Active polypeptide and application thereof in preparation of product with whitening effect
By preparing the extract through enzymatic hydrolysis and combining it with efficient separation and verification technologies, the problem of unclear activity mechanisms of bioactive peptides in the field of skin whitening has been solved, and the stability and efficacy have been improved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-31
- Publication Date
- 2026-03-24
AI Technical Summary
The existing bioactive peptides in the field of skin whitening have unclear mechanisms of action, vague targets, insufficient stability, and lack systematic efficacy verification, resulting in inconsistent application effects.
The extract was prepared by enzymatic hydrolysis, and a molecular weight fractionation system was established by ultrafiltration. Combined with gel chromatography and solid-phase extraction, the peptide sequence was resolved by HPLC-Q-Exactive-MS technology. A multidimensional network of 'component-target-pathway' was constructed for network pharmacology, and key targets were screened and molecular docking was performed for verification.
It has enabled the precise separation and efficacy verification of bioactive peptides, identified key targets, and improved the stability and effectiveness of whitening products.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of cosmetics, and more particularly to active polypeptides and their application in the preparation of products with whitening effects. Background Technology
[0002] Bioactive peptides are a class of short-chain polypeptides composed of 2-20 amino acids, which can be obtained from natural proteins through enzymatic hydrolysis, fermentation, or chemical synthesis. Unlike traditional proteins, bioactive peptides often contain functional fragments with specific physiological functions in their structure. In recent years, due to their wide range of health benefits, they have become a research hotspot in the fields of functional foods, pharmaceuticals, and cosmetics.
[0003] However, there are still some key problems with the bioactive peptides currently used in the skin whitening field: most products have unclear mechanisms of action, vague targets, insufficient stability, and lack systematic efficacy verification, resulting in inconsistent actual application effects. Summary of the Invention
[0004] In view of this, the present invention provides active peptides and their application in the preparation of products with whitening effects. The present invention employs an enzymatic hydrolysis method to prepare the extract, establishes a molecular weight fractionation system based on ultrafiltration, determines the optimal active component through in vitro screening, and further uses gel permeation chromatography to finely separate the target fraction. Solid-phase extraction combined with HPLC-Q-Exactive-MS technology is used to resolve the peptide sequence, and a multidimensional "component-target-pathway" network is constructed based on network pharmacology. After screening key targets, molecular docking is used to verify the interaction mode. Finally, candidate peptides are synthesized in solid phase, and an evaluation system is established to verify their efficacy.
[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:
[0006] This invention provides an active polypeptide having:
[0007] (1) An amino acid sequence as shown in any of SEQ ID NO:1 to SEQ ID NO:14; or
[0008] (2) An amino acid sequence obtained by substituting, deleting, or adding one or more amino groups to the amino acid sequence shown in (1), and which has the same or similar function as the amino acid sequence shown in (1); or
[0009] (3) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (1) or (2).
[0010] The present invention also provides a nucleic acid fragment encoding the above-mentioned active polypeptide.
[0011] The present invention also provides a method for preparing the above-mentioned active polypeptide, comprising the following steps:
[0012] S1: Provides raw materials containing the target protein;
[0013] S2: The raw material is hydrolyzed using a protease to obtain a hydrolysate;
[0014] S3: The enzymatic hydrolysate is separated and purified to obtain the active polypeptide.
[0015] In some embodiments of the present invention, the raw material in the above preparation method includes: sika deer antlers.
[0016] This invention also provides the application of the above-mentioned active polypeptide, the above-mentioned nucleic acid fragment, and the active polypeptide obtained by the above-mentioned preparation method in the preparation of products with whitening effects.
[0017] In some embodiments of the present invention, in the above applications, the whitening effect includes: inhibiting the activity of tyrosinase.
[0018] In some embodiments of the present invention, in the above applications, the whitening effect includes: reducing melanin synthesis.
[0019] In some embodiments of the present invention, in the above applications, the product includes one or more of cosmetics, topical skin preparations, and medical dressings.
[0020] The present invention also provides cosmetics comprising: the above-described active polypeptide, the above-described nucleic acid fragment and / or the active polypeptide obtained by the above-described preparation method, and cosmetically acceptable excipients.
[0021] The present invention also provides topical skin preparations and / or medical dressings, comprising: the above-described active polypeptide, the above-described nucleic acid fragment and / or the active polypeptide obtained by the above preparation method, and a cosmetically acceptable carrier, medical carrier or medical device integrating the active ingredient.
[0022] This invention employs an enzymatic hydrolysis method to prepare the extract, establishes a molecular weight fractionation system based on ultrafiltration, and determines the optimal active component through in vitro screening. The target fraction is then further separated using gel permeation chromatography. Solid-phase extraction combined with HPLC-Q-Exactive-MS is used to resolve the peptide sequence, and a multidimensional "component-target-pathway" network is constructed based on network pharmacology. Key targets are screened, and molecular docking is used to verify the interaction patterns. Finally, candidate peptides are synthesized in solid phase, and an evaluation system is established to verify their efficacy. Attached Figure Description
[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.
[0024] Figure 1 It shows the whitening activity of VI, DI, and DB-I;
[0025] Figure 2 It shows the whitening activity of V-II, V-III, V-IV, and VV;
[0026] Figure 3 Show the elution peaks of components V-IV;
[0027] Figure 4 Show VG-III liquid chromatography-mass spectrometry (LC-MS) plot;
[0028] Figure 5 This indicates potential targets for skin whitening activity;
[0029] Figure 6 PPI network diagram;
[0030] Figure 7 Component-target network diagram;
[0031] Figure 8 Showing the core target point map;
[0032] Figure 9 It shows monomeric whitening activity;
[0033] Figure 10 The whitening activity of the monomer zebrafish is shown; wherein: A is nonapeptide-1, B is SEQ ID NO:9, and C is SEQ ID NO:10. Detailed Implementation
[0034] This invention discloses active polypeptides and their application in the preparation of products with whitening effects.
[0035] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.
[0036] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.
[0037] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.
[0038] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.
[0039] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.
[0040] In Examples 1 to 6 of this invention, all raw materials and reagents used can be purchased from the market.
[0041] The present invention will be further illustrated below with reference to the embodiments:
[0042] Example 1: Extraction of deer-derived polypeptides
[0043] (1) Extraction of deer antler polypeptides
[0044] After washing and air-drying the whole deer antler, crush it and pass it through an 80-mesh sieve. Add distilled water at a material-to-liquid ratio of 1:10 g / mL, reflux and extract for 1.5 h. Extract twice in total. Combine the extracts, centrifuge at 3600 r / min for 15 min, and collect the supernatant to obtain antler protein (Piloseantler protein, PAP).
[0045] The enzymatic hydrolysis conditions were set at 50℃ and pH 9.0. 2.0% alkaline protease (200 U / mg) was added to the deer antler protein extract and stirred in a constant temperature magnetic stirrer for 4 hours. During this period, 1 mol / L NaOH was added to maintain the solution at the optimal pH of the enzyme. After the enzymatic hydrolysis was completed, the solution was transferred to boiling water at 100℃ for 15 minutes to inactivate the enzyme. The solution was then centrifuged at 3600 r / min for 15 minutes, and the supernatant was collected to obtain the deer antler enzymatic hydrolysate (VAEH).
[0046] (2) Extraction of deer hide polypeptides
[0047] Take an appropriate amount of deer hide, clean it thoroughly, remove subcutaneous fat, and cut it into 1cm x 1cm pieces. Stir in a 5% NaCO3 aqueous solution at a ratio of 1:15 g / mL for 24 hours. Rinse repeatedly with distilled water, then add a 5% NaCl aqueous solution at a ratio of 1:8 g / mL and stir at room temperature for 24 hours to remove salt-soluble proteins. Wash 3-4 times with distilled water at 30℃, vacuum package, and refrigerate for later use. Take the treated deer hide, add 10 times the amount of water, and pressure cook at 120 kPa for 2 hours, then at 8000 r·min. -1 Centrifuge for 10 min and collect the supernatant to obtain deerskin protein (DP).
[0048] The enzymatic hydrolysis conditions were set at 60℃ and pH 9.0. 5.0% alkaline protease (200 U / mg) was added to the deer hide protein extract and stirred in a constant temperature magnetic stirrer for 6 hours. During this period, 1 mol / L NaOH was added to maintain the solution at the optimal pH of the enzyme. After the enzymatic hydrolysis was completed, the solution was transferred to boiling water at 100℃ for 15 minutes to inactivate the enzyme. The solution was then centrifuged at 3600 r / min for 15 minutes, and the supernatant was collected to obtain deer hide enzymatic hydrolysate (DEH).
[0049] (3) Extraction of deer bone polypeptides
[0050] After removing non-bone materials such as meat and fascia from the deer bones, the bones are processed using a crusher to break them down to a particle size of 20-30 mm. The crushed deer bones are then dried to a moisture content of 10-15%. The dried deer bones are then subjected to secondary pulverization to 80 mesh to obtain deer bone powder. The processed deer bones are then mixed with distilled water at a material-to-liquid ratio of 1:10 g / mL and refluxed for 2 hours. This extraction is repeated twice. The extracts are combined and centrifuged at 3600 rpm for 15 minutes. The supernatant is collected to obtain deer bone protein (DBP).
[0051] The enzymatic hydrolysis conditions were set at 60℃ and pH 10.0. 4.0% alkaline protease (200 U / mg) was added to the deer bone protein extract and stirred in a constant temperature magnetic stirrer for 6 hours. During this period, 1 mol / L NaOH was added to maintain the solution at the optimal pH of the enzyme. After the enzymatic hydrolysis was completed, the solution was transferred to boiling water at 100℃ for 15 minutes to inactivate the enzyme. The solution was then centrifuged at 3600 r / min for 15 minutes, and the supernatant was collected to obtain deer bone enzymatic hydrolysate (DBEH).
[0052] Example 2 Purification of deer-derived peptides
[0053] Preliminary separation of deer antler hydrolysate, deer hide hydrolysate, and deer bone hydrolysate was performed using ultrafiltration membranes with different molecular weight cutoffs. The total components of the deer antler hydrolysate were labeled VI, the total components of the deer hide hydrolysate DI, and the total components of the deer bone hydrolysate DB-I. The whitening activity of these components was screened, and the results are shown below. Figure 1 As shown in Table 1, the results indicate that VI has the best whitening activity. VI was freeze-dried and stored in a -80℃ freezer for further experiments.
[0054] Table 1
[0055]
[0056] Filtering of VI was performed using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa. Fractions with a molecular weight >3 kDa were collected and labeled V-II. The filtrate was then filtered again using an ultrafiltration membrane with a molecular weight cutoff of 1 kDa. Fractions with a molecular weight between 1 and 3 kDa were collected and labeled V-III. Fractions with a molecular weight <3 kDa were also collected and labeled V-IV. Fractions with a molecular weight <1 kDa were also collected and labeled VV. All collected fractions were freeze-dried and stored at -80°C for further experiments.
[0057] The whitening activity of each component obtained above was tested, and the results are shown in the figure. Figure 2 As shown in Table 2, components V-IV were the best. The components with the best activity were collected and stored in a -80°C freezer for further experiments.
[0058] Table 2
[0059]
[0060] Skin whitening activity assay: First, add the test sample, PBS solution, and 100 U / mL tyrosinase solution to a 96-well plate according to Table 3. Mix the sample and tyrosinase thoroughly and incubate in a 37℃ water bath for 10 min. Then, add 120 μL of levodopa solution to each well sequentially, controlling the reaction time for each well to 5 min. Immediately measure the absorbance (A) at a wavelength of 475 nm using a microplate reader. Tyrosinase inhibition rate (%) = [1-(AA-AB) / (AC-AD)]×100%.
[0061] Table 3 Whitening Active System
[0062]
[0063] To further purify the ultrafiltration fractions, Sephadex G-25 gel chromatography was used to further separate and purify the ultrafiltration peptides. The ultrafiltration fractions were separated using a Sephadex G-25 gel column with ultrapure water as the eluent, and the eluent was collected at a constant flow pump speed of 17.5 rpm. Each elution peak appearing at 220 nm was collected using an automated fraction collector. The elution peaks of fractions V-IV were named VG-I, VG-II, VG-III, and VG-IV, respectively. The results are shown in [Figure number missing]. Figure 3 After activity screening, the optimal active fraction for whitening was determined to be VG-III. The optimal active fraction was collected and stored in a -80℃ freezer for further experiments.
[0064] Example 3 Identification of Deer Antler Polypeptides
[0065] To determine the peptide sequence of VG-III, each component was purified using a C18 solid-phase extraction column, and the 5% and 30% methanol eluates were collected and stored under nitrogen blowing. The amino acid sequence of the peptide was analyzed using high-performance liquid chromatography-quadrupole electrostatic field orbital trap mass spectrometry (HPLC-Q-Exactive-MS). The HPLC-MS chromatogram of VG-III is shown below. Figure 4 The liquid chromatography-mass spectrometry (LC-MS) data were input into PeptideProphet software for evaluation, and peptide sequences with 5-15 amino acids were collected. The results are shown in Table 4.
[0066] Table 4. Peptide Sequences
[0067]
[0068] Example 4: Network Pharmacology and Molecular Docking
[0069] (1) Screening of active ingredient targets
[0070] The components analyzed above were used as active ingredients. The pharmmapper website was used to retrieve target markers, selecting the top 300 targets for each component as active ingredient targets. This database was set to "human species," and the names of target proteins were filtered using "probability > 0."
[0071] (2) Acquisition of functionally relevant targets
[0072] Skin whitening-related active target genes were retrieved from the GeneCard database (https: / / www.genecards.org / ) and the OMIM database (https: / / www.omim.org / ) using "pigmentation" as the keyword. After integration and deduplication, the top 2500 targets were collected and then normalized in UniProt (http: / / www.uniprot.org / ), selecting "human species" to obtain a list of targets related to each function. Common targets between active ingredients and predicted targets of active functions were extracted and represented by a Venn diagram (see [link to Venn diagram]). Figure 5 .
[0073] (3) Construction of protein-protein interaction (PPI) network
[0074] PPI network construction and functional enrichment analysis were performed on the above targets using STRINGdb (https: / / string-db.org / ). The species was designated as "human species" with a confidence level of 0.4. The protein-protein interaction data were then imported into Cytoscape 3.7.2 (https: / / cytoscape.org / ) to construct the PPI network. The PPI network diagram is shown below. Figure 6 .
[0075] (3) Construction of disease-target-pathway network
[0076] This invention utilizes the aforementioned information on active ingredients (components) and key protein targets (targets) to construct an ingredient-target network. Subsequently, Cytoscape 3.7.2 was used to visualize the network, and network topology analysis was performed using the CytoNCA plugin, resulting in an ingredient-whitening activity-target network graph with 132 nodes and 512 edges. Each edge represents the interaction between ingredient-whitening activity targets, denoted by a degree value. The node size represents the degree value; a higher degree value indicates more target targets connected to the node. (See...) Figure 7 The analysis parameters included degree centrality, intermediate centrality, and proximity centrality. Core target points were selected by taking the intersection of twice the median degree value and twice the median betweenness value. (See...) Figure 8 .
[0077] (4) Molecular docking and visualization
[0078] Molecular docking technology was used to evaluate the ability and affinity of the active ingredient for selected key protein targets. The 3D structures of the protein targets were plotted and saved as mol2 files. Crystal structures of the core protein targets were extracted from the protein database (PDB, https: / / www1.rcsb.org / ), and molecular docking was performed using AutoDock, with a binding energy of -5.0 kcal / mol as the selection criterion. The docking results between the active compound and the protein targets were observed using PyMOL software (version 3.0, https: / / PyMOL.org / ). The molecular docking binding energy results are shown in Table 5.
[0079] Table 5 Molecular docking results
[0080]
[0081] Example 5: Peptide Synthesis
[0082] Using Fmoc-protected amino acids as raw materials and polystyrene resin as a solid-phase carrier, solid-phase synthesis was carried out using the Fmoc solid-phase synthesis strategy.
[0083] Example 6: Whitening Activity of Deer-Derived Peptides
[0084] Tyrosinase inhibition rate experiments were performed on the peptide chains synthesized in the solid phase according to SEQ ID NO:9 and SEQ ID NO:10. The positive control was nonapeptide-1. The results are shown in [Figure number missing]. Figure 9 As shown in Table 7, an experiment was conducted to investigate the inhibitory effect of zebrafish embryo melanin. Zebrafish 6 hours after fertilization were randomly selected and placed in 6-well plates, 15 fish per well. The drug concentration was 0.1%, and the volume per well was 3 mL. A blank control group (C) and a positive control (nonapeptide-1) were also included. The plates were incubated at 28°C in the dark for 45 hours. Ten zebrafish from each experimental group were randomly selected and photographed under a dissecting microscope. Advanced image processing software was used to analyze and collect data. The melanin signal intensity (S) in the zebrafish head was analyzed, and the whitening effect of the samples was calculated and determined according to the formula. The results are shown in Table 7. Figure 10 Table 6. SEQ ID NO:10 was determined to be a skin-whitening peptide.
[0085] Whitening effect (%) =
[0086] Table 6 Whitening Efficacy
[0087]
[0088] Table 7
[0089]
[0090] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. An active polypeptide, characterized in that, It has the following characteristics: (1) An amino acid sequence as shown in any of SEQ ID NO:1 to SEQ ID NO:14; or (2) An amino acid sequence obtained by substituting, deleting, or adding one or more amino groups to the amino acid sequence shown in (1), and which has the same or similar function as the amino acid sequence shown in (1); or (3) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (1) or (2).
2. A nucleic acid fragment, characterized in that, Encoding the active polypeptide as described in claim 1.
3. The method for preparing the active polypeptide as described in claim 1, characterized in that, Includes the following steps: S1: Provides raw materials containing the target protein; S2: The raw material is hydrolyzed using a protease to obtain a hydrolysate; S3: The enzymatic hydrolysate is separated and purified to obtain the active polypeptide.
4. The preparation method according to claim 3, characterized in that, The raw materials include: sika deer antlers.
5. The application of the active polypeptide as described in claim 1, the nucleic acid fragment as described in claim 2, and the active polypeptide obtained by the preparation method as described in claim 3 or 4 in the preparation of products with whitening effects.
6. The application as described in claim 5, characterized in that, The whitening effects include: inhibiting the activity of tyrosinase.
7. The application as described in claim 5 or 6, characterized in that, The whitening effects include reducing melanin synthesis.
8. The application as described in any one of claims 5 to 7, characterized in that, The products include one or more of the following: cosmetics, topical skin preparations, and medical dressings.
9. A cosmetic product, characterized in that, include: The active polypeptide as described in claim 1, the nucleic acid fragment as described in claim 2, and / or the active polypeptide obtained by the preparation method as described in claim 3 or 4, and cosmetically acceptable excipients.
10. Topical skin preparations and / or medical dressings, characterized in that, include: The active polypeptide as described in claim 1, the nucleic acid fragment as described in claim 2, and / or the active polypeptide obtained by the preparation method as described in claim 3 or 4, as well as a cosmetically acceptable carrier, medical carrier, or medical device integrating the active ingredient.