Polypeptide with antioxidant activity and application thereof
The high-purity peptides prepared by chemical solid-phase synthesis have solved the safety and activity problems of existing antioxidants, achieving a highly efficient antioxidant protection effect, and are suitable for the fields of pharmaceuticals, food and cosmetics.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-12
- Publication Date
- 2026-04-03
AI Technical Summary
Existing antioxidants have safety risks, poor stability, and low bioavailability. Furthermore, antioxidant peptides have low screening efficiency, insufficient activity, or unclear protective effects at the cellular level.
A series of structurally confirmed peptides were provided, prepared by chemical solid-phase synthesis, with a purity ≥95.0%, and applied in pharmaceuticals, food preservatives, cosmetics and antioxidant auxiliaries, including amino acid sequences QY, YG, SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, exhibiting excellent antioxidant activity.
The large-scale preparation of high-purity peptides has been achieved, which have significant chemical antioxidant capacity and cell protection effects, good biosafety, and are suitable for the preparation of drugs, food and cosmetics, especially for liver damage prevention and anti-aging functions.
Smart Images
Figure CN121779488A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of biomedicine, food science, and daily chemical technology, specifically to a group of structurally confirmed novel antioxidant peptides and their classification and applications in pharmaceuticals, food preservation, and cosmetics. Background Technology
[0002] Oxidative stress refers to the pathological process in which excessive production of reactive oxygen species (ROS) or decreased scavenging capacity in the body leads to their accumulation and oxidative damage. Oxidative stress is closely related to a variety of human diseases, including cardiovascular disease, neurodegenerative diseases, inflammation, and liver damage. Furthermore, in the food and cosmetic industries, oxidation is a major cause of product spoilage and reduced efficacy.
[0003] Currently, antioxidants used in the market are mainly divided into synthetic antioxidants and natural antioxidants. While synthetic antioxidants (such as BHT and BHA) are low-cost and effective, long-term high-dose use may pose potential safety risks. Natural antioxidants (such as vitamin C and vitamin E) have better safety profiles, but often suffer from poor stability and low bioavailability. Bioactive peptides, as a class of biomolecules with antioxidant functions, have become a hot topic in antioxidant research in recent years due to their small molecular weight, easy absorption, large structural modification space, and good biocompatibility. However, existing antioxidant peptides often suffer from low screening efficiency, insufficient activity, or unclear cellular-level protective effects. Therefore, developing novel antioxidant peptides with novel structures, excellent activity, and rigorous structural verification has significant application value. Summary of the Invention
[0004] To achieve the above objectives, the present invention provides the following technical solutions: In a first aspect, the present invention provides a polypeptide with antioxidant activity, the amino acid sequence of which is selected from one of the following sequences: (1) QY; (2) YG; (3) the amino acid sequence is shown in SEQ ID NO: 1; (4) the amino acid sequence is shown in SEQ ID NO: 2; (5) the amino acid sequence is shown in SEQ ID NO: 3.
[0005] Furthermore, the polypeptide is prepared by a chemical solid-phase synthesis method; the polypeptide is in the form of trifluoroacetate or acetate.
[0006] Furthermore, the polypeptide has the following purity characteristics as determined by high performance liquid chromatography and mass spectrometry analysis: (1) purity of sequence QY ≥ 95.0%; (2) purity of sequence YG ≥ 95.0%; (3) purity of sequence SEQ ID NO: 1 ≥ 95.0%; (4) purity of sequence SEQ ID NO: 2 ≥ 95.0%; (5) purity of sequence SEQ ID NO: 3 ≥ 95.0%.
[0007] Secondly, the present invention provides the use of the polypeptide in the preparation of medicaments for the prevention or treatment of diseases related to oxidative stress injury, particularly for the preparation of medicaments for the prevention or treatment of liver injury. The polypeptide is preferably selected from QY, YG, SEQ ID NO: 1, or SEQ ID NO: 2.
[0008] Thirdly, the present invention provides the use of the polypeptide in the preparation of non-pharmaceutical antioxidants, food preservatives, or chemical antioxidant auxiliaries. The polypeptide is preferably SEQ ID NO: 3.
[0009] Fourthly, the present invention provides the use of the polypeptide in the preparation of cosmetics with antioxidant and anti-aging functions. The polypeptide is preferably selected from QY, YG, SEQ ID NO: 1, or SEQ ID NO: 2.
[0010] Fifthly, the present invention provides an antioxidant composition comprising any of the peptides described above and a pharmaceutically, food-, or cosmetically acceptable carrier or excipient.
[0011] The beneficial effects of this invention are as follows: the polypeptide sequences provided by this invention are short, and can be prepared on a large scale using mature solid-phase synthesis technology, with stable processes and controllable quality. Experimental data show that the polypeptide shown in SEQ ID NO: 3 has superior chemical antioxidant capacity compared to the positive control GSH. Experimental data also show that polypeptides QY, YG, SEQ ID NO: 1, and SEQ ID NO: 2 have significant antioxidant protective effects at the cellular level, effectively resisting oxidant-induced cell damage, and exhibiting good biocompatibility. Attached Figure Description
[0012] Figure 1 The HPLC chromatogram (a) and mass spectrum (b) of peptide Pep 4 (QY) are shown. Figure 2 The HPLC chromatogram (a) and mass spectrum (b) of peptide Pep 5 (YG) are shown. Figure 3 The HPLC chromatogram (a) and mass spectrum (b) of peptide Pep 10 (SEQ ID NO: 1) are shown. Figure 4The HPLC chromatogram (a) and mass spectrum (b) of peptide Pep 11 (SEQ ID NO: 2) are shown. Figure 5 The HPLC chromatogram (a) and mass spectrum (b) of peptide Pep 16 (SEQ ID NO: 3) are shown. Figure 6 The diagram shows the sequence information and in vitro chemical antioxidant activity of the polypeptide of the present invention; wherein, (a) is the sequence information and prediction score table, (b) is the result of DPPH free radical scavenging activity determination, and (c) is the result of ABTS free radical cation scavenging activity determination. Figure 7 The bar chart shows the effect of different concentrations of peptide Pep 4 (QY) on the survival rate of t-BHP-induced HepG2 cells. Figure 8 The bar chart shows the effect of different concentrations of peptide Pep 5 (YG) on the survival rate of t-BHP-induced HepG2 cells. Figure 9 The bar chart shows the effect of different concentrations of peptide Pep 11 (SEQ ID NO: 2) on the survival rate of t-BHP-induced HepG2 cells. Figure 10 The bar chart shows the effect of different concentrations of peptide Pep 10 (SEQ ID NO: 1) on the survival rate of t-BHP-induced HepG2 cells. Detailed Implementation
[0013] The present invention will be further described in detail below with reference to specific embodiments.
[0014] Example 1: Synthesis and Structural Confirmation of Peptides The peptides described in this invention were prepared using the standard Fmoc solid-phase synthesis method (SPPS). The synthesized crude peptides were purified by preparative reversed-phase high-performance liquid chromatography (RP-HPLC) with an acetonitrile-water system (containing 0.1% trifluoroacetic acid) as the mobile phase. The main peak fraction was collected and freeze-dried to obtain a white powder product. Purity was determined by analytical HPLC, and molecular weight was determined by electrospray ionization mass spectrometry (ESI-MS).
[0015] The detection data for each peptide are as follows: (1) Pep 4 (QY) Appearance: White lyophilized powder. Solubility: Easily soluble in water. HPLC analysis: As shown Figure 1 As shown in (b), the chromatographic column was Kromasil C18, the retention time (RT) was 9.668 min, and the peak area normalized purity was 95.39%. MS analysis: as shown Figure 1 As shown in (a), the molecular formula is C14H19N3O5, the theoretical molecular weight is 309.32, and the measured mass-to-charge ratio (m / z) is 310.05 [M+H]+, which is consistent with the theoretical value.
[0016] (2) Pep 5 (YG) Appearance: White lyophilized powder. Solubility: Easily soluble in water. HPLC analysis: As shown Figure 2 (b) shows the chromatographic column used was Kromasil C18, the retention time (RT) was 8.159 min, and the peak area normalized purity was 96.28%. MS analysis: as shown. Figure 2 As shown in (a), the molecular formula is C11H14N2O4, the theoretical molecular weight is 238.24, and the measured mass-to-charge ratio (m / z) is 239.05 [M+H]+, which is consistent with the theoretical value.
[0017] (3) Pep 10 (SEQ ID NO: 1) Appearance: White lyophilized powder. HPLC analysis: as shown. Figure 3 (b) shows the chromatographic column used was Symmetrix ODS-R, the retention time (RT) was 9.126 min, and the peak area normalized purity was 96.42%. MS analysis: as shown. Figure 3 As shown in (a), the molecular formula is C36H38N4O9, the theoretical molecular weight is 670.71, and the measured mass-to-charge ratio (m / z) is 671.4 [M+H]+, which is consistent with the theoretical value.
[0018] (4) Pep 11 (SEQ ID NO: 2) Appearance: White lyophilized powder. Solubility: Easily soluble in water. HPLC analysis: As shown Figure 4 As shown in (b), the chromatographic column was a Symmetrix ODS-R, the retention time (RT) was 10.048 min, and the peak area normalized purity was 95.14%. MS analysis: as shown Figure 4 As shown in (a), the molecular formula is C29H35N5O7, the theoretical molecular weight is 569.61, and the measured mass-to-charge ratio (m / z) is 570.5 [M+H]+, which is consistent with the theoretical value.
[0019] (5) Pep 16 (SEQ ID NO: 3) Appearance: White lyophilized powder. HPLC analysis: as shown. Figure 5 (b) shows the chromatographic column used: Sinochrom ODS-BP, retention time (RT) of 9.223 min, and peak area normalized purity of 95.80%. MS analysis: as shown. Figure 5 As shown in (a), the molecular formula is C53H70N14O16, and the theoretical molecular weight is 1183.25. The measured main ion peaks include 606.5 [M+2H]2+ and 404.8 [M+3H]3+. Calculations confirmed that the synthesized polypeptide conforms to the expected structure.
[0020] Example 2: In vitro chemical antioxidant activity evaluation was conducted using DPPH (2,2-biphenyl-1-picrylhydrazine) and ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) free radical scavenging assays to evaluate the antioxidant activity of the peptides, with glutathione (GSH) as a positive control. The peptide sequence information and predicted scores involved in this example are as follows: Figure 6 As shown in (a).
[0021] The results are as follows Figure 6 (b) and Figure 6 As shown in (c): Pep 16 (SEQ ID NO: 3) exhibited the strongest activity in both experiments. Particularly in the ABTS experiment ( Figure 6 (c) Its Trolox equivalent (TE) value was significantly higher than that of other peptides and superior to the positive control GSH at the same concentration. This indicates that Pep 16 has extremely high electron transfer or hydrogen atom transfer capabilities and is a highly efficient antioxidant.
[0022] Pep 4, Pep 5, Pep 10, and Pep 11 also exhibited significant free radical scavenging capabilities, with a good dose-response relationship between their scavenging rate and concentration.
[0023] Example 3: Protective effect of peptides against oxidative damage in HepG2 cells. To evaluate the antioxidant and protective effects of peptides in biological systems, this example uses the human hepatocellular carcinoma cell line (HepG2) as an in vitro model and oxidative stress damage is induced by tert-butyl hydrogen peroxide (t-BHP).
[0024] HepG2 cells were seeded in 96-well plates and, after adhesion, were divided into a control group, a damage group (treated with t-BHP only), and a protection group (treated with peptides for 24 h + t-BHP). Peptide concentrations were set at 0.25, 0.5, 1.0, and 2.0 mg / mL. Cell viability was determined using the CCK-8 assay after treatment.
[0025] Pep 4 (QY): such as Figure 7 As shown, compared with the damaged group, the cell viability of the Pep 4 pretreatment group increased significantly with increasing concentration. At a concentration of 2.0 mg / mL, the cell viability recovered to over 100% (p<0.0001), indicating that Pep 4 can significantly enhance the cell's resistance to t-BHP oxidative damage and is non-toxic to cells at the tested concentration.
[0026] Pep 5 (YG): such as Figure 8As shown, it exhibits a significant dose-dependent protective effect. At concentrations of 1.0 mg / mL and 2.0 mg / mL, cell viability was significantly higher than in the injury group, indicating that it has good cytoprotective activity.
[0027] Pep 10 (SEQ ID NO: 1): such as Figure 9 As shown, the Pep 10 pretreatment group also exhibited resistance to t-BHP-induced cytotoxicity and significantly improved cell viability.
[0028] Pep 11 (SEQ ID NO: 2): such as Figure 10 As shown, the high concentration group (2.0 mg / mL) significantly improved the survival rate of damaged cells, demonstrating an effective antioxidant protective effect.
[0029] Experimental results showed that Pep 4, Pep 5, Pep 10, and Pep 11 not only possess chemical antioxidant capabilities, but more importantly, they can effectively protect hepatocytes from damage caused by the strong oxidant t-BHP through pretreatment. Combined with their high cell viability at effective concentrations, this indicates that these peptides have good biocompatibility and are suitable for development into antioxidant drugs or functional health foods. Although Pep 16 has a strong chemical scavenging ability, it is mainly recommended for use as a highly effective antioxidant without biological contact in this experimental system.
[0030] In summary, the antioxidant peptides provided by this invention have well-defined structures and high purity, and based on their different activity characteristics, they can be applied to the pharmaceutical, food, and chemical fields respectively.
Claims
1. A polypeptide with antioxidant activity, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO:
3.
2. The polypeptide according to claim 1, characterized in that, The polypeptide was prepared by chemical solid-phase synthesis; the polypeptide was in the form of trifluoroacetate or acetate.
3. The polypeptide according to claim 1, characterized in that, The polypeptide has the following purity characteristics as determined by high performance liquid chromatography and mass spectrometry analysis: the purity of sequence SEQ ID NO: 3 is ≥95.0%.
4. Use of the polypeptide according to any one of claims 1 to 3 in the preparation of non-pharmaceutical antioxidants, food preservatives or chemical antioxidant auxiliaries.
5. An antioxidant composition, characterized in that, It contains the polypeptide according to any one of claims 1 to 3, as well as a carrier or excipient acceptable in the food science or chemical industry.