A short-chain antioxidant peptide with antioxidant activity and its applications

Abstract

This invention discloses a short-chain peptide with antioxidant activity and its applications. Four short-chain polypeptides, P1-P4, were constructed, among which the P3 peptide chain Tyr-Trp-Cys-Cys-Arg (YWCCR) exhibits the most outstanding antioxidant activity and excellent biocompatibility. This invention demonstrates, through cell damage and protection models, as well as UV and D-gal-induced mouse aging models, that this peptide chain can significantly enhance the activity of SOD and CAT in cells and in the liver and kidneys, and inhibit the overexpression of inflammatory factors IL-6 and TNF-α. Simultaneously, Western blot analysis shows that it can regulate redox homeostasis by activating signaling pathways such as AMPK, inhibiting the production of reactive oxygen species and pro-inflammatory factors, and effectively repairing organ and tissue damage. The antioxidant peptide of this invention has a simple preparation process and good biocompatibility, showing broad application prospects in the preparation of anti-aging drugs, functional foods, or liver and kidney protection products.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a novel short-chain antioxidant peptide and its role in anti-aging and organ protection. Background Technology

[0002] In recent years, with the accelerating aging of the global population, aging and the resulting chronic physiological decline have become core issues urgently needing to be addressed in the biomedical field. Research has shown that oxidative stress is a key factor leading to aging and various chronic diseases. Antioxidant peptides (AOPs), as potential alternatives to natural antioxidants, have attracted considerable attention. Antioxidant peptides possess good biocompatibility and can actively repair oxidative damage and effectively delay the aging process by scavenging free radicals and regulating intracellular antioxidant defense systems.

[0003] Currently developed antioxidant peptides often have complex amino acid sequences, resulting in high synthesis costs and limited stability. Short peptides with simple sequences exhibit varying antioxidant properties, making it difficult to achieve multiple benefits such as systemic anti-aging and localized skin protection. There are currently no antioxidant peptides that combine a short amino acid sequence, strong biological activity, and clear signaling pathway regulation, posing challenges to the development of anti-aging drugs and functional cosmetics. Summary of the Invention

[0004] The purpose of this invention is to provide a short-chain antioxidant peptide with antioxidant activity and its application, which addresses the shortcomings of existing technologies. This antioxidant peptide has a short amino acid sequence, strong biological activity, and a clear signaling pathway regulatory effect, and can be applied to organ protection and anti-sunburn treatment.

[0005] To achieve the purpose of this invention, the following solution is adopted: A short-chain antioxidant peptide with antioxidant activity, wherein the amino acid sequence of the short-chain antioxidant peptide is one of the following four amino acid sequences: SEQ ID NO 1: Tyr-Glu-Cys-Cys-Arg (YECCR), SEQ ID NO 2: Tyr-Glu-Cys-Cys-Glu (YECCE), SEQ ID NO 3: Tyr-Trp-Cys-Cys-Arg (YWCCR), SEQ ID NO 4: Tyr-Trp-Cys-Cys-Glu (YWCCE).

[0006] The preferred amino acid sequence of the short-chain antioxidant peptide is Tyr-Trp-Cys-Cys-Arg (YWCCR).

[0007] Linear peptide chains, cyclic peptide chains, or pharmaceutically acceptable salts thereof, or derivatives formed by chemical modification, comprising the short-chain antioxidant peptides.

[0008] The application of the aforementioned short-chain antioxidant peptides in the preparation of products with antioxidant, anti-aging, or skin protection properties.

[0009] The use of the linear peptide chain, cyclic peptide chain, or pharmaceutically acceptable salt thereof, or derivative thereof formed by chemical modification, in the preparation of products with antioxidant, anti-aging, or skin-protecting properties.

[0010] The product is used to prevent or treat damage caused by oxidative stress.

[0011] The application described herein refers to damage selected from D-galactose (D-gal)-induced systemic oxidative damage and ultraviolet (UVB)-induced tissue damage.

[0012] The aforementioned application, specifically the systemic oxidative damage, includes D-galactose-induced liver and kidney pathological damage.

[0013] In the aforementioned application, the ultraviolet-induced tissue damage specifically refers to photoaging damage of the skin.

[0014] In the aforementioned application, the short-chain antioxidant peptide activates the AMPK signaling pathway in the body, thereby upregulating the expression of downstream antioxidant enzymes and inhibiting the release of inflammatory factors.

[0015] Compared with the prior art, the advantages of the present invention are: (1) The antioxidant peptides prepared by the present invention have short amino acid sequences, simple preparation process, low cost and good reproducibility.

[0016] (2) The antioxidant peptides prepared by the present invention not only have a significant protective effect on the liver and kidneys in vivo, but also have a repairing effect on skin collagen damage.

[0017] (3) The antioxidant peptides prepared by the present invention can precisely act on the AMPK target and regulate the downstream antioxidant pathway.

[0018] (4) The antioxidant peptides prepared by this invention have excellent stability and high biosafety. Attached Figure Description

[0019] Figure 1 The structures and MALDI-TOF-MS images of the antioxidant peptides obtained in this invention are shown in (a) for YECCR peptide chain, (b) for YECCE peptide chain, (c) for YWCCR peptide chain, and (d) for YWCCE peptide chain.

[0020] Figure 2(a) is a graph showing the DPPH antioxidant properties of the antioxidant peptides obtained in this invention. Figure 2 (b) is a graph showing the antioxidant properties of ABTS. Figure 2 (c) is a graph showing the antioxidant properties of FRAP.

[0021] Figure 3 (a) is a pH stability test diagram of the antioxidant peptides obtained in this invention. Figure 3 (b) is the temperature stability test chart. Figure 3 (c) is the UV stability test graph.

[0022] Figure 4 This is a graph showing the cell viability of the antioxidant peptides obtained in this invention.

[0023] Figure 5 This is a cell liveness / death diagram of the antioxidant peptides obtained in this invention.

[0024] Figure 6 This is the hemolysis data of the antioxidant peptides obtained in this invention.

[0025] Figure 7 (a) is a graph showing the effect of the antioxidant peptides obtained in this invention on the intracellular antioxidant enzyme SOD. Figure 7 (b) is a diagram analyzing the effects of the antioxidant enzyme CAT.

[0026] Figure 8 (a) is a graph showing the effect of the antioxidant peptides obtained in this invention on the intracellular inflammatory factor TNF-α. Figure 8 (b) is a diagram analyzing the role of the inflammatory factor IL-6.

[0027] Figure 9 This is a diagram illustrating the protective effect of the antioxidant peptides obtained in this invention against sunburn in mice.

[0028] Figure 10 (a) is a graph showing the effect of the antioxidant peptides obtained in this invention on the liver antioxidant enzyme CAT. Figure 10 (b) is a diagram analyzing the role of the liver antioxidant enzyme SOD. Figure 10 (c) is a diagram analyzing the role of the inflammatory factor IL-6 in the liver. Figure 10 (d) is a diagram analyzing the effects of TNF-α in the liver.

[0029] Figure 11 (a) is a graph showing the effect of the antioxidant peptides obtained in this invention on the renal antioxidant enzyme CAT. Figure 11 (b) is a diagram analyzing the role of the renal antioxidant enzyme SOD. Figure 11 (c) is a diagram analyzing the role of the inflammatory factor IL-6 in the kidneys. Figure 11 (d) is a diagram analyzing the effects of TNF-α in the kidneys.

[0030] Figure 12 This is a graph showing the effect of the antioxidant peptides obtained in this invention on the AMPK / SIRT1 / Nrf2 signaling pathway. Detailed Implementation

[0031] The present invention will be described in detail below with reference to specific embodiments.

[0032] Example 1: Preparation of antioxidant peptides The solid-phase synthesis of peptides P1-P4 using the Fmoc method follows these steps: Dichlorotriphenylmethyl chloride resin is swollen in DCM for 0.5-3 h. The first amino acid of each of the four antioxidant peptides P1-P4, all tyrosine (Tyr, Y), is added, and the reaction is carried out in DMF solution containing DIEA for 2 h. After the reaction, unreacted groups are blocked with methanol, and the Fmoc protecting groups are removed with 20% piperidine solution. Subsequently, following the order of the four antioxidant peptide amino acids, glutamic acid (Glu, E), cysteine ​​(Cys, C), cysteine ​​(Cys, C), and arginine (Arg, R) were added sequentially to YECCR; glutamic acid (Glu, E), cysteine ​​(Cys, C), cysteine ​​(Cys, C), and glutamic acid (Glu, E) were added sequentially to YECCE; tryptophan (Trp, W), cysteine ​​(Cys, C), cysteine ​​(Cys, C), and arginine (Arg, R) were added sequentially to YWCCR; and tryptophan (Trp, W), cysteine ​​(Cys, C), cysteine ​​(Cys, C), and glutamic acid (Glu, E) were added sequentially to YWCCE. PyBoP, HoBt, and DIEA were added to the DMF simultaneously with each amino acid addition, and the reaction was carried out for 2 hours each time. After amino acid coupling was completed, the peptides were cleaved using a cleavage reagent (trifluoroacetic acid:water:triisopropylsilane = 95:1.25:1.25, v / v / v), precipitated in ice-cold anhydrous ether, dissolved in water, and then freeze-dried.

[0033] The results are as follows Figure 1 The results showed that the synthesized peptides were homogeneous, and the [M+H]⁺ molecular weight ion peaks of P1-P4 detected by MALDI-TOF-MS were 744.84 Da, 717.77 Da, 810.94 Da, and 774.84 Da, respectively. The peptides were of high purity and showed only a single chromatographic peak, making them suitable for subsequent experiments.

[0034] Example 2: In vitro antioxidant activity of antioxidant peptides The antioxidant peptides prepared in this embodiment were tested for antioxidant activity using DPPH, ABTS, and FRAP methods. (1) DPPH method: DPPH working solution (absorbance = 0.89) was prepared with ethanol, and peptide solutions of different concentrations (0.01 mg / mL - 0.1 mg / mL) were prepared. Then, the working solution and the sample were mixed at a volume ratio of 1:2 and reacted in the dark for 30 minutes. The absorbance value was measured at 517 nm. (2) ABTS method: ABTS diammonium salt stock solution and K2S2O8 stock solution were mixed at a ratio of 1:1 and reacted in the dark for 12 hours to obtain ABTS working solution. Then, the peptide solution was mixed at a ratio of 4:1 and reacted for 10 minutes. The absorbance value was measured at 734 nm. (3) FRAP method: TPTZ (10 mM), FeCl3 (20 mM), and sodium acetate (0.3 M) were mixed in a ratio of 1:1:10 (v / v) to obtain a working solution. This solution was then mixed with the sample at a ratio of 24:1 and reacted at 37°C for 5 minutes. The absorbance was measured at 593 nm. The results are as follows: Figure 2 The results show that the antioxidant peptide (P3) prepared in this embodiment has the best antioxidant activity.

[0035] Example 3: Stability of antioxidant peptides The antioxidant peptides prepared in this embodiment were prepared at a concentration of 0.5 mg / mL and subjected to thermal stability tests (4 ℃, 25 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃), pH stability tests (2, 5, 7, 9, 12), and UV stability tests (exposure to UV light for 5, 10, 30, 60, 120 minutes). Their free radical scavenging ability was determined using the ABTS method to evaluate the stability of the antioxidant peptides. The results are as follows: Figure 3 The results showed that all four peptide chains exhibited superior stability, with the P3 peptide chain showing the best stability.

[0036] Example 4: Cytotoxicity of Antioxidant Peptides The antioxidant peptides prepared in this embodiment were tested for cytotoxicity using the MTT assay. The antioxidant peptide solution was incubated in cells for 24, 48, and 72 hours, respectively. Cell viability was measured using an enzyme-linked immunosorbent assay (ELISA) reader. The cells were then stained with AM / PI and observed for cell viability under an inverted fluorescence microscope.

[0037] The results are as follows Figure 4 and 5 As shown, Figure 4 A represents the cytotoxicity test. Figure 4 B represents a three-day cytotoxicity test; cell survival was good, and [the cell structure was] combined with [the cell toxicity]. Figure 5 The staining results showed that the cells in the drug-treated group had good morphology. Therefore, the antioxidant peptide prepared in this embodiment has no cytotoxicity and has a high cell survival rate, indicating that it has good biocompatibility.

[0038] Example 5: Hemolytic activity of antioxidant peptides Fresh blood was repeatedly washed with PBS buffer to remove plasma and leukocytes until the supernatant was clear. Then, red blood cells were mixed with equal volumes of four peptide chains and incubated at 37 °C. After centrifugation at 3000 rpm for 3 min, the supernatant was collected and its absorbance was measured at 540 nm. Ultrapure water was used as a complete hemolysis control to assess the degree of hemolysis induced by the peptides. The centrifuged cell pellet was fixed with 2.5% glutaraldehyde and dehydrated by a gradient ethanol exchange. Finally, the effect of the peptides on the blood cell membrane structure was visually analyzed using scanning electron microscopy (SEM).

[0039] The results are as follows Figure 6 As shown, the hemolysis rate of all four peptide chains was below 5%, and the blood cell structure remained intact under SEM, indicating that the antioxidant peptides are safe against hemolysis.

[0040] Example 6: Experimental Model of Intracellular Damage and Protection by Antioxidant Peptides HepG2 cells were seeded in 6-well plates and divided into blank group, control group, glutathione group, and antioxidant peptide protection group. After treatment with the drug for 24 h, H2O2 was added and incubated for 4 h. Then the cells were lysed and the supernatant was collected. The cells were detected by CAT, SOD detection kit and ELISA respectively.

[0041] The results are as follows Figure 7 Figure 8 As shown, P3 peptide significantly increased intracellular SOD and CAT activity and downregulated the overexpression of pro-inflammatory factors such as IL-6 and TNF-α, even outperforming the traditional antioxidant peptide GSH.

[0042] Example 7: Experimental model of sunburn protection in mammals by antioxidant peptides An ultraviolet (UVB)-induced sunburn model was established in mice. The day before the experiment, the hair on the back of the mice (approximately 2 × 3 cm) was thoroughly shaved using an electric shaver. The mice were treated with 0.3 ml of sample before the experiment, and then irradiated in a self-made UV chamber. Results are as follows: Figure 9 As shown, the skin of mice treated with the antioxidant peptide prepared in this embodiment was relatively smooth and intact, and no obvious erythema or inflammatory damage was observed, indicating that the antioxidant peptide can effectively alleviate skin oxidative damage.

[0043] Example 8: Experiment on the protective effect of antioxidant peptides against galactose aging in mammals A galactose (D-gal)-induced aging mouse model was established. Mice were randomly divided into 6 groups, with 3 animals in each group. The model group and other experimental groups were induced by intraperitoneal injection of 150 mg / kg body weight of D-gal solution, while the normal group received an equal volume of physiological saline daily. After 14 days, mice in groups P3-1, P3-2, and P3-3 were injected with 150, 300, and 450 mg / kg of the treatment solution, respectively, for 16 days. Mice in the normal and model groups were orally administered an equal volume of physiological saline. Twenty-four hours after the last administration, mice were anesthetized and sacrificed. Blood was centrifuged, and serum was collected. Liver and kidney samples were weighed separately. Some samples were immediately stored in 4% paraformaldehyde, then embedded in paraffin and subjected to histopathological staining analysis. The remaining samples were quantitatively analyzed for superoxide dismutase (SOD), catalase (CAT), and inflammatory factors (IL-6, IL-α). The experimental results are as follows: Figure 10 and 11 As shown, this antioxidant peptide increased the activity of SOD and CAT in the liver and kidneys, and inhibited the overexpression of inflammatory factors IL-6 and TNF-α.

[0044] Example 9: Western blot experiment of antioxidant peptides After lysing the liver and kidneys with protease inhibitors, protein extracts were obtained. The supernatant was collected by centrifugation, and protein concentration was determined to ensure equal protein levels in the samples. The protein samples were denatured by heating in a buffer at 95°C, and proteins were separated by SDS-PAGE gel electrophoresis. After electrophoresis, the proteins were transferred to a PVDF membrane. The proteins in the gel were transferred at 100 V for 1 h using transfer buffer to transfer them to a PVDF or nitrocellulose membrane. After transfer, the membrane was blocked with a buffer containing 5% skim milk powder to avoid non-specific antibody binding. The primary antibody was diluted 1:1000 and incubated overnight at 4°C. The membrane was washed three times with TBST buffer, and the secondary antibody was diluted 1:10000 and incubated at room temperature for 1 h. The membrane was washed again with TBST. Chemiluminescence detection was performed using an ECL luminescent reagent, and protein bands were recorded by X-ray film or an imaging system. Results are as follows: Figure 12 As shown, antioxidant peptides can exert their antioxidant effects by precisely activating the AMPK signaling pathway, thereby regulating the Nrf2 and SIRT1 signaling pathways.

[0045] It should be understood that those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A short-chain antioxidant peptide with antioxidant activity, characterized in that, The amino acid sequence of the short-chain antioxidant peptide is one of the following four amino acid sequences: SEQ ID NO 1: Tyr-Glu-Cys-Cys-Arg (YECCR), SEQ ID NO 2: Tyr-Glu-Cys-Cys-Glu (YECCE), SEQ ID NO 3: Tyr-Trp-Cys-Cys-Arg (YWCCR), SEQ ID NO 4: Tyr-Trp-Cys-Cys-Glu (YWCCE).

2. The short-chain antioxidant peptide according to claim 1, characterized in that, The preferred amino acid sequence of the short-chain antioxidant peptide is Tyr-Trp-Cys-Cys-Arg (YWCCR).

3. A linear peptide chain, a cyclic peptide chain, or a pharmaceutically acceptable salt thereof, or a derivative thereof, comprising the short-chain antioxidant peptide of claim 1 or 2, or formed by chemical modification.

4. The use of the short-chain antioxidant peptide according to claim 1 or 2 in the preparation of products with antioxidant, anti-aging or skin protection properties.

5. The use of the linear peptide chain, cyclic peptide chain, or pharmaceutically acceptable salt thereof, or derivative thereof, as described in claim 3, in the preparation of products having antioxidant, anti-aging, or skin-protecting properties.

6. The application according to claim 4 or 5, characterized in that, The product is used to prevent or treat damage caused by oxidative stress.

7. The application according to claim 6, characterized in that, The damage was selected from D-galactose (D-gal)-induced systemic oxidative damage and ultraviolet (UVB)-induced tissue damage.

8. The application according to claim 7, characterized in that, The systemic oxidative damage specifically includes liver pathological damage and kidney pathological damage induced by D-galactose.

9. The application according to claim 7, characterized in that, The ultraviolet-induced tissue damage specifically refers to photoaging damage of the skin.

10. The application according to claim 6, characterized in that, The short-chain antioxidant peptides activate the AMPK signaling pathway in the body, thereby upregulating the expression of downstream antioxidant enzymes and inhibiting the release of inflammatory factors.

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