Shell nacre layer source antioxidant peptide as well as preparation method and application thereof

Abstract

The invention discloses a shell nacre layer source antioxidant peptide as well as a preparation method and application thereof. The shell nacre antioxidant peptides FYFPKYGR and GYFPSYY provided by the invention both have relatively strong cell antioxidant activity. In order to explore the action mechanism of the peptide, a conjugate of the peptide and Keap1 is subjected to visual analysis and research to find that the conjugate can generate hydrogen bonds and hydrophobic interaction with part of binding sites in a Keap1-Kelch structural domain, and the conjugate is proved to be capable of competitively binding with Keap1 so as to release Nrf2 and activate a Keap1-Nrf2-ARE pathway. In conclusion, the shell nacre antioxidant peptide 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 nacre.

Description

Technical Field

[0001] The invention relates to the technical field of active peptides, and in particular to shell nacre-derived antioxidant peptides and a preparation method and application thereof. Background Art

[0002] The pearl layer of shells is one of the components of shells. The structure of shells from the outside to the inside is the stratum corneum, prismatic layer and nacre. The nacre is formed by the secretion of a large amount of nacre by the epithelial cells of the mantle, which is the same as the formation of pearls. Therefore, the nacre is homologous to pearls and its composition is similar to that of pearls. In addition to basic components such as calcium carbonate and organic matter, it also contains trace elements, amino acids, vitamins, polysaccharides, proteins and other ingredients. Therefore, the pearl layer of shells is expected to be used as a functional substitute for pearls in health products and skin care products. While reducing product costs, it can also achieve high-value utilization of discarded shells and prevent resource waste. In recent years, the research on shell nacre extracts mainly includes: anti-aging, osteogenesis and improvement of LPS-induced depression and anxiety, etc. There are few studies on the antioxidant activity of shell nacre extracts, so it is of research significance to develop it into antioxidant active peptides. Summary of the invention

[0003] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide an antioxidant peptide derived from the nacre layer of shells.

[0004] Another object of the present invention is to provide a method for preparing the above-mentioned shell nacre-derived antioxidant peptides.

[0005] Another object of the present invention is to provide the application of the above-mentioned shell nacre-derived antioxidant peptides.

[0006] The purpose of the present invention is achieved through the following technical solutions: An antioxidant peptide derived from shell nacre, having any of the following amino acid sequences: (1) Phe-Tyr-Phe-Pro-Lys-Tyr-Gly-Arg (FYFPKYGR); (2) Gly-Tyr-Phe-Pro-Ser-Tyr-Tyr (GYFPSYY).

[0007] Furthermore, the shell nacre-derived antioxidant peptides are prepared by shell nacre protein hydrolysis, solid phase synthesis or genetic engineering technology.

[0008] A shell nacre protein hydrolyzate comprising the above-mentioned shell nacre-derived antioxidant peptides.

[0009] A solid phase synthesis product comprising the above-mentioned shell nacre-derived antioxidant peptide.

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

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

[0014] A pharmaceutical composition comprises the shell nacre-derived antioxidant 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-derived antioxidant 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-derived antioxidant 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.

[0018] Compared with the prior art, the present invention has the following advantages and effects: The antioxidant peptides FYFPKYGR and GYFPSYY from the pearl layer of shells have strong cellular antioxidant activity, EC 50 The results showed that the two antioxidant peptides from shell nacre (250 μg / mL) could increase the survival rate of cells in the AAPH injury group (81.75±0.61%) to 85.71±1.56%~86.27±0.60%, and the protective effect was close to that of GSH (91.17±0.07%). In order to explore the mechanism of action, we visualized the conjugates of the two antioxidant peptides with Keap1. The results showed that both antioxidant peptides could generate hydrogen bonds and hydrophobic interactions with some binding sites in the Keap1-Kelch domain, proving that both pearl layer-derived antioxidant peptides could competitively bind to Keap1, thereby releasing Nrf2 and activating the Keap1-Nrf2-ARE pathway. In summary, the two shell nacre antioxidant peptides are 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 1 is 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: In A and C, different letters 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: In A, different letters 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 the shell nacre antioxidant peptide FYFPKYGR and Keap1.

[0022] Figure 4 This is a visualization of the docking results of the shell nacre antioxidant peptide GYFPSYY and Keap1. DETAILED DESCRIPTION

[0023] The present invention is described below by specific embodiments. The technical means not particularly described in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material components and dosages in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention.

[0024] Unless otherwise indicated, it should be understood that each individual element in a list and each combination of individual elements in the list will be interpreted as a different embodiment. For example, a list of embodiments represented as "A, B, or C" should be interpreted to include embodiments "A", "B", "C", "A or B", "A or C", "B or C", or "A, B, or C".

[0025] In the present disclosure, the singular forms of the articles "a," "an," and "the" also include the corresponding plural references, and a reference to a specific value includes at least that specific value unless the context clearly indicates otherwise. Thus, for example, a reference to "a substance" is a reference to at least one of that substance and equivalents thereof.

[0026] The terms including ordinal numbers such as "first" and "second" can be used to explain various components or fluids, but these components and fluids are not limited by these terms. Therefore, without departing from the teachings of the present disclosure, these terms are only used to distinguish the component / fluid from another component / fluid.

[0027] When items are described by using the conjunction terms "... and / or...," etc., the description should be understood to include any one of the associated listed items and all combinations of one or more thereof.

[0028] In general, the use of the term "about" indicates an approximate value that may vary depending on the desired properties obtained by the disclosed subject matter, and will be interpreted in a context-dependent manner based on function. Therefore, one of ordinary skill in the art will be able to interpret a certain degree of difference on a case-by-case basis. In some cases, the number of significant figures used when expressing a particular value can be a representative technique for determining the difference allowed by the term "about". In other cases, a gradient in a range of values ​​can be used to determine the range of differences allowed by the term "about". Further, all ranges in this disclosure are inclusive and combinable, and reference to the values ​​stated in the range includes each value within the range.

[0029] In the present invention, the term "comprising" or "containing" means that various components can be used together in the composition of the present invention. Therefore, the terms "consisting mainly of" and "consisting of" are included in the terms "comprising" or "containing".

[0030] Example 1 1. Synthesis of antioxidant peptides from nacre Based on the sequences FYFPKYGR and GYFPSYY, the shell nacre antioxidant peptide was synthesized by solid phase synthesis. The purity of the synthetic peptide was ≥98% as determined by HPLC.

[0031] 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.

[0032] 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.

[0033] 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-(AUCt -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.

[0034] 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 4 100 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.

[0035] 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.

[0036] 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 carried out molecular docking of shell nacre antioxidant peptides with Keap1.

[0037] 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.

[0038] 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).

[0039] Effects of antioxidant peptides from shell nacre 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 within this concentration range, shell nacre antioxidant peptides had no toxicity to HepG2 cells. 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. Both antioxidant peptides showed a certain cellular antioxidant capacity, and the effect was close to that of GSH at the same concentration. Figure 1As shown in C, the intracellular antioxidant activity EC 50 The values ​​were 32.9 μg / mL and 37.989 μg / mL respectively. The results of CAA showed that both FYFPKYGR and GYFPSYY had good antioxidant capacity.

[0040] (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.

[0041] Depend on Figure 2 As shown in Figure B, the cell survival rate was concentration-dependent with the peptide concentration. After the addition of the antioxidant peptide FYFPKYGR, the cell survival rate was improved to a certain extent compared with the injury group, especially when the concentration was 250 μg / mL, the cell survival rate was significantly higher than that of the injury group; and at a low concentration (25 μg / mL), the injury protection effect was not significantly different from that of the positive control GSH group ( p >0.05). Similarly, after adding 25 μg / mL of GYFPSYY, the cell survival rate was improved to a certain extent, and the current effect was not significantly different from that of GSH at the same concentration ( p >0.05). In summary, both antioxidant peptides showed certain ability to protect against cell damage.

[0042] (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 FYFPKYGR and GYFPSYY with Keap1 are shown in Figure 3 , Figure 4The information of hydrogen bonds and hydrophobic interactions generated by it is shown in Table 1. The binding energy of FYFPKYGR with Keap1 is -10.9, and 13 hydrogen bonds are formed at the 10 binding sites on Keap1: GLY603, ALA556, ASN382, ARG415, TYR525, GLY509, GLY574, VAL606, ILE559 and GLY364, of which 6 sites: GLY603, ALA556, ASN382, ARG415, TYR525 and GLY509 are located in the Kelch domain; in addition, FYFPKYGR also forms 6 hydrophobic interactions with the 5 binding sites of Keap1: SER508, TYR334, TYR572, TYR525 and ALA556, and these 5 binding sites are all from the Kelch domain. The binding energy of GYFPSYY with Keap1 is -10.6, and it generates five hydrogen bonds with the five binding sites of Keap1, SER508, TYR572, GLY509, LEU557 and SER383, of which SER508 and TYR572 are from the Kelch domain; in addition, GYFPSYY also generates four hydrophobic interactions with three binding sites, ALA556, TYR334 and TYR525, all of which are located in the Kelch domain.

[0043] In summary, both FYFPKYGR and GYFPSYY formed multiple hydrogen bonds and hydrophobic interactions with multiple binding sites of Keap1, which enabled 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 active site of the protein. At the same time, most of the binding sites for generating hydrogen bonds and hydrophobic interactions are located in the Kelch domain of Keap1, and they can occupy the connection site of Nrf2 by binding to the Kelch domain of Keap1 protein. Therefore, these two antioxidant peptides are likely to competitively bind to Keap1 to release Nrf2, thereby exhibiting antioxidant activity in cells.

[0044] Table 1 The results of the interaction between shell nacre antioxidant peptides and Keap1

[0045] 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. An antioxidant peptide derived from shell nacre, characterized in that: It has any of the following amino acid sequences: (1) Phe-Tyr-Phe-Pro-Lys-Tyr-Gly-Arg; (2) Gly-Tyr-Phe-Pro-Ser-Tyr-Tyr.

2. The shell nacre-derived antioxidant peptide according to claim 1, characterized in that: The shell nacre-derived antioxidant 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-derived antioxidant peptides as claimed in claim 1.

4. A solid phase synthesis product comprising the antioxidant peptide derived from the nacre layer of shells as claimed in claim 1.

5. A salt, characterized in that: The salt is a pharmaceutically acceptable salt of the shell nacre-derived antioxidant 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-derived antioxidant 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-derived antioxidant 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-derived antioxidant 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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