Shell nacre active peptides with cellular antioxidant activity and their applications

Abstract

The present invention discloses shell nacre active peptides with cellular antioxidant activity and applications thereof. The shell nacre antioxidant peptide GVPVYVAGY provided by the present invention has strong cellular antioxidant activity with an EC50 of 6.715 μg / mL. At the same time, it can increase the survival rate of cells in the AAPH damage group to 91.14±1.83%. In order to explore the mechanism of action, we conducted a visual analysis of the conjugate of the peptide and Keap1. The study found that GVPVYVAGY can generate hydrogen bonds and hydrophobic interactions with some binding sites in the Keap1-Kelch domain, proving that it can competitively bind to Keap1, thereby releasing Nrf2 and activating the Keap1-Nrf2-ARE pathway. In summary, GVPVYVAGY is expected to be used in the field of cosmetics, providing a theoretical basis for the high-value utilization of shell nacre.

Description

Technical Field

[0001] The present invention relates to the technical field of active peptides, in particular to shell nacre active peptides with cell antioxidant activity and applications thereof. Background Art

[0002] Reactive oxygen species (ROS), such as superoxide anion (•O 2− ), hydroxyl radicals (•OH), and hydrogen peroxide (H2O2) are highly reactive molecules formed endogenously (physiologically) or exogenously (non-physiologically) during aerobic biological oxidation. In this state, reactive oxygen species also play certain functions, such as acting as growth factors and intercellular signaling regulators. Under normal circumstances, due to the action of the body's antioxidant defense system, the production and elimination of ROS are in a relatively balanced state. However, with the ever-accelerating pace of life, unhealthy daily lifestyles, anxiety, excessive mental stress, and malnutrition can all lead to the massive production of ROS, resulting in excessive accumulation of ROS and triggering oxidative stress in the body. Oxidative stress is a major risk factor for a series of chronic diseases. Therefore, the development and research of antioxidants has always attracted much attention. Summary of the Invention

[0003] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a shell nacre active peptide with cellular antioxidant activity.

[0004] Another object of the present invention is to provide a method for preparing the above-mentioned shell nacre active peptide having cellular antioxidant activity.

[0005] Another object of the present invention is to provide the application of the above-mentioned shell nacre active peptide having cellular antioxidant activity.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A shell nacre active peptide with cellular antioxidant activity has the following amino acid sequence: Gly-Val-Pro-Val-Tyr-Val-Ala-Gly-Tyr (GVPVYVAGY).

[0008] Furthermore, the shell nacre active peptide is prepared by shell nacre protein hydrolysis, solid phase synthesis or genetic engineering technology.

[0009] A shell nacre protein hydrolyzate containing the shell nacre active peptide.

[0010] The invention relates to a solid phase synthesis product comprising the above shell nacre active peptide.

[0011] A nucleic acid encoding the above-mentioned shell nacre active peptide.

[0012] A biological material comprising the above nucleic acid, which is a recombinant DNA, an expression cassette, a transposon, a vector or a host cell.

[0013] A salt, which is a pharmaceutically acceptable salt of the shell nacre active peptide.

[0014] Furthermore, the pharmaceutically acceptable salt is acetate, hydrochloride, phosphate, sulfate, methanesulfonate, or toluenesulfonate.

[0015] A pharmaceutical composition comprises the shell nacre active peptide or salt and at least one pharmaceutically acceptable excipient.

[0016] Furthermore, the excipient is a carrier and / or a solvent.

[0017] Application of the above shell nacre active peptides, shell nacre protein hydrolysates, solid phase synthesis products, nucleic acids, biomaterials, salts or pharmaceutical compositions in the preparation of antioxidants.

[0018] The use of the above-mentioned shell nacre active peptides, shell nacre protein hydrolysates, solid-phase synthesis products, nucleic acids, biomaterials, salts or pharmaceutical compositions in the preparation of drugs for protecting cells from oxidative damage.

[0019] The present invention has the following advantages and effects compared to the prior art:

[0020] The antioxidant peptide GVPVYVAGY from the pearl layer of shells has strong cellular antioxidant activity, EC 50 The activity was 6.715 μg / mL, close to that of GSH (EC 50 =2.804 μg / mL). Furthermore, the shell nacre antioxidant peptide GVPVYVAGY (250 μg / mL) increased the survival rate of cells in the AAPH-injured group (81.75±0.61%) to 91.14±1.83%, a protective effect similar to that of GSH (91.17±0.07%). To explore the mechanism of action, we performed a visual analysis of the GVPVYVAGY-Keap1 conjugate. This study revealed that GVPVYVAGY forms hydrogen bonds and hydrophobic interactions with certain binding sites within the Keap1-Kelch domain, demonstrating that GVPVYVAGY competitively binds to Keap1, thereby releasing Nrf2 and activating the Keap1-Nrf2-ARE pathway. In summary, GVPVYVAGY has potential for application in cosmetics, providing a theoretical basis for the high-value utilization of shell nacre. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1is the effect of shell nacre antioxidant peptides on the antioxidant activity of cells; A is the effect of different concentrations of shell nacre antioxidant peptides on the survival rate of HepG2 cells, B and C are the CAA values ​​and EC values ​​of different concentrations of shell nacre antioxidant peptides, respectively. 50 Note: Different letters in A and C represent significant differences ( p <0.05); In B, for the comparison between different samples with the same concentration, different capital letters represent significant differences ( p <0.05), when comparing different concentrations of the same sample, different lowercase letters represent significant differences ( p <0.05).

[0022] Figure 2 is the protective effect of shell nacre antioxidant peptides on cell oxidative damage; A is the effect of different concentrations of AAPH on cell survival rate, and B is the effect of different concentrations of shell nacre antioxidant peptides on the viability of cells induced by AAPH oxidative damage; Note: Different letters in A represent significant differences ( p <0.05); In B, * indicates significant difference compared with the injury group ( p <0.05), compared with the same concentration of GSH, different letters represent significant differences ( p <0.05).

[0023] Figure 3 This is a visualization of the docking results of shell nacre antioxidants and Keap1. DETAILED DESCRIPTION

[0024] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0025] The medicine of the present invention also includes other pharmaceutically acceptable carriers or excipients.

[0026] As used herein, a "pharmaceutically acceptable" ingredient is a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic response), ie, at a reasonable benefit / risk ratio.

[0027] As used herein, a "pharmaceutically acceptable carrier" is a pharmaceutically acceptable solvent, suspending agent or excipient used to deliver the active peptide, drug, or pharmaceutical composition of the present invention to animals or humans. The carrier can be liquid or solid.

[0028] As mentioned herein, "pharmaceutically acceptable carriers or excipients" include, but are not limited to, preservatives, wetting agents, emulsifiers, dispersants, and the like.

[0029] The medicament of the present invention can be used as sole medicament, or can be used in conjunction with one or more other medicaments that have synergistic and / or synergistic effects with the substance of the present invention on physiological activity. Combination therapy can be achieved by administering the individual therapeutic components simultaneously, sequentially or separately.

[0030] The actual dosage level and route of administration of the active ingredient (i.e., active peptide) in the medicaments of the present invention can be varied to obtain an amount of active substance effective for achieving the desired therapeutic response in a specific patient. The dosage level should be selected based on the activity of the specific active substance, the route of administration, the severity of the condition being treated, and the condition and medical history of the patient being treated. However, it is common practice in the art to start the dosage of the active substance at a level below that required for the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0031] Example 1

[0032] 1. Synthesis of antioxidant peptides from nacre

[0033] Based on the sequence GVPVYVAGY, an antioxidant peptide from shell nacre was synthesized by solid phase synthesis. The purity of the synthesized peptide was ≥98% as determined by HPLC.

[0034] 2. Evaluation of the antioxidant activity of nacreous antioxidant peptides based on a HepG2 cell model

[0035] 2.1 Cell culture

[0036] Cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin antibiotic mixture at 37°C in a 5% CO2 incubator. The medium was changed every other day and the cells were passaged when they reached 80% to 90% confluence. HepG2 cells were used at passages 15 to 30.

[0037] 2.2 Analysis of the toxicity of nacreous antioxidant peptides on HepG2 cells

[0038] The effects of the samples on the viability of HepG2 cells were determined by MTT assay. 4Cells were inoculated into 96-well plates at a volume of 100 µL per well and cultured at 37°C and 5% CO2 for 24 h. 100 µL of culture medium containing different concentrations of the sample to be tested was added to the sample group, and fresh culture medium without sample was added to the control group, and culture was continued for 48 h. The culture medium was discarded, 100 µL of 0.5 mg / mL MTT solution was added, and the cells were incubated in the dark for 4 h. The MTT was discarded, and DMSO solution of the culture medium containing different concentrations of the sample to be tested was added, and the cells were shaken for 10 min to dissolve all the blue crystals. The absorbance was measured at 490 nm, and the cell viability was calculated according to the following formula: Cell viability / %=A t / A o *100%, where At is the absorbance value of the sample group and A0 is the absorbance value of the control group.

[0039] 2.3 Intracellular antioxidant activity assay

[0040] HepG2 cells were grown at a cell density of 6 × 10 4 Each well of a black 96-well plate was seeded with 100 µL of culture medium. After 24 hours of culture, the culture medium was discarded, and 50 µL of the test sample solution and 50 µL of 50 µM DCFH-DA working solution were added to the treated sample groups. 50 µL of sterile water and 50 µL of 50 µM DCFH-DA working solution were added to the control and blank groups. After incubation at 37°C for 1 hour, the culture medium was discarded, and the cells were washed with 100 µL of PBS. 100 µL of 600 µM 2,2'-Azobis (2-methylpropionamidine) dihydrochloride (AAPH) working solution (prepared in HBSS) was added to the treated sample groups and control groups, and 100 µL of Hank's Balanced Salt Solution (HBSS) was added to the blank group. The microplate reader was preheated to 37°C, and the fluorescence value was measured at an excitation wavelength of 485 nm and a measurement wavelength of 528 nm. The fluorescence value was measured every 5 minutes for a total of 1 hour. The fluorescence decay curve was obtained and the integral area under the fluorescence intensity-time curve (AUC) was used to calculate CAA and EC according to the following formula. 50 : CAA=[1-(AUC t -AUC o ) / (AUC c -AUC o )]*100%, where AUC t : Fluorescence curve area of ​​sample treatment group; AUC c : Fluorescence curve area of ​​the control group; AUC o: Fluorescence curve area of ​​the blank group. Trolox was used as a standard antioxidant to calculate the Trolox equivalent (TEAC μM TE / g lyophilized powder). GSH at the same concentration was used as a positive control.

[0041] 2.4 Protective effect of shell nacre antioxidant peptides on HepG2 cells oxidatively damaged by AAPH

[0042] HepG2 cells were grown at a cell density of 2 × 10 4 Cells were seeded into 96-well plates in a volume of 100 µL. After 48 hours of incubation, 50 µL of AAPH solution was added to each well to achieve final concentrations of 0.1, 0.4, 0.8, 1, 1.5, and 2 mM. After 24 hours of incubation, 100 µL of 0.5 mg / mL MTT solution was added and incubated in the dark for 4 hours. Cell viability was determined according to the procedure in 2.2. The AAPH concentration that achieved a cell viability of 80% was used as the oxidative damage concentration for HepG2 cells.

[0043] Plate cells according to the above procedure and culture for 24 hours. Discard the old culture medium, add fresh culture medium to the blank and damaged groups, and add 100 µL of culture medium containing various concentrations of the test sample to the sample group. After another 24 hours of culture, add 50 µL of fresh culture medium to the blank group and 50 µL of AAPH to the damaged and sample groups to achieve a cell viability of 80% in the damaged group. After 24 hours of culture, add 100 µL of 0.5 mg / mL MTT solution and incubate in the dark for 4 hours. Determine cell viability according to the procedure in 2.5.2 to investigate the protective effect of antioxidant peptides against oxidatively damaged HepG2 cells. Use the same concentration of GSH as a positive control.

[0044] 3. Molecular docking analysis

[0045] Molecular docking is a computer simulation method that focuses on studying molecular interactions and predicting their binding patterns and affinities. It can be used to investigate the structure-activity relationship, binding sites, and mechanisms of action of active substances. The Keap1-Nrf2 pathway is a key endogenous antioxidant signaling pathway. Activation of this pathway can reduce oxidative damage caused by exogenous stimuli. Keap1 is a key protein in the Keap1-Nrf2 pathway. Molecular docking simulates the binding of ligands to Keap1. The lower the binding energy, the more stable the complex. Therefore, the binding energy of Keap1 obtained through molecular docking can be used as a screening tool for antioxidant peptides. We performed molecular docking on the nacre antioxidant peptide GVPVYVAGY with Keap1.

[0046] 4. Statistical Analysis

[0047] Each experiment was conducted in parallel three times, and SPSS and WPS Excel software were used for data processing. P <0.05 is statistically significant. Graphics were generated using Origin 2021 and Grahpad Prism 8.

[0048] 5. Results:

[0049] (1) Determination of intracellular antioxidant activity (CAA)

[0050] The CAA assay uses a fluorescent probe (2',7'-dichlorofluorescein diacetate, or DCFH-DA) to monitor the inhibition of peroxyl radical-induced intracellular oxidation. 2',7'-Dichlorofluorescein diacetate (DCFH-DA) is a nonpolar, nonionic form of 2',7'-dichlorofluorescein (DCFH) that readily crosses cell membranes. Upon entry into cells, DCFH-DA is deacetylated by endogenous cellular esterases, leaving the more readily oxidizable DCFH form. 2,2'-Azo(2-amidopropane) dihydrochloride (AAPH) is added to the system as a free radical generator to generate peroxyl radicals. The presence of an antioxidant quenches these free radicals, preventing DCFH from being oxidized to the fluorescent 2',7'-dichlorofluorescein (DCF).

[0051] Effect of GVPVYVAGY on the viability of HepG2 cells Figure 1 As shown in Figure A, the cell survival rate was above 90% when treated with different shell nacre antioxidant peptides at concentrations of 0.1 to 1 mg / mL, indicating that GVPVYVAGY had no toxicity to HepG2 cells within this concentration range. Figure 1 As shown in Figure B, the CAA values ​​of all samples and GSH increased with increasing concentration, showing a concentration-dependent manner. Among them, the CAA values ​​corresponding to concentrations of 5, 25, and 50 μg / mL were not significantly different from those of GSH at the same concentration ( p >0.05), indicating that the antioxidant capacity at this time is equivalent to that of the positive control. GVPVYVAGY has the best cellular antioxidant activity, such as Figure 1 As shown in C, its EC 50 The activity was 6.715 μg / mL, close to that of GSH (EC 50 =2.804 μg / mL). The results of CAA showed that GVPVYVAGY has good antioxidant capacity.

[0052] (2) Protective effect of shell nacre antioxidant peptides on oxidative damage in HepG2 cells

[0053] Oxidative damage caused by AAPH can lead to the death of some HepG2 cells. Before exploring the protective effect of shell nacre antioxidant peptides on oxidative damage of HepG2 cells, the AAPH concentration for modeling was determined. The AAPH concentration corresponding to a survival rate of 80% was selected as the modeling concentration, such as Figure 2 As shown in Figure A, the cell survival rate decreased with the increase of AAPH concentration. When the AAPH concentration was 1.5 mM, the survival rate was 80%, so this concentration was selected as the AAPH concentration for the subsequent experimental model group.

[0054] Depend on Figure 2 As shown in Figure B, the cell survival rate was concentration-dependent with the peptide concentration. At low, medium, and high concentrations of GVPVYVAGY, the cell survival rate was significantly higher than that of the injury group ( p <0.05, and the protective effect had no significant difference with the positive control GSH ( p >0.05). GVPVYVAGY showed a certain ability to protect against cell damage.

[0055] (3) Using molecular docking technology to study the mechanism of action of antioxidant peptides from shell nacre

[0056] The 3D and 2D images of the docking results of GVPVYVAGY and Keap1 are shown in the figure below. Figure 3 The information of hydrogen bonds and hydrophobic interactions generated is shown in Table 1. The binding energy of GVPVYVAGY to Keap1 is -11.2. It generates a total of 15 hydrogen bonds with 12 binding sites of Keap1, namely ARG415, SER508, TYR572, GLY462, SER555, GLY464, VAL606, ASN387, SER383, GLY367, ASN414 and VAL463. Among them, ARG415, SER508, TYR572, GLY462 and SER555 are the binding sites in the reported Kelch domain. In addition, GVPVYVAGY also generates four hydrophobic interactions with Keap1 at the four binding sites ALA556, TYR334, TYR525 and ALA366. Three of the binding sites: ALA556, TYR334 and TYR525 are also from the binding sites in the Kelch domain.

[0057] In summary, GVPVYVAGY forms multiple hydrogen bonds and hydrophobic interactions with multiple binding sites of Keap1. These hydrogen bonds and hydrophobic interactions enable them to maintain a stable conformation near the active site of Keap1, which helps maintain the overall structure of the complex and prevents the ligand from easily detaching from the protein active site. At the same time, the binding sites for generating hydrogen bonds and hydrophobic interactions are mostly located in the Kelch domain of Keap1. It can occupy the Nrf2 binding site by binding to the Kelch domain of the Keap1 protein. Therefore, GVPVYVAGY is likely to competitively bind to Keap1 to release Nrf2, thereby exhibiting antioxidant activity in cells.

[0058] Table 1 Interaction results between shell nacre antioxidant peptides and Keap1

[0059]

[0060] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A shell nacre active peptide having cellular antioxidant activity, characterized in that: Its amino acid sequence: Gly-Val-Pro-Val-Tyr-Val-Ala-Gly-Tyr.

2. The shell nacre active peptide having cellular antioxidant activity according to claim 1, characterized in that: The shell nacre active peptide is prepared by shell nacre protein hydrolysis, solid phase synthesis or genetic engineering technology.

3. A shell nacre protein hydrolyzate comprising the shell nacre active peptide according to claim 1.

4. A solid phase synthesis product comprising the shell nacre active peptide according to claim 1.

5. A salt, characterized in that: The salt is a pharmaceutically acceptable salt of the shell nacre active peptide described in claim 1.

6. The salt according to claim 5, characterized in that: The pharmaceutically acceptable salt is acetate, hydrochloride, phosphate, sulfate, methanesulfonate, or toluenesulfonate.

7. A pharmaceutical composition, characterized in that: The invention comprises the shell nacre active peptide as claimed in claim 1 and at least one pharmaceutically acceptable excipient.

8. The pharmaceutical composition according to claim 7, characterized in that: The excipient is a solvent.

9. Use of the shell nacre active peptide according to claim 1, the shell nacre protein hydrolyzate according to claim 3, the solid phase synthesis product according to claim 4 or the salt according to any one of claims 5 to 6 in the preparation of antioxidant cosmetics.

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