Shell nacre active peptide with cell antioxidant activity and application of shell nacre active peptide

Abstract

The invention discloses a shell nacre active peptide with cell antioxidant activity and application thereof. The shell nacre antioxidant peptide GVPVYVAGY provided by the invention has relatively strong cell antioxidant activity, and EC50 is 6.715 [mu] g / mL. Meanwhile, the survival rate of AAPH damaged group cells can be increased to 91.14 + / -1.83%. In order to explore the action mechanism of the GVPVYVAGY peptide, a conjugate of peptide and Keap1 is subjected to visual analysis, and research finds that the GVPVYVAGY can generate hydrogen bonds and hydrophobic interaction with part of binding sites in a Keap1-Kelch structural domain, and proves that the GVPVYVAGY peptide can be competitively bound with Keap1, so that Nrf2 is released, and a Keap1-Nrf2-ARE pathway is activated. In conclusion, the GVPVYVAGY is expected to be applied to the field of food or cosmetics, and a theoretical basis is provided for high-value utilization of the shell pearl layer.

Description

Technical Field

[0001] The 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), and are produced during aerobic biological oxidation. In this state, reactive oxygen species also play certain functions, such as acting as growth factors and intercellular signal regulators. Under normal circumstances, due to the role of the body's antioxidant defense system, the production and elimination of ROS are in a relatively balanced state. However, with the continuous acceleration of the pace of life, unhealthy lifestyles, anxiety, excessive mental stress and malnutrition in daily life can lead to the massive production of ROS, resulting in excessive accumulation of ROS and triggering oxidative stress in the body. Oxidative stress is the main 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 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 cell 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: A shell nacre active peptide with cellular antioxidant activity, which has the following amino acid sequence: Gly-Val-Pro-Val-Tyr-Val-Ala-Gly-Tyr (GVPVYVAGY).

[0007] Furthermore, the shell nacre active peptide is prepared by shell nacre protein hydrolysis, solid phase synthesis or genetic engineering technology.

[0008] A shell nacre protein hydrolyzate comprising the shell nacre active peptide.

[0009] A solid phase synthesis product comprising the above shell nacre active peptide.

[0010] A nucleic acid encoding the above-mentioned shell nacre active peptide.

[0011] A biological material, comprising the above-mentioned nucleic acid, is a recombinant DNA, an expression cassette, a transposon, a vector or a host cell.

[0012] A salt, which is a pharmaceutically acceptable salt of the shell nacre active peptide.

[0013] Furthermore, the pharmaceutically acceptable salt is acetate, hydrochloride, phosphate, sulfate, methanesulfonate, or toluenesulfonate.

[0014] A pharmaceutical composition comprises the shell nacre active peptide or salt and at least one pharmaceutically acceptable excipient.

[0015] Furthermore, the excipient is a carrier and / or a solvent.

[0016] 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.

[0017] The use of the above-mentioned shell nacre active peptides, shell nacre protein hydrolysate, solid phase synthesis product, nucleic acid, biomaterial, salt or pharmaceutical composition in the preparation of cell oxidative damage protection drugs.

[0018] Compared with the prior art, the present invention has the following advantages and effects: 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). At the same time, the shell nacre antioxidant peptide (250 μg / mL) GVPVYVAGY can increase the survival rate of AAPH-damaged cells (81.75±0.61%) to 91.14±1.83%, and the protective effect is close to that of GSH (91.17±0.07%). In order to explore the mechanism of action, we visualized the conjugate of GVPVYVAGY 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 GVPVYVAGY can release Nrf2 and activate the Keap1-Nrf2-ARE pathway by competitively binding to Keap1. In summary, GVPVYVAGY is expected to be used in the field of food or cosmetics, providing a theoretical basis for the high-value utilization of shell nacre. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 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), for comparisons between different concentrations of the same sample, different lowercase letters represent significant differences ( p <0.05).

[0020] 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 AAPH-induced oxidative damage cells; 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).

[0021] Figure 3 This is a visualization of the docking results of shell nacre antioxidants and Keap1. DETAILED DESCRIPTION

[0022] 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 used to limit the present invention.

[0023] The medicine of the present invention also includes other pharmaceutically acceptable carriers or excipients.

[0024] 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) at a reasonable benefit / risk ratio).

[0025] As described herein, "pharmaceutically acceptable carrier" is a pharmaceutically or food-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.

[0026] As described herein, "pharmaceutically acceptable carriers or excipients" include, but are not limited to, preservatives, wetting agents, emulsifiers, dispersants and the like.

[0027] The drug of the present invention can be used as the sole drug, or can be used in combination with one or more other drugs 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.

[0028] The actual dosage level and mode of administration of the active ingredient (i.e., active peptide) in the medicament of the present invention can be varied so that the amount of active substance obtained can effectively obtain the desired therapeutic response for a specific patient. The dosage level must 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 to be treated. However, it is practiced in the art to start the dosage of the active substance from a level lower than that required to obtain the desired therapeutic effect and gradually increase the dosage until the desired effect is obtained.

[0029] Example 1 1. Synthesis of antioxidant peptides from nacre Based on the sequence GVPVYVAGY, the shell nacre antioxidant peptide was synthesized by solid phase synthesis. The purity of the synthetic peptide was ≥98% as determined by HPLC.

[0030] 2. Evaluation of the antioxidant activity of nacreous antioxidant peptides based on HepG2 cell model 2.1 Cell culture The cells were cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin antibiotic mixture in a 37°C, 5% CO2 incubator. The medium was changed every other day and the cells were passaged when they grew to 80% to 90%. The HepG2 cell generations used were 15 to 30.

[0031] 2.2 Analysis of the toxicity of pearl nacre antioxidant peptides on HepG2 cells The MTT method was used to determine the effect of the samples on the viability of HepG2 cells. 4 100 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 incubated for 4 h in the dark. MTT was discarded, and DMSO solution of the culture medium containing different concentrations of the sample to be tested was added, and the blue crystals were completely dissolved by shaking for 10 min. The absorbance was measured at 490 nm, and the cell survival rate was calculated according to the following formula: Cell survival rate / %=A t / A o*100%, where At is the absorbance of the sample group and A0 is the absorbance of the control group.

[0032] 2.3 Intracellular antioxidant activity assay HepG2 cells were grown at a cell density of 6 × 10 4 100 µL of 50 µM DCFH-DA working solution were added to the sample treatment group, and 50 µL of sterile water and 50 µL of 50 µM DCFH-DA working solution were added to the control group and blank group. After incubation at 37℃ for 1 h, 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) dihydrochlori (AAPH) working solution (prepared with HBSS) was added to the sample treatment group and control group, 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 ​​the sample treatment group; AUC c : Fluorescence curve area of ​​the control group; AUC o : Fluorescence curve area of ​​blank group. At the same time, Trolox was used as standard antioxidant to calculate Trolox equivalent (TEAC μM TE / g lyophilized powder). GSH of the same concentration was used as positive control.

[0033] 2.4 Protective effect of pearl nacre antioxidant peptides on AAPH-induced oxidative damage in HepG2 cells HepG2 cells were grown at a cell density of 2 × 10 4100 μL of AAPH solution was added to each well of a 96-well plate after culturing for 48 h, and the final concentration of each well was 0.1, 0.4, 0.8, 1, 1.5, and 2 mM. After culturing for 24 h, 100 μL of 0.5 mg / mL MTT solution was added and incubated for 4 h in the dark. The cell viability was determined according to the steps in 2.2. The AAPH concentration used when the cell viability was 80% was selected as the HepG2 oxidative damage concentration.

[0034] According to the above steps, the plates were plated and cultured for 24 hours, the old culture medium was discarded, fresh culture medium was added to the blank group and the damaged group, and 100 μL of culture medium containing different concentrations of the sample to be tested was added to the sample group. After further culture for 24 hours, 50 μL of fresh culture medium was added to the blank group, and 50 μL of AAPH was added to the damaged group and the sample group. The cell survival rate of the damaged group was 80%, and 100 μL of 0.5 mg / mL MTT solution was added for 24 hours, and the cells were incubated in the dark for 4 hours. The cell survival rate was determined according to the steps in 2.5.2 to explore the protective effect of antioxidant peptides on oxidatively damaged HepG2 cells. The same concentration of GSH was used as a positive control.

[0035] 3. Molecular docking analysis Molecular docking is a computer simulation method that focuses on studying the interactions between molecules and predicting their binding modes and affinities. It can be used to study the structure-activity relationship, binding sites and mechanisms of action of active substances. The Keap1-Nrf2 pathway is a key signaling pathway for endogenous antioxidants. The activation of this pathway can reduce oxidative damage caused by exogenous stimuli. Keap1 is a key protein in the Keap1-Nrf2 pathway. Molecular docking is used to simulate the binding of ligands to Keap1. The lower the binding energy, the more stable the complex. Therefore, the binding energy obtained based on molecular docking with Keap 1 can be used as one of the screening methods for antioxidant peptides. We molecularly docked the shell nacre antioxidant peptide GVPVYVAGY with Keap 1.

[0036] 4. Statistical Analysis Each experiment was conducted in parallel three times, and SPSS and WPS Excel software were used for data processing. P <0.05 is statistically different, Origin 2021 and Grahpad Prism 8 graphics.

[0037] 5. Results: (1) Determination of intracellular antioxidant activity (CAA) 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 non-polar and non-ionic form of 2',7'-dichlorofluorescein (DCFH) that can easily cross the cell membrane. When DCFH-DA enters the cell, it is deacetylated by endogenous cellular esterases and leaves behind the more easily oxidized DCFH form. At this point, 2,2'-azo(2-amidopropane) dihydrochloride (AAPH) is added to the system as a free radical generator to generate peroxyl radicals, and when antioxidants are present, they can quench the free radicals, thereby preventing DCFH from being oxidized to fluorescent 2',7'-dichlorofluorescein (DCF).

[0038] Effect of GVPVYVAGY on the survival rate 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 the increase of 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. 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 had good antioxidant capacity.

[0039] (2) Protective effect of shell nacre antioxidant peptides on oxidative damage in HepG2 cells Oxidative damage of AAPH can cause the death of some HepG2 cells. Before exploring the protective effect of shell nacre antioxidant peptides on oxidative damage of HepG2 cells, the AAPH modeling concentration was determined first. The AAPH concentration corresponding to a survival rate of 80% was selected as the modeling concentration, such as Figure 2 As shown in 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.

[0040] Depend on Figure 2As 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 was not significantly different from that of the positive control GSH ( p >0.05). GVPVYVAGY showed a certain ability to protect against cell damage.

[0041] (3) Using molecular docking technology to study the mechanism of action of antioxidant peptides from nacre The 3D and 2D images of the docking results of GVPVYVAGY and Keap1 are shown in Figure 3 The information of hydrogen bonds and hydrophobic interactions generated is shown in Table 1. The binding energy of GVPVYVAGY with Keap1 is -11.2. It generates a total of 15 hydrogen bonds with the 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 reported binding sites in the Kelch domain. In addition, GVPVYVAGY also generates four hydrophobic interactions with Keap1 at the four binding sites of ALA556, TYR334, TYR525 and ALA366. Three of the binding sites: ALA556, TYR334 and TYR525 are also from the binding sites in the Kelch domain.

[0042] In summary, GVPVYVAGY forms multiple hydrogen bonds and hydrophobic interactions with multiple binding sites of Keap1, which enable them to maintain a stable conformation near the active site of Keap1, which helps to maintain the overall structure of the complex and prevent the ligand from easily detaching from the active site of the protein. At the same time, most of the binding sites that generate hydrogen bonds and hydrophobic interactions are located in the Kelch domain of Keap1, which can occupy the connection site of Nrf2 by binding to the Kelch domain of Keap1 protein. Therefore, GVPVYVAGY is likely to competitively bind to Keap1 to release Nrf2, thereby exhibiting antioxidant activity in cells.

[0043] Table 1 The results of the interaction between shell nacre antioxidant peptides and Keap1

[0044] 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 equivalent replacement methods and are included in the protection scope of the present invention.

Claims

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

2. The shell nacre active peptide with 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 as claimed in 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 or salt 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 carrier and / or a solvent.

9. Use of the shell nacre active peptide described in claim 1, the shell nacre protein hydrolyzate described in claim 3, the solid phase synthesis product described in claim 4, the salt described in any one of claims 5-6 or the pharmaceutical composition described in any one of claims 7-8 in the preparation of antioxidants.

10. Use of the shell nacre active peptide described in claim 1, the shell nacre protein hydrolyzate described in claim 3, the solid phase synthesis product described in claim 4, the salt described in any one of claims 5-6 or the pharmaceutical composition described in any one of claims 7-8 in the preparation of a drug for protecting cells from oxidative damage.

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