East asian buthus martensii koch anti-atherosclerosis polypeptide and application thereof
By screening and synthesizing the East Asian scorpion peptide fragment 5, and especially by preparing anti-atherosclerotic peptides through solid-phase synthesis, the problem of unclear structure and function of East Asian scorpion peptides has been solved, and significant antioxidant and anti-lipid deposition effects have been achieved, providing a theoretical basis for the development of atherosclerotic drugs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- NANJING UNIV OF TRADITIONAL CHINESE MEDICINE
- Filing Date
- 2026-03-11
- Publication Date
- 2026-05-12
AI Technical Summary
In the current technology, the structure and function of East Asian scorpion peptides are not well understood, which limits their clinical application and molecular mechanism exploration in the field of anti-atherosclerosis. Furthermore, existing lipid-lowering therapies have limited effects on controlling the progression of atherosclerotic plaques.
We screened and synthesized polypeptide fragments from the East Asian scorpion, especially peptide 5, and prepared anti-atherosclerotic polypeptides using solid-phase synthesis. We then revealed their antioxidant and anti-lipid deposition mechanisms and applied them to the preparation of anti-atherosclerotic drugs.
The anti-atherosclerotic peptides from the East Asian scorpion significantly reduce oxidative damage and lipid deposition, providing a theoretical basis for drug development and exhibiting significant anti-atherosclerotic effects.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of polypeptide technology of traditional Chinese medicine, specifically relating to an anti-atherosclerotic polypeptide of East Asian scorpion and its application. Background Technology
[0002] Atherosclerosis is an inflammatory disease characterized by arterial stenosis caused by elevated blood lipids. Commonly used lipid-lowering therapies are ineffective in controlling the progression of atherosclerotic plaques. Inflammation plays a significant role in the progression of atherosclerotic plaques, especially inflammation mediated by oxidative stress and lipid deposition. Developing anti-inflammatory drugs targeting oxidative stress and lipid deposition may be an effective way to reverse atherosclerosis.
[0003] As a traditional Chinese medicine, the East Asian scorpion has the effects of attacking toxins and dispersing nodules, calming wind and relieving spasms, and clearing the meridians and relieving pain. Modern medical research focuses on its protein and polypeptide active ingredients. Studies have shown that polypeptides in the East Asian scorpion have anti-inflammatory, antibacterial, neuroprotective, and cell proliferation-promoting effects. It may be used to control the inflammatory response of atherosclerosis and exert an anti-atherosclerotic effect.
[0004] Peptides are a class of small, bioactive molecules formed by 2-10 amino acids linked by peptide bonds, and they hold significant value in drug development and biomedical research. In particular, peptides are small in molecular weight and highly water-soluble, allowing for direct absorption and utilization by the intestines without the complex digestion process of proteins. For individuals with weak gastrointestinal function, peptides can effectively improve protein utilization and alleviate malnutrition. Clinically, they are commonly used for postoperative rehabilitation and nutritional support in the elderly and patients with digestive system diseases. The anti-inflammatory effects of *Scorpio scorpio* hold promise for developing anti-atherosclerotic peptides; however, current understanding of the structure and function of single *Scorpio scorpio* peptides is limited, restricting their clinical application and exploration of molecular mechanisms. Summary of the Invention
[0005] To address the problems existing in the prior art, this invention provides an anti-atherosclerotic polypeptide from the East Asian scorpion and its application, providing a theoretical basis for the development of anti-atherosclerotic polypeptide drugs and revealing its potential antioxidant and anti-lipid deposition mechanisms.
[0006] This invention is achieved through the following technical solution:
[0007] In a first aspect, the present invention provides an anti-atherosclerotic polypeptide from the East Asian scorpion, wherein the East Asian scorpion anti-atherosclerotic polypeptide is one of peptide 1, peptide 2, peptide 3, peptide 4, peptide 5, peptide 6, and peptide 7.
[0008] The amino acid sequence of peptide 1 is shown in SEQ ID NO.1;
[0009] The amino acid sequence of peptide 2 is shown in SEQ ID NO.2;
[0010] The amino acid sequence of peptide 3 is shown in SEQ ID NO.3;
[0011] The amino acid sequence of peptide 4 is shown in SEQ ID NO.4;
[0012] The amino acid sequence of peptide 5 is shown in SEQ ID NO. 5;
[0013] The amino acid sequence of peptide 6 is shown in SEQ ID NO. 6;
[0014] The amino acid sequence of peptide 7 is shown in SEQ ID NO.7.
[0015] Furthermore, the East Asian scorpion anti-atherosclerotic polypeptide was synthesized using a solid-phase synthesis method.
[0016] In a second aspect, the present invention provides the application of the East Asian scorpion anti-atherosclerotic polypeptide in the preparation of an anti-atherosclerotic drug, wherein the East Asian scorpion anti-atherosclerotic polypeptide is one of peptide 1, peptide 2, peptide 3, peptide 4, peptide 5, peptide 6, and peptide 7.
[0017] Furthermore, the East Asian scorpion anti-atherosclerotic polypeptide is peptide segment 5.
[0018] Compared with the prior art, the beneficial effects achieved by the present invention are as follows:
[0019] The anti-atherosclerotic peptide fragments (especially peptide 5) obtained by screening in this invention can significantly reduce oxidative damage and lipid deposition; this provides a theoretical basis for the pharmaceutical development of the anti-atherosclerotic peptides of Scorpion scorpion and reveals its potential anti-atherosclerotic mechanism. Attached Figure Description
[0020] Figure 1 Fluorescent probe diagrams showing the antioxidant effects of 10 candidate peptides on the RAW264.7 cell model;
[0021] Figure 2 The graph shows the fluorescence statistics of 10 candidate peptides against the MCF probe in the RAW264.7 cell model.
[0022] Figure 3 Oil Red O staining image of lipid deposition in RAW264.7 after intervention with peptides 4, 5 and 6;
[0023] Figure 4 The Oil Red O intensity statistics of lipid deposition in RAW264.7 were obtained by intervening in peptides 4, 5 and 6. Detailed Implementation
[0024] The present invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the following examples are generally performed under conventional conditions or as recommended by the manufacturer.
[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. All reagents and materials used in this invention are readily available through conventional means, and unless otherwise specified, they shall be used in accordance with conventional methods in the art or as per the product instructions.
[0026] Example 1
[0027] Polypeptide extract from East Asian pincers:
[0028] The samples of *Scorpion scorpion* were frozen in liquid nitrogen, then ground. 100 mg of the ground sample was placed in a 1.5 mL centrifuge tube, and 1 mL of a 70% methanol-water solution (V:V) containing L-2-chlorophenylalanine (4 μg / mL) was added. The mixture was then vortexed for 1 min. After vortexing, the sample solution was pre-cooled at -40°C for 2 min, then ground in a grinder (60 Hz, 2 min), and sonicated for 60 min. The mixture was then placed in a -40°C freezer and allowed to stand for 30 min. Afterward, it was centrifuged at 12000 rpm for 10 min (4°C), and the supernatant was filtered through a 0.22 μm organic phase membrane. The mixture was then allowed to stand overnight at 4°C. Finally, it was centrifuged at 12000 rpm for 10 min (4°C), and the supernatant was filtered through a 0.22 μm organic phase membrane to obtain the *Scorpion scorpion* polypeptide extract.
[0029] Example 2
[0030] The peptide extract of East Asian scorpion from Example 1 was analyzed by liquid chromatography-mass spectrometry. The peptides obtained by liquid chromatography-mass spectrometry analysis were further screened by NCBI. The bioactivity probability of peptides was predicted by NCBI and the online website PeptideRanker, and the toxicity of peptides was predicted by ToxIBTL to obtain candidate peptides.
[0031] The liquid chromatography conditions were as follows: column: ACQUITY UPLC HSS T3, 100 mm × 2.1 mm, 1.8 μm; mobile phase A: aqueous solution containing 0.1% formic acid; mobile phase B: acetonitrile; gradient elution flow rate: 0.35 mL / min; column temperature: 45℃; injection volume: 5 μL; PDA scan range: 210-400 nm; gradient elution program: 0-2 min, 95% mobile phase A; 2-4 min, 95-70% mobile phase A; 4-8 min, 70-50% mobile phase A; 8-10 min, 50-20% mobile phase A; 10-14 min, 20-0% mobile phase A; 14-15 min, 0% mobile phase A; 15-16 min, 95% mobile phase A.
[0032] The mass spectrometry conditions were as follows: ion source: HESI; mass spectrometry signal acquisition: positive and negative ion scanning modes; data acquisition mode: DDA; Full MS / dd-MS2 scanning mode; mass spectrometry parameters are shown in Table 1 below:
[0033] Table 1 Mass Spectrometry Parameters
[0034]
[0035] Progenesis QI v3.0 Analysis: The raw data were processed using the metabolomics analysis software Progenesis QI v3.0 (Nonlinear Dynamics, Newcastle, UK). This processing included baseline correction, peak detection, integration calculation, retention time adjustment, peak alignment, and data normalization. For compound identification, the TCM database was used for qualitative analysis based on precise mass numbers, secondary mass spectrometry fragment information, and isotopic distribution characteristics to ensure accuracy and reliability. For substances identified in the QI library, after merging and deduplicating substances in both positive and negative ion modes, the total relative peak area of the metabolites was set to 100% to obtain qualitative and quantitative results. Compounds contained in the East Asian scorpion were collected, and information such as the name, molecular formula, and structural formula of each compound was summarized, including information extracted from the raw data that could be used for analysis. Subsequent analyses were based on this information. Finally, 43 peptides and amino acids were obtained. NCBI and the online website PeptideRanker were used to predict the bioactivity probability of peptides, and ToxIBTL was used to predict peptide toxicity, further identifying candidate peptides. Through screening, 10 candidate peptides (peptide 1-peptide 10) were obtained, corresponding to sequences SEQ ID NO.1-SEQ ID NO.10 respectively. The information table of the 10 candidate peptides is shown in Table 2 below.
[0036] Table 2. Information on candidate peptides obtained from screening in Example 2
[0037] .
[0038] Example 3
[0039] The 10 candidate peptides screened in Example 2 were synthesized using a solid-phase synthesis method:
[0040] The solid-phase synthesis method described above is as follows:
[0041] (1) Solvent treatment: DMF and methanol were soaked overnight in G3 pore molecular sieve before use to remove impurities and water;
[0042] (2) Full swelling of resin: Weigh 2.0 g blank Wang resin into a clean and dry reaction tube, add 15 mL DMF, and activate at room temperature for 30 min;
[0043] (3) Adding the first amino acid: At room temperature, filter out the solvent from step (2) through a sand core filter, add 1 mmol of 5 times the molar excess of the first C-terminal amino acid, 5 times the molar excess of DMAP, 5 times the molar excess of DIC, and DMF as the solvent. React at room temperature for 3 hours. After the reaction is complete, wash with DMF 5 times, 5-6 mL each time. Then add a pyridine and acetic anhydride mixture with a volume ratio of 1:1 and react for 30 minutes. After the reaction is complete, wash with DMF 5 times, 5-6 mL each time.
[0044] (4) Removal of Fmoc protecting group: Remove the solvent in step (3) by filtration, add 10 mL of 20% piperidine DMF solution to the resin, stir with N2 for 10 min and filter out the solution, add another 10 mL of 20% piperidine DMF solution, stir with N2 for 5 min and filter out the solution again. Repeat this operation twice, then stir with DMF 4 times and wash with methanol 2 times, 5-6 mL each time.
[0045] (5) Detection of ninhydrin removal effect: Take 15 mg of resin, wash it three times with methanol, add one drop each of ninhydrin, KCN and phenol solution, heat at 105-110 ℃ for 5 min. If it turns dark blue, it is a positive reaction, indicating that the removal is complete and the next step can be carried out. If it is colorless, it means that the protecting group has not been completely removed, and the above deprotection operation needs to be repeated.
[0046] (6) Removal of the second amino acid and Fmoc protecting group: Weigh 3 times the molar excess of the second C-terminal amino acid, 3 times the molar excess of HBTU, and 3 times the molar excess of HOBT into a reaction tube. Add an appropriate amount of DMF solution to completely dissolve them, then add 10 times the molar excess of DIEA. React at room temperature for 40 min, and wash with DMF 5 times, 5-6 mL each time. Take a small amount of resin and test it with ninhydrin reagent. If it turns colorless, add 10 mL of 20% piperidine DMF solution to remove Fmoc. Repeat this process twice, for 10 min and 5 min respectively. Then wash with DMF 4 times and methanol 2 times, 5-6 mL each time. Take a small amount of resin and test it with ninhydrin reagent. If it turns blue, proceed to the next step of the reaction.
[0047] (7) Repeat step (6) in this manner until the last amino acid at the N-terminus is synthesized, remove the Fmoc protecting group, and then dry the mixture.
[0048] (8) Resin shedding and pure product separation detection: The peptide was cut with trifluoroacetic acid cutting solution (95% TFA: 2% TIS: 2% EDT: 1% H2O) for 2 h. The reaction solution was filtered to obtain a trifluoroacetic acid solution of the peptide. The lysis solution was dried as much as possible with nitrogen gas, then precipitated with ether, centrifuged, and then washed with ether 4 times to obtain a white solid. After being dissolved in pure water, it was desalted and purified by HPLC, and then lyophilized to precipitate crystals to obtain the target peptide.
[0049] The 10 candidate peptides listed in Table 2 were all synthesized using the methods described in steps (1) to (8) above.
[0050] Example 4
[0051] (1) Culture of mouse mononuclear macrophage leukemia cells (RAW264.7): Cells were cultured in 10% fetal bovine serum + 90% DMEM medium and placed in a 37 ℃, 5% CO2 cell culture incubator. The cell culture medium was changed every 2 days. When the cells reached about 90% confluence, they were passaged and the logarithmic growth phase cells were used for subsequent experiments.
[0052] (2) The cells in the logarithmic growth phase were seeded at a density of 2000 cells per well in a 96-well plate. After 24 h, the RAW264.7 oxidative damage model was created using 300 μM H2O2. Ten candidate peptides were used to treat the cells at concentrations of 1 µM, 5 µM, and 20 µM, respectively. The positive control group was treated with VC (100 mg / L), and the blank control group was treated with 100 μL / well culture medium. The cells were incubated with 10 µM DCF probe for 30 minutes (H2DCFDA fluorescent probe detection ROS kit). After washing with PBS three times, the cells were photographed using a fluorescence microscope and the fluorescence intensity was calculated.
[0053] The fluorescent probe diagrams of the antioxidant effects of 10 candidate peptides on the RAW264.7 cell model are shown below. Figure 1 As shown, by Figure 1 It can be seen that the 10 polypeptides have different degrees of antioxidant activity;
[0054] The fluorescence statistics of 10 candidate peptides against the MCF probe in the RAW264.7 cell model are shown in the figure below. Figure 2 As shown, by Figure 2 It can be seen that some peptides have a slight effect or the effect varies greatly with the dose (peptide 1, peptide 2, peptide 3, peptide 7, peptide 8, peptide 9, peptide 10). Peptide 4, peptide 5, and peptide 6 were selected for further verification.
[0055] Example 5
[0056] (1) The culture method for mouse mononuclear macrophage leukemia cells (RAW264.7) was the same as step (1) in Example 4; (2) The cells in the logarithmic growth phase were seeded at a density of 2000 cells per well in a 96-well plate. The RAW264.7 cells were stimulated with 75ug / ml of oxidized low-density lipoprotein (ox-LDL) for 24 h. The establishment of the model was confirmed by Oil Red O staining. A normal culture negative control group, a low-density lipoprotein positive control group, a VC positive drug group, and three peptide groups: peptide 4 (1 µM), peptide 5 (1 µM), and peptide 6 (1 µM) were set up. The lipid deposition level was determined by comparing the Oil Red O staining depth with the model group under a bright field microscope at 20x magnification. The anti-lipid deposition effect was evaluated by statistical analysis.
[0057] Peptide 4, peptide 5, and peptide 6 intervention; Oil Red O staining image of lipid deposition in RAW264.7. Figure 3 As shown, the negative control group had no Oil Red O staining, while the ox-LDL model group had deep Oil Red staining and abundant lipid deposition. The positive drug VC partially alleviated lipid deposition. Peptide 4, peptide 5, and peptide 6 were stained less than those in the model group, indicating varying degrees of anti-lipid deposition effects.
[0058] The statistical graph of Oil Red O intensity of lipid deposition in RAW264.7 was obtained by intervening in peptides 4, 5, and 6. Figure 4 As shown, peptides 4, 5, and 6 all exhibit anti-lipid deposition effects, with peptide 5 showing the strongest effect. Peptide 5, derived from the East Asian scorpion, significantly inhibits lipid deposition of oxidized low-density lipoprotein RAW264.7, demonstrating anti-atherosclerotic activity and potentially leading to the development of related drugs.
Claims
1. A polypeptide from the East Asian scorpion that combats atherosclerosis, characterized in that, The East Asian scorpion anti-atherosclerotic polypeptide is one of peptide 4, peptide 5, and peptide 6; The amino acid sequence of peptide 4 is shown in SEQ ID NO.4; The amino acid sequence of peptide 5 is shown in SEQ ID NO. 5; The amino acid sequence of peptide 6 is shown in SEQ ID NO.
6.
2. The East Asian scorpion anti-atherosclerotic polypeptide according to claim 1, characterized in that, The East Asian scorpion anti-atherosclerotic polypeptide was synthesized using a solid-phase synthesis method.
3. The application of the East Asian scorpion anti-atherosclerotic polypeptide according to claim 1 in the preparation of anti-atherosclerotic drugs, characterized in that, The East Asian scorpion anti-atherosclerotic polypeptide is one of peptide 4, peptide 5, and peptide 6.
4. The application according to claim 3, characterized in that, The East Asian scorpion anti-atherosclerotic polypeptide is peptide segment 5.