A clematis peptide with tyrosinase inhibitory activity and antioxidant activity, and a preparation method and application thereof
By preparing a white peony peptide with the amino acid sequence KCR, the stability and safety issues of tyrosinase inhibitors and antioxidants in existing technologies have been solved, achieving effective tyrosinase inhibition and antioxidant effects, which can be applied in cosmetics and pharmaceutical preparations.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- LUDONG UNIVERSITY
- Filing Date
- 2026-06-15
- Publication Date
- 2026-07-21
AI Technical Summary
In the existing technology, conventional drugs such as ascorbic acid and arbutin are used to treat pigmentation diseases but have problems with poor stability or the production of harmful metabolites. There is a lack of highly efficient, stable and safe tyrosinase inhibitors and antioxidants.
By hydrolyzing Cynanchum atratum, a peptide with the amino acid sequence KCR was screened out. The peptide, which has tyrosinase inhibitory activity and antioxidant activity, was prepared by a double enzymatic hydrolysis method and a solid-phase synthesis method. It can be used to prepare skin cosmetics and pharmaceutical preparations.
Baiwei peptide KCR effectively inhibits tyrosinase activity and scavenges free radicals, exhibiting whitening and antioxidant effects. It is suitable for preparing functional cosmetics and treating pigmentation disorders.
Smart Images
Figure CN122427232A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a small molecule peptide, its preparation method and application, specifically to a white peony peptide with tyrosinase inhibitory activity and antioxidant activity, its preparation method and application, belonging to the field of bioactive peptide technology. Background Technology
[0002] Skin problems have long been a critical issue in the medical and cosmetic fields. The factors contributing to skin problems are complex and diverse, primarily including oxidative stress, environmental exposure, and abnormal pigment metabolism. Tyrosinase, as a key rate-limiting enzyme in melanin synthesis, directly affects melanin production. It catalyzes the hydroxylation of monophenols to ortho-diphenols, which are then oxidized to ortho-quinones, ultimately leading to melanin production. While the melanin system plays a vital physiological role in protecting against ultraviolet radiation and maintaining skin homeostasis, abnormal activation of tyrosinase can lead to excessive melanin deposition, resulting in pigmentary disorders such as age spots and freckles, and even being closely related to the development of malignant melanoma. Furthermore, oxidative stress can upregulate melanin production, further exacerbating skin dullness and pigmentation. Therefore, specifically inhibiting tyrosinase activity and enhancing antioxidant capacity to regulate melanin production has become a crucial strategy for preventing and treating pigmentary skin diseases.
[0003] Currently, ascorbic acid, kojic acid, and arbutin are the main clinical treatments for the aforementioned pigmentary disorders. However, these conventional drugs have significant drawbacks: ascorbic acid has poor stability and is easily oxidized and inactivated; while kojic acid and arbutin are widely used in functional cosmetics, they produce harmful metabolites. Therefore, developing novel, highly effective, stable, and safe skin-repairing active ingredients has significant clinical application value and market potential.
[0004] In recent years, bioactive peptides have attracted widespread attention as a novel therapeutic agent. Among them, food-derived tyrosinase inhibitory peptides and antioxidant peptides have become research hotspots due to their high biocompatibility and low toxicity. (Bai Wei) Cynanchum atratum Bunge As a traditional Chinese medicinal herb, it has been proven to possess various biological activities. Current research mainly focuses on its C... 21 The bioactivity of steroidal and polysaccharide components is studied, but there is limited research on the bioactive peptides obtained from their hydrolysis. Summary of the Invention
[0005] To address the shortcomings of existing technologies, the present invention aims to: hydrolyze *Cynanchum paniculatum* and screen out *Cynanchum paniculatum* peptides with good tyrosinase inhibitory and antioxidant activities from the active peptides obtained by hydrolysis.
[0006] To achieve the above objectives, the present invention adopts the following technical solution: A white peony peptide with tyrosinase inhibitory activity and antioxidant activity, wherein the amino acid sequence of the white peony peptide is KCR.
[0007] A method for preparing the aforementioned cynanchum acinum peptide, which possesses both tyrosinase inhibitory and antioxidant activities, employs a dual-enzymatic hydrolysis method, as detailed below: (1) The dried roots and rhizomes of Cynanchum paniculatum were ground into powder and passed through a cell sieve. The powder was then mixed with ultrapure water to prepare a homogenate of Cynanchum paniculatum. (2) Adjust the pH of the Baiwei homogenate to 4.8, add cellulase and hydrolyze for 120 min, centrifuge and collect the supernatant, adjust the pH of the supernatant to 7.0, add neutral protease and hydrolyze for 180 min, centrifuge and collect the supernatant. (3) The supernatant was ultrafiltered using an ultrafiltration tube with a molecular weight cutoff of 5 kDa, and the filtrate with a molecular weight of <5 kDa was collected, which contained the white peony peptide with the amino acid sequence KCR.
[0008] Preferably, in step (2), the amount of cellulase added is 50 U / g and the amount of neutral protease added is 8000 U / g, based on the weight of the Baiwei powder.
[0009] Another preparation method for the aforementioned tyrosinase inhibitory and antioxidant peptides uses a solid-phase synthesis method, with Fmoc-protected amino acids as raw materials and polystyrene resin as a solid-phase carrier, to synthesize peptides with the amino acid sequence KCR.
[0010] The aforementioned application of white peony peptide, which has tyrosinase inhibitory and antioxidant activities, in the preparation of skin cosmetics with whitening and / or antioxidant functions.
[0011] The aforementioned application of Baiwei peptide, which has tyrosinase inhibitory and antioxidant activities, in the preparation of pharmaceutical formulations for treating pigmentation disorders.
[0012] The advantages of this invention are: This invention discovers and confirms that the hydrolysate of Cynanchum paniculatum contains polypeptide components with tyrosinase inhibitory activity and antioxidant activity. Among them, Cynanchum paniculatum peptide with the amino acid sequence KCR can effectively inhibit the activity of tyrosinase and scavenge DPPH free radicals and ABTS free radicals, and has whitening and antioxidant effects. It can be used to prepare skin cosmetics with whitening and / or antioxidant functions as well as pharmaceutical preparations for treating pigmentation diseases. Attached Figure Description
[0013] Figure 1 This is a 3D diagram of the optimal binding conformation for docking the candidate active peptide KCR with the tyrosinase protein molecule. Figure 2 This is a curve showing the inhibitory activity of white peony peptide KCR on tyrosinase; Figure 3 This is a graph showing the scavenging ability of Baiwei peptide KCR against DPPH free radicals. Figure 4 This is a graph showing the scavenging ability of Baiwei peptide KCR against ABTS free radicals. Detailed Implementation
[0014] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.
[0015] I. Obtaining Baiwei polypeptide components by hydrolysis of Baiwei A method for obtaining Baiwei polypeptide components by hydrolyzing Baiwei includes the following steps: (1) Processing raw materials and preparing homogenate The dried roots and rhizomes of Cynanchum paniculatum were ground into powder and passed through an 80-mesh cell sieve. The powder was then mixed with ultrapure water at a ratio of 1g:20ml to prepare a Cynanchum paniculatum homogenate.
[0016] (2) Preparation of Baiwei polypeptide by enzymatic hydrolysis Adjust the pH of the *Cynanchum paniculatum* homogenate obtained in step (1) to 4.8, add cellulase at a ratio of 50 U / g (based on the weight of *Cynanchum paniculatum* powder), and inactivate the enzyme after enzymatic hydrolysis at 50℃ (within the range of 50-55℃) for 120 min. After the hydrolysate cools to room temperature, centrifuge at 7000 r / min for 15 min and collect the supernatant. Adjust the pH of the supernatant to 7.0, add neutral protease at a ratio of 8000 U / g (based on the weight of *Cynanchum paniculatum* powder), and inactivate the enzyme after enzymatic hydrolysis at 50℃ (within the range of 50-55℃) for 180 min. After the hydrolysate cools to room temperature, centrifuge at 5000 r / min for 10 min and collect the supernatant.
[0017] (3) Ultrafiltration separation and purification of Baiwei polypeptide The supernatant collected in step (2) was subjected to ultrafiltration using an ultrafiltration tube with a molecular weight cutoff of 5 kDa. The filtrate with a molecular weight < 5 kDa was collected to obtain the Baiwei polypeptide component.
[0018] II. Identification and Analysis of Baiwei Peptide Sequence The sequences of the previously obtained Baiwei peptide components were determined by LC-MS / MS, and the amino acid sequences of the peptides were determined by comparison analysis using the UniProt database.
[0019] In this step, a total of 168 sequences were obtained through peptide profiling.
[0020] III. Screening for Baiwei peptides with both good bioactivity and safety The 168 sequences obtained from the previous identification were used for activity prediction, novelty screening, toxicity assessment, allergenicity analysis, cell membrane permeability prediction, digestibility prediction, and stability prediction.
[0021] First, the Peptide Ranker platform was used to score the bioactivity of all peptides. Peptides with a score ≥0.5 were screened, and peptides with a score <0.5 were not further screened.
[0022] Then, peptide sequences were compared using the UniProt and BIOPEP databases to exclude known active peptides and ensure the novelty of the screened peptides.
[0023] Subsequently, the ToxinPred tool was used to assess the potential toxicity and physicochemical properties of the peptides, excluding peptides that might be cytotoxic. The AlgPred 2.0 tool was used to predict the potential allergenicity of the peptides, screening for non-allergenic peptides. The CPPpred tool was used to analyze the cell membrane permeability of the peptides, screening for peptides with a score >0.5. The PeptideCutter tool was used to predict the digestibility of the peptides, screening for peptides that do not have sites that can be cleaved by pepsin, chymotrypsin, and trypsin. The PLifePred tool was used to predict the plasma half-life of the peptides, screening for peptides with a half-life >800s, which were considered to have good stability in the blood.
[0024] The above screening criteria ensure that the bioactive peptides obtained in the final screening have both good bioactivity and safety, and can be used for subsequent synthesis, validation and product development.
[0025] Of the 168 sequences identified previously, 112 had a Peptide Ranker score ≥ 0.5, and 56 had a score < 0.5.
[0026] Of the 112 sequences with a Peptide Ranker score ≥ 0.5, 96 were previously reported sequences and the remaining 16 were new sequences.
[0027] Among the 16 new sequences, a multi-dimensional screening based on toxicity, allergenicity, cell membrane permeability, digestibility, and stability was conducted, and a peptide with the amino acid sequence KCR was finally selected for molecular docking analysis with tyrosinase.
[0028] The physicochemical properties of the peptide KCR are detailed in Table 1.
[0029] Table 1 Physicochemical properties of peptide KCR
[0030] IV. Molecular docking screening The crystal structure of tyrosinase (PDB ID: 2Y9X) was obtained from the PDB database (http: / / www.rcsb.org / ). The protein structure was preprocessed using Pymol software (removing water molecules and existing small molecule ligands), and the preprocessed tyrosinase protein was used as the acceptor for molecular docking.
[0031] For the previously screened candidate active peptide KCR, its initial three-dimensional structure was constructed and energy minimization was performed. The most stable spatial conformation of the peptide was obtained through conformation optimization, which can be used as a ligand for molecular docking.
[0032] Molecular docking was performed using AutoDock Vina, and the binding sites between candidate active peptides and tyrosinase proteins were further analyzed.
[0033] The optimal binding conformation 3D diagram of the candidate active peptide KCR docking with the tyrosinase protein molecule is shown below. Figure 1 .
[0034] Depend on Figure 1 It can be seen that the binding free energy of the candidate active peptide KCR to tyrosinase is -6.8 kcal / mol, showing a strong binding ability; the candidate active peptide KCR forms 10 hydrogen bonds with THR308, ASP353, GLU356, ASP357 and LYS376.
[0035] V. In vitro inhibitory activity of Baiwei peptide KCR against tyrosinase Using Fmoc-protected amino acids as raw materials and polystyrene resin as a solid-phase carrier, a peptide with the amino acid sequence KCR was synthesized by solid-phase synthesis method, denoted as Baiwei peptide KCR.
[0036] The white peony peptide KCR was prepared into a series of concentration gradient solutions of 0.125 mg / ml, 0.25 mg / ml, 0.5 mg / ml, 1 mg / ml and 2 mg / ml respectively.
[0037] Four reaction systems were set up, specifically: Group A (sample blank group): PBS + PBS + tyrosinase + levodopa; Group B (blank group): PBS + PBS + PBS + Levodopa; Group C (Experimental Group): Baiwei peptide KCR + PBS + Tyrosinase + Levodopa; Group D (enzyme blank group): Baiwei peptide KCR + PBS + PBS + levodopa.
[0038] The reaction process is as follows: First, add 40 μL of Baiwei peptide KCR solution or phosphate buffer (PBS) to each reaction system, then add 80 μL of PBS, followed by 40 μL of tyrosinase solution or PBS at a concentration of 500 U / ml. Mix well and incubate at room temperature in the dark for 5 min. Then add 40 μL of 2.5 mM levodopa solution and continue incubating at room temperature in the dark for 5 min.
[0039] After incubation, the absorbance of each group at 475 nm was measured.
[0040] Calculate the tyrosinase inhibition rate (%) using the following formula:
[0041] The calculated inhibition rates of different concentrations of Baiwei peptide KCR on tyrosinase are shown in the figure. Figure 2 .
[0042] Depend on Figure 2 It can be seen that the IC50 of Baiwei peptide KCR 50 The concentration was 0.338 mg / ml (0.8335 mM), and it exhibited good inhibitory activity against tyrosinase.
[0043] V. The scavenging ability of Baiwei peptide KCR against DPPH free radicals The solid-phase synthesized white peony peptide KCR was prepared into a series of concentration gradient solutions of 0.025 mg / ml, 0.05 mg / ml, 0.1 mg / ml, 0.5 mg / ml, 1 mg / ml and 2 mg / ml.
[0044] Three reaction systems were set up, specifically: Group A (Experimental Group): White Peony Peptide KCR+DPPH; Group B (background group): Baiwei peptide KCR + anhydrous ethanol; Group C (control group): PBS + DPPH.
[0045] The reaction process is as follows: First, add 100 μL of Baiwei peptide KCR solution or PBS to each reaction system, mix it with 100 μL of anhydrous ethanol or 0.2 mM DPPH solution, and incubate at room temperature in the dark for 30 min.
[0046] After incubation, the absorbance of each group at 517 nm was measured.
[0047] Calculate the DPPH clearance rate (%) using the following formula:
[0048] The calculation results of the scavenging rate of DPPH free radicals by different concentrations of Baiwei peptide KCR are shown in the figure. Figure 3.
[0049] Depend on Figure 3 It can be seen that the IC50 of Baiwei peptide KCR 50 It has a concentration of 0.141 mg / ml (0.3477 mM) and exhibits good scavenging ability against DPPH free radicals, i.e., it has good antioxidant activity.
[0050] VI. The scavenging ability of Baiwei peptide KCR against ABTS free radicals The solid-phase synthesized white peony peptide KCR was prepared into a series of concentration gradient solutions of 0.125 mg / ml, 0.25 mg / ml, 0.5 mg / ml, 0.75 mg / ml, 1 mg / ml and 2 mg / ml.
[0051] The assay method for determining the scavenging ability of Baiwei peptide KCR against ABTS free radicals was performed according to the instructions of the commercially available kit.
[0052] The calculation results of the scavenging rate of ABTS free radicals by different concentrations of Baiwei peptide KCR are shown in the figure. Figure 4 .
[0053] Depend on Figure 4 It can be seen that the IC50 of Baiwei peptide KCR 50 It has a concentration of 0.270 mg / ml (0.6658 mM) and exhibits good scavenging ability against ABTS free radicals, i.e., it has good antioxidant activity.
[0054] In summary, the white peony peptide KCR screened in this invention possesses good tyrosinase inhibitory and antioxidant activities, and can be used as a tyrosinase inhibitor and / or antioxidant in functional cosmetics and / or pharmaceutical fields. Specifically: (1) It can be used to prepare skin cosmetics with whitening and / or antioxidant functions; (2) It can be used to prepare pharmaceutical preparations for treating pigmentation disorders.
[0055] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.
Claims
1. A white peony peptide possessing tyrosinase inhibitory activity and antioxidant activity, characterized in that, The amino acid sequence of the white peony peptide is KCR.
2. The method for preparing the white peony peptide with tyrosinase inhibitory activity and antioxidant activity as described in claim 1, characterized in that, The double-enzyme hydrolysis method is used, as detailed below: (1) The dried roots and rhizomes of Cynanchum paniculatum were ground into powder and passed through a cell sieve. The powder was then mixed with ultrapure water to prepare a homogenate of Cynanchum paniculatum. (2) Adjust the pH of the Baiwei homogenate to 4.8, add cellulase and hydrolyze for 120 min, centrifuge and collect the supernatant, adjust the pH of the supernatant to 7.0, add neutral protease and hydrolyze for 180 min, centrifuge and collect the supernatant. (3) The supernatant was ultrafiltered using an ultrafiltration tube with a molecular weight cutoff of 5 kDa, and the filtrate with a molecular weight of <5 kDa was collected, which contained the white peony peptide with the amino acid sequence KCR.
3. The preparation method according to claim 2, characterized in that, In step (2), the amount of cellulase added is 50 U / g based on the weight of Baiwei powder.
4. The preparation method according to claim 2, characterized in that, In step (2), the amount of neutral protease added is 8000 U / g based on the weight of Baiwei powder.
5. The method for preparing the white peony peptide with tyrosinase inhibitory activity and antioxidant activity as described in claim 1, characterized in that, A solid-phase synthesis method was adopted, using Fmoc-protected amino acids as raw materials and polystyrene resin as a solid-phase carrier to synthesize peptides with the amino acid sequence KCR.
6. The use of the white pith peptide with tyrosinase inhibitory activity and antioxidant activity as described in claim 1 in the preparation of skin cosmetics with whitening and / or antioxidant functions.
7. The use of the white peony peptide with tyrosinase inhibitory activity and antioxidant activity as described in claim 1 in the preparation of a pharmaceutical formulation for treating pigmentation disorders.