Shell nacre-derived antioxidant peptides and their preparation method and application

Abstract

The present invention discloses shell nacre-derived antioxidant peptides, and a preparation method and application thereof. The shell nacre antioxidant peptides FYFPKYGR and GYFPSYY provided by the present invention both have strong cellular antioxidant activity. In order to explore the mechanism of action, we conducted a visual analysis of the conjugates of the peptide and Keap1. The study found that it 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, shell nacre antioxidant peptides are 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-derived antioxidant peptides and a preparation method and application thereof. Background Art

[0002] Nacre is a component of shells. From the outside in, the shell's structure consists of the stratum corneum, prismatic layer, and nacre. The nacre is formed by the secretion of large amounts of nacre from the mantle epithelial cells, following the same process used to form pearls. Therefore, nacre is homologous to pearls and shares similar compositional properties. In addition to essential components such as calcium carbonate and organic matter, it also contains trace elements, amino acids, vitamins, polysaccharides, proteins, and other ingredients. Therefore, nacre has the potential to serve as a functional alternative to pearls in health and skincare products, reducing product costs while also enabling the high-value utilization of discarded shells and preventing resource waste. In recent years, research on nacre extracts has focused on anti-aging, osteogenesis, and improving LPS-induced depression and anxiety. However, limited research has examined the antioxidant activity of nacre extracts, paving the way for their development into antioxidant 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 shell nacre.

[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 use of the above-mentioned shell nacre-derived antioxidant peptides.

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

[0007] An antioxidant peptide derived from shell nacre, having any of the following amino acid sequences:

[0008] (1) Phe-Tyr-Phe-Pro-Lys-Tyr-Gly-Arg (FYFPKYGR);

[0009] (2) Gly-Tyr-Phe-Pro-Ser-Tyr-Tyr (GYFPSYY).

[0010] Furthermore, the shell nacre-derived antioxidant peptide is prepared by shell nacre protein hydrolysis, solid phase synthesis or genetic engineering technology.

[0011] A shell nacre protein hydrolyzate comprising the above-mentioned shell nacre-derived antioxidant peptides.

[0012] A solid phase synthesis product comprising the above-mentioned shell nacre-derived antioxidant peptide.

[0013] A nucleic acid encoding the above-mentioned shell nacre-derived antioxidant peptide.

[0014] A biological material comprising the above nucleic acid, which is a recombinant DNA, an expression cassette, a transposon, a vector or a host cell.

[0015] A salt, which is a pharmaceutically acceptable salt of the shell nacre-derived antioxidant peptide.

[0016] Furthermore, the pharmaceutically acceptable salt is acetate, hydrochloride, phosphate, sulfate, methanesulfonate, or toluenesulfonate.

[0017] A pharmaceutical composition comprises the shell nacre-derived antioxidant peptide or salt and at least one pharmaceutically acceptable excipient.

[0018] Furthermore, the excipient is a carrier and / or a solvent.

[0019] Application of the 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.

[0020] The use 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 drugs for protecting cells from oxidative damage.

[0021] The present invention has the following advantages and effects compared to the prior art:

[0022] The antioxidant peptides FYFPKYGR and GYFPSYY from shell nacre have strong cellular antioxidant activity, EC 50 The concentrations of the two nacre-derived peptides were 32.9 μg / mL and 37.989 μg / mL, respectively. Furthermore, the two nacre-derived antioxidant peptides (250 μg / mL) were able to increase the survival rate of cells in the AAPH-injured group (81.75±0.61%) to 85.71±1.56%–86.27±0.60%, achieving a protective effect similar to that of GSH (91.17±0.07%). To explore the underlying mechanism, we performed a visual analysis of the binding of these two antioxidant peptides to Keap1. The results revealed that both peptides could form hydrogen bonds and hydrophobic interactions with certain binding sites within the Keap1-Kelch domain, demonstrating that both nacre-derived antioxidant peptides can competitively bind to Keap1, thereby releasing Nrf2 and activating the Keap1-Nrf2-ARE pathway. In summary, the two nacre-derived antioxidant peptides have potential for application in cosmetics, providing a theoretical basis for the high-value utilization of nacre. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] 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), when comparing different concentrations of the same sample, different lowercase letters represent significant differences ( p <0.05).

[0024] 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: 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).

[0025] Figure 3 This is a visualization of the docking results of the shell nacre antioxidant peptide FYFPKYGR and Keap1.

[0026] Figure 4 This is a visualization of the docking results of the shell nacre antioxidant peptide GYFPSYY and Keap1. DETAILED DESCRIPTION

[0027] The present invention is described below by specific embodiments. The technical means not particularly described in the present invention are methods well known to those skilled in the art. In addition, the embodiments should be understood to be illustrative rather than limiting the scope of the present invention, and the spirit and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications of the material components and amounts in these embodiments, without departing from the spirit and scope of the present invention, also fall within the scope of protection of the present invention.

[0028] Unless otherwise indicated, it should be understood that each individual element in a list and each combination of individual elements in that list will be interpreted as a different embodiment. For example, a list of embodiments expressed 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."

[0029] In this disclosure, the singular forms of the articles "a," "an," and "the" also include the corresponding plural reference, and a reference to a specific value includes at least that specific value unless the context clearly dictates otherwise. Thus, for example, a reference to "a substance" is a reference to at least one of that substance and equivalents thereof.

[0030] Terms including ordinal numbers such as "first" and "second" may 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.

[0031] When items are described by using the conjunction terms "... and / or..." etc., the description should be understood to include any one and all combinations of one or more of the associated listed items.

[0032] In general, the use of the term "about" indicates an approximate value that can 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 important 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 a value stated in a range includes every value within that range.

[0033] 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".

[0034] Example 1

[0035] 1. Synthesis of antioxidant peptides from nacre

[0036] Based on the sequences FYFPKYGR and GYFPSYY, the nacre antioxidant peptide was synthesized by solid phase synthesis. The purity of the synthesized peptide was ≥98% as determined by HPLC.

[0037] 2. Evaluation of the antioxidant activity of nacreous antioxidant peptides based on a HepG2 cell model

[0038] 2.1 Cell culture

[0039] 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.

[0040] 2.2 Analysis of the toxicity of nacreous antioxidant peptides on HepG2 cells

[0041] The effects of the samples on the viability of HepG2 cells were determined by MTT assay. 4 Cells 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.

[0042] 2.3 Intracellular antioxidant activity assay

[0043] HepG2 cells were grown at a cell density of 6 × 10 4Each 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.

[0044] 2.4 Protective effect of shell nacre antioxidant peptides on HepG2 cells oxidatively damaged by AAPH

[0045] 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.

[0046] 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.

[0047] 3. Molecular docking analysis

[0048] 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 relationships, binding sites, and mechanisms of action of active substances. The Keap1-Nrf2 pathway is a key endogenous antioxidant signaling pathway, and 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 an antioxidant peptide derived from nacre of shells and Keap1.

[0049] 4. Statistical Analysis

[0050] 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.

[0051] 5. Results:

[0052] (1) Determination of intracellular antioxidant activity (CAA)

[0053] 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).

[0054] 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 increasing 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 1 As 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.

[0055] (2) Protective effect of shell nacre antioxidant peptides on oxidative damage in HepG2 cells

[0056] 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.

[0057] Depend on Figure 2As shown in Figure B, the cell survival rate is 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 damaged group, especially when the concentration was 250 μg / mL, the cell survival rate was significantly higher than that of the damaged group; and at a low concentration (25 μg / mL), the damage 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.

[0058] (3) Using molecular docking technology to study the mechanism of action of antioxidant peptides from shell nacre

[0059] The 3D and 2D images of the docking results of FYFPKYGR and GYFPSYY with Keap1 are shown in the figure below. Figure 3 、 Figure 4 The information on hydrogen bonds and hydrophobic interactions is shown in Table 1. The binding energy of FYFPKYGR to Keap1 is -10.9. It forms 13 hydrogen bonds with 10 binding sites on Keap1: GLY603, ALA556, ASN382, ARG415, TYR525, GLY509, GLY574, VAL606, ILE559, and GLY364. Six of these sites, GLY603, ALA556, ASN382, ARG415, TYR525, and GLY509, are located in the Kelch domain. In addition, FYFPKYGR forms six hydrophobic interactions with five binding sites on Keap1: SER508, TYR334, TYR572, TYR525, and ALA556. All five binding sites are located in the Kelch domain. The binding energy of GYFPSYY with Keap1 is -10.6, and it generates five hydrogen bonds with Keap1's five binding sites 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 all located in the Kelch domain: ALA556, TYR334 and TYR525.

[0060] In summary, both FYFPKYGR and GYFPSYY form 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 prevent 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. They can occupy the Nrf2 binding site by binding to the Kelch domain of the Keap1 protein. Therefore, these two antioxidant peptides are likely to competitively bind to Keap1 to release Nrf2, thereby exhibiting antioxidant activity in cells.

[0061] Table 1 Interaction results between shell nacre antioxidant peptides and Keap1

[0062]

[0063] The above embodiments are preferred implementations of the present invention, but the implementations 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 equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. An antioxidant peptide derived from shell nacre, characterized by: Its amino acid sequence is shown as any one of the following: (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 peptide according to claim 1.

4. A solid phase synthesis product comprising the antioxidant peptide derived from the nacre of a shell 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 antioxidant peptide derived from the nacre layer of shells 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-derived antioxidant peptide of claim 1, the shell nacre protein hydrolyzate of claim 3, the solid-phase synthesis product of claim 4, or the salt of any one of claims 5 to 6 in the preparation of antioxidant cosmetics.

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