen peptide (SEQ ID NO:1) from Example 2 in resisting UVA-induced photoaging, Hs68 cells were first irradiated with 20 J / cm² UVA, and then treated with different concentrations of fish scale collagen peptide (SEQ ID NO:1) from Example 2. X-gal staining was used to observe cell aging. In senescent cells, the activity of β-galactosidase (SA-β-gal) was enhanced. X-gal, acting as a receptor, was converted by SA-β-gal to form a blue precipitate, which can be used as an indicator of senescent cells.

[0104] Please refer to Figures 12A and 12B, which respectively show the staining of SA-β-gal positive cells in human dermal fibroblasts after UVA irradiation and treatment with fish scale collagen peptides according to an embodiment of the present invention, as shown in Figure 1201 (Figure 12A) and Figure 1203 (Figure 12B), which shows the percentage of SA-β-gal positive cells. Figure number *** represents p < 0.001 compared with the UVA irradiation control group, and figure number ### represents p < 0.001 compared with the control group.

[0105] Figures 12A and 12B show that after UVA irradiation, the percentage of Hs68 cells stained with X-gal (SA-β-gal positive) significantly increased from 38.10% to 70.33%, indicating that 20 J / cm² UVA irradiation induced photoaging in Hs68 cells. Further treatment with 0.1, 0.5, 1, and 2 μM of the fish scale collagen peptide (SEQ ID NO:1) from Example 2 significantly reduced the percentage of X-gal-stained cells to 38.93%, 35.75%, 31.33%, and 28.67%, respectively, demonstrating that the fish scale collagen peptide (SEQ ID NO:1) from Example 2 can effectively delay cellular senescence in Hs68 cells induced by UVA irradiation and indeed has a preventive effect against photoaging.

[0106] In summary, the above embodiments demonstrate that the fish type I collagen-derived peptides of the present invention can promote antioxidant activity, promote extracellular matrix production, whiten skin, and prevent photoaging, and can be applied to topical formulations.

[0107] It should be added that the present invention uses specific fish species, specific sequences, specific culture media, specific processes, or specific evaluation methods to illustrate the fish type I collagen-derived peptides, topical components containing them, and their use in preparing topical components. However, those skilled in the art will understand that other fish species, other sequences, other culture media, other processes, or other evaluation methods can also be used to illustrate the fish type I collagen-derived peptides, topical components containing them, and their use in preparing topical components without departing from the spirit and scope of the present invention, and are not limited to those described above. For example, the fish scale collagen peptide (SEQ ID NO:1) of Example 2 can be selectively linked with other functional sequences, or the N-terminus or C-terminus can be selectively truncated, but it contains a sequence of 27 amino acid residues as shown in the thick box in Figure 1.

[0108] According to the above embodiments, the advantages of the fish type I collagen-derived peptide and the external composition containing it are that the fish type I collagen-derived peptide is used as the active ingredient and can be used to prepare external compositions that promote antioxidant activity, promote extracellular matrix generation, whiten skin and prevent photoaging, thereby increasing the added value of fish scales and expanding their application areas.

[0109] While the invention has been disclosed above with reference to several specific embodiments, other embodiments are also possible. Therefore, the spirit and scope of the appended claims should not be limited to the embodiments described herein.

[0110] 101: Comparison Image 103: Box 201: Dot Print Analysis Chart 301, 401, 503, 601, 701, 801, 901, 1003, 1005, 1103, 1203: Bar chart 501,1101: Western Ink Dot Analysis Diagram 1001, 1201: Photos

[0111] Domestic storage information (please note in order of storage institution, date, and number) none Overseas storage information (please note in the order of storage country, institution, date, and number) none A0101_OR_PSEQ.xml

Claims

1. A type I collagen-derived peptide from fish, comprising an amino acid sequence as shown in SEQ ID NO:

1.

2. The fish type I collagen-derived peptide as claimed in claim 1, wherein the fish type I collagen-derived peptide is derived from the scales of the grouper genus (Epinephelus).

3. A topical composition comprising a fish type I collagen-derived peptide as an active ingredient, wherein the fish type I collagen-derived peptide is composed of an amino acid sequence as shown in SEQ ID NO:

1.

4. The topical composition as described in claim 3, wherein one dosage form of the topical composition includes liquid, powder, emulsion and aerosol.

5. The use of a fish type I collagen-derived peptide in the preparation of a topical composition for promoting extracellular matrix generation, wherein the topical composition uses the fish type I collagen-derived peptide as an active ingredient, and the fish type I collagen-derived peptide is composed of the amino acid sequence shown in SEQ ID NO: 1, thereby promoting the generation of elastin.

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