Ocean-source mottled bamboo shark polypeptide with antioxidant activity
Four small molecule peptides were screened from striped bamboo shark protein using virtual enzymatic digestion and molecular docking technology. This solved the problem of low efficiency in the preparation of antioxidant peptides in traditional methods, and enabled the efficient screening of peptides with significant antioxidant activity, which have the potential to prevent chronic diseases.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- QINGDAO UNIV OF SCI & TECH
- Filing Date
- 2025-09-01
- Publication Date
- 2026-05-01
AI Technical Summary
The protein resources of the marine organism striped bamboo shark have not been fully developed in the current technology. Traditional methods for preparing antioxidant peptides are cumbersome and inefficient, making it difficult to efficiently screen out peptides with significant antioxidant activity.
Four small molecule peptides were screened from the protein of the striped bamboo shark using virtual enzymatic hydrolysis and molecular docking techniques. Virtual enzymatic hydrolysis was performed using ExPASy PeptideCutter, and molecular docking was performed using Autodock Vina to screen out peptides with significant antioxidant activity. The solubility and toxicity of these peptides were evaluated using ToxinPred and a peptide property calculator, and their activity was finally verified through chemical synthesis.
This method enables efficient screening of peptides with significant antioxidant activity, improves the screening efficiency of antioxidant peptides, discovers novel antioxidants with potential for preventing chronic diseases, and avoids the loss problems of traditional methods.
Smart Images
Figure CN121949508A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the virtual screening of antioxidant peptide sequences from marine-derived striped bamboo sharks, belonging to the field of marine biomedical technology. Background Technology
[0002] When reactive oxygen species (ROS) (such as hydrogen peroxide H2O2, superoxide anion O2) are present in cells - and hydroxyl radical OH - When excessive amounts of antioxidants (such as oxidative stress and reactive oxygen species) accumulate, the body experiences oxidative stress, causing oxidative damage to cells and tissues, ultimately leading to various chronic diseases such as diabetes, cardiovascular disease, and obesity. Antioxidants can maintain redox homeostasis in cells and tissues by scavenging reactive oxygen species (ROS), reducing oxidative stress and alleviating various chronic diseases. Compared to chemically synthesized antioxidants, natural antioxidants have better safety profiles. For example, some natural antioxidant peptides are gaining popularity due to their high safety and easy digestibility.
[0003] Antioxidant peptides are a class of small, bioactive peptides containing 2-20 amino acid sequences, widely found in proteins of various plants and animals. Many methods exist for preparing antioxidant peptides, such as traditional enzymatic hydrolysis, microbial fermentation, and chemical synthesis. However, the cumbersome separation processes during preparation can lead to peptide loss, thus necessitating the development of a more efficient method. The rapid development of bioinformatics technologies (such as virtual enzymatic hydrolysis and molecular docking) has brought new insights to bioactive peptide research. Molecular docking is a computer-aided method that simulates the interaction between receptors and ligands, while virtual enzymatic hydrolysis is a computer-assisted method for rapidly digesting proteins. These computer-aided technologies have been increasingly applied to the preparation of various bioactive peptides in recent years, and large-scale screening of bioactive peptides is expected through molecular docking and virtual enzymatic hydrolysis.
[0004] The striped bamboo shark, as a marine organism, possesses untapped protein resources, potentially harboring novel antioxidant peptides. Virtual enzymatic digestion can rapidly break down striped bamboo shark proteins into polypeptide sequences, which can then be efficiently extracted using molecular docking. This bioinformatics-based method for screening bioactive peptides offers advantages over traditional methods, including higher efficiency and shorter timeframes. Therefore, combining virtual screening with molecular docking holds promise for the rapid discovery of novel marine-derived antioxidant peptides. Summary of the Invention
[0005] In response to the aforementioned existing technologies, and in order to develop antioxidant peptides, this invention utilizes virtual enzymatic hydrolysis and molecular docking techniques to virtually screen four small molecule polypeptides with significant antioxidant activity from striped bamboo shark protein, and elucidates the mechanism of action of antioxidant peptides through molecular docking results.
[0006] This invention is achieved through the following technical solution:
[0007] Antioxidant peptides include the following four types, with the following amino acid sequences:
[0008] ESK, as shown in SEQ ID NO.1;
[0009] TGER, as shown in SEQ ID NO.2;
[0010] DQER, as shown in SEQ ID NO.3;
[0011] EDR, as shown in SEQ ID NO.4.
[0012] The above four antioxidant peptides are used as or in the preparation of antioxidants, and as or in the preparation of drugs that can scavenge reactive oxygen species (ROS) and reduce oxidative stress.
[0013] The antioxidant peptides of this invention were virtually screened from striped bamboo shark proteins using virtual enzymatic hydrolysis and molecular docking, as follows:
[0014] (1) Virtual enzymatic hydrolysis of striped bamboo shark protein
[0015] This invention selects three gastrointestinal proteases—pepsin, trypsin, and chymotrypsin—for virtual enzymatic digestion. The virtual enzymatic digestion system used is ExPASy PeptideCutter (https: / / web.expasy.org / peptide_cutter / ), and the protein sequences of the intermediate cartilage layer protein (accession no. NCBI: XP_043530492.1), collagen α-1 (accession no. NCBI: XP_043547174.1), and collagen α-2 (accession no. NCBI: XP_043547172) from the striped bamboo shark cartilage from the National Center for Biotechnology Information (NCBI) (https: / / www.ncbi.nlm.nih.gov / ).
[0016] (2) Screening potential antioxidant peptides by molecular docking
[0017] Peptides released from striped bamboo shark-related proteins during virtual enzymatic digestion were analyzed using molecular docking technology. These peptides were used as ligands for molecular docking, with Kelch-like ECH-associated protein 1 (Keap1) as the acceptor. The crystal structure of Keap1 (PDB ID: 2FLU) was obtained from the RCSB protein database (https: / / www.rcsb.org / ). AutodockVina was used as the molecular docking tool. Peptide structures were drawn using Chemdraw 19.0 software. Finally, potential antioxidant peptides were screened based on VINA values.
[0018] (3) Prediction of the solubility and toxicity of antioxidant peptides
[0019] The toxicity of the 10 peptides with the lowest Vina values (Top 10) was predicted using the online tool ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / design.php), and the solubility of the Top 10 peptides was assessed using the peptide property calculator (http: / / www.innovagen.com / ).
[0020] (4) Synthesis and activity determination of antioxidant peptides
[0021] Based on the Vina value obtained from docking, the top 10 peptides were screened for chemical synthesis, and their antioxidant activity was verified by measuring the scavenging activity of DPPH and ABTS.
[0022] Experiments have shown that the Top 10 peptides we screened all have varying degrees of ability to scavenge DPPH and ABTS. Among them, peptides ESK, TGER, DQER, and EDR have significant antioxidant activity and have the potential to be used as antioxidant functional products / antioxidant drugs.
[0023] The positive and beneficial effects of this invention are:
[0024] This invention represents a revolution in traditional methods for screening marine-derived antioxidant peptides. It utilizes virtual enzymatic hydrolysis instead of traditional enzymatic hydrolysis and leverages molecular docking technology to achieve rapid screening of antioxidant peptides, thereby improving the efficiency of the screening process. Furthermore, the antioxidant peptides screened by this invention possess potential value in preventing various chronic diseases.
[0025] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description
[0026] Figure 1 Determination of the DPPH free radical scavenging ability of the Top 10 peptides.
[0027] Figure 2 Determination of the ABTS free radical scavenging ability of the Top 10 peptides.
[0028] Figure 3 Molecular docking results of antioxidant peptide TGER and Keap1.
[0029] Figure 4 Molecular docking results of antioxidant peptide DQER with Keap1.
[0030] Figure 5 Results of molecular docking between antioxidant peptide EDR and Keap1.
[0031] Figure 6 Results of molecular docking between antioxidant peptide ESK and Keap1. Detailed Implementation
[0032] The following detailed embodiments further illustrate the present invention. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.
[0033] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.
[0034] Example 1: Virtual Screening of Antioxidant Peptides from Striped Bamboo Shark
[0035] We used the virtual enzymatic digestion system ExPASy PeptideCutter to virtually digest the protein sequences of the striped bamboo shark using three proteases: pepsin, trypsin, and chymotrypsin, obtaining a total of 387 polypeptide sequences. These 387 polypeptides were then analyzed and docked with the receptor Keap1 to screen for potent antioxidant peptides. ChemDraw 19.0 and Autodock Vina were used as the polypeptide structure plotting and docking software, respectively. The Vina docking box coordinates were set to x=5, y=9, z=1, with a grid spacing of [missing information]. Box size set Other parameters were left at their default values. Finally, the binding degree of the peptide to Keap1 was determined based on the Vina value, and the top 10 peptides with the highest Vina values were screened out. The solubility and toxicity of these peptides were then predicted, and the results are shown in Table 1.
[0036] Antioxidant activity determination of the peptides screened in Example 2
[0037] The specific method for DPPH determination is as follows: Take 2 mL of a 1 mg / mL polypeptide solution in a test tube, add 2 mL of a 0.04 mg / mL DPPH solution, incubate in a light-protected water bath at 37℃ for 30 min, and finally measure the absorbance value A1 at 517 nm. Anhydrous ethanol instead of DPPH solution is the control group A2, and distilled water instead of polypeptide solution is the blank group A0. The calculation formula is: DPPH scavenging rate (%) = [1 - (A1 - A2) / A0] × 100%.
[0038] The specific method for determining ABTS is as follows: Mix an equal volume of 7 mM ABTS aqueous solution with an equal volume of 2.45 mM potassium persulfate aqueous solution. Dilute the ABTS solution with 50 mM phosphate-buffered saline (PBS) at pH 7.4. The absorbance at 734 nm is 0.70, and then the antioxidant activity is evaluated. Take 100 μL of a 1 mg / mL sample and react it with 300 μL of diluted ABTS solution at room temperature for 1 h. Finally, measure the absorbance (As) at 734 nm. Use an equal volume of 50 mM PBS at pH 7.4 as a control group (Ac). The ABTS scavenging activity is calculated using the formula: ABTS scavenging rate (%) = [(Ac-As) / Ac] × 100%.
[0039] Example 3: Synthesis and Activity Verification of Peptides
[0040] All of the top 10 peptides were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The antioxidant activity of the peptides was verified according to the method in Example 2, and the activity assay results are as follows: Figure 1 and Figure 2 As shown.
[0041] Table 1. Information on the selected peptides
[0042]
[0043] As shown in Table 1, virtual enzymatic digestion of the cartilage intermediate layer proteins and collagen of the striped bamboo shark yielded a total of 387 polypeptide sequences. By docking these polypeptides with Keap1, the top 10 polypeptides with the highest Vina values were screened. Prediction of the toxicity and solubility of the top 10 Vina value polypeptides revealed that these polypeptides were non-toxic and possessed good water solubility. Figure 1 and Figure 2As shown, by measuring the antioxidant activity of the Top 10 peptides, it was found that all 10 peptides exhibited varying degrees of DPPH and ABTS scavenging ability. In particular, peptides ESK, TGER, DQER, and EDR showed extremely strong antioxidant activity, with DPPH scavenging abilities of 93%, 97%, 95%, and 97%, respectively, and ABTS scavenging abilities of 87%, 81%, 86%, and 91%, respectively, indicating that these four peptides have the potential to act as antioxidants. To further explore the reasons for the activity of antioxidant peptides, we analyzed the molecular docking results of these four peptides with the strongest antioxidant activity in order to elucidate their mechanism of action.
[0044] Hydrophobic and charged amino acids (such as Tyr334, Arg380, Asn382, Arg415, Arg483, Tyr525, Ala556, Tyr572, and Ser602) are the main amino acids affecting the binding of Nrf2 to Keap1. Nrf2 can assist cells in defending against tissue damage caused by oxidative stress; therefore, antioxidants can exert their antioxidant effects by regulating the Keap1-Nrf2 signaling pathway. The molecular docking results of the antioxidant peptides TGER, DQER, EDR, and ESK are shown below. Figures 3-6 As shown, peptide TGER forms hydrogen bonds with amino acids Arg380, Asn414, Arg415, and Arg483 of Keap1, respectively, and peptide DQER also forms hydrogen bonds with amino acid residues Arg380, Asn414, Arg415, and Arg483 of Keap1. Similarly, peptides EDR and ESK bind to amino acids Arg380, Asn382, Asn414, Arg415, and Phe577, or Arg483 and Ser508 of Keap1, respectively, through hydrogen bonds. This indicates that the four antioxidant peptides can competitively bind to key amino acids in Keap1, thereby inhibiting the interaction between Keap1 and Nrf2, and ultimately activating and releasing Nrf2. This reveals that the antioxidant peptides TGER, DQER, EDR, and ESK may exert their antioxidant activity by affecting the interaction between Keap1 and Nrf2.
[0045] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.
Claims
1. A marine-derived striped bamboo shark polypeptide with antioxidant activity, characterized in that, Its amino acid sequence is ESK, as shown in SEQ ID NO.
1.
2. A marine-derived striped bamboo shark polypeptide with antioxidant activity, characterized in that, Its amino acid sequence is TGER, as shown in SEQ ID NO.
2.
3. A marine-derived striped bamboo shark polypeptide with antioxidant activity, characterized in that, Its amino acid sequence is: DQER, as shown in SEQ ID NO.
3.
4. A marine-derived striped bamboo shark polypeptide with antioxidant activity, characterized in that, Its amino acid sequence is: EDR, as shown in SEQ ID NO.
4.
5. The use of the antioxidant peptide according to any one of claims 1 to 4 as or in the preparation of antioxidants.
6. The use of the antioxidant peptide according to any one of claims 1 to 4 in the preparation of a medicament that scavenges reactive oxygen species (ROS) and reduces oxidative stress.