Antioxidant peptides, eggshell membrane hydrolysate containing antioxidant peptides and application thereof

Abstract

The invention discloses antioxidant peptides, eggshell membrane hydrolysates containing antioxidant peptides and applications thereof. The amino acid sequence of the antioxidant peptide is any one of the following: NDADYPWPH, DFGPGPM, FPMRT, KPLCPP, DKDGPFRL, CRPPSLGR. The antioxidant peptides of the invention have strong antioxidant activity, and possess one or more of ABTS free radical scavenging activity, ORAC activity, DPPH free radical scavenging activity, total antioxidant activity TAA, and cellular antioxidant activity. Among them, NDADYPWPH has the highest ORAC activity and ABTS free radical scavenging activity, which are 8.64 times and 3.42 times of reduced glutathione, respectively. NDADYPWPH and KPLCPP simultaneously possess high ABTS, ORAC and cellular antioxidant activities. The antioxidant peptides of the invention are obtained by hydrolysis of eggshell membranes, separation and purification, peptideomics and bioinformatics screening, and are all new antioxidant peptides that have not been reported, have the characteristics of small molecular weight, being directly absorbed by the human body, targeting and regulating cellular antioxidant pathways, and can be widely used in the fields of food, medicine, health products and cosmetics.

Description

Technical Field

[0001] The invention relates to the technical field of biological small molecule active peptides, in particular to antioxidant peptides, eggshell membrane hydrolysates containing the antioxidant peptides and applications thereof. Background Art

[0002] Cellular oxidative stress and defense disorders can cause a variety of cell dysfunctions and inflammatory diseases, such as osteoarthritis, cardiovascular disease, cancer, etc. Among them, osteoarthritis, as a chronic disease with an increasing incidence, is particularly closely related to oxidative stress. Antioxidants can inhibit oxidative stress by reducing the production of free radicals and peroxides in the body. Chemically synthesized antioxidants such as BHT (butylated hydroxytoluene) and BHA (butylated hydroxyanisole) pose certain safety risks to human health and may cause attention deficit, allergies and dermatitis, and even cancer. Natural antioxidants are in urgent need because of their high efficiency and no toxic side effects. Antioxidant peptides are one of the natural and efficient sources of antioxidants.

[0003] Eggshells are the main byproducts of the egg industry, and about 8 million tons of them are discarded every year around the world, causing serious environmental pollution and waste of resources. Eggshell membranes adhere to the inner surface of eggshells, and the amount of waste is huge. Studies have shown that eggshell membrane hydrolyzates have antioxidant and anti-inflammatory activities. There have been reports on the research of eggshell membranes: for example, Chinese patent CN 103275205 B discloses a method for producing water-soluble egg membrane active peptides; Chinese patent CN 108323764 B discloses a method for preparing eggshell membrane polypeptides with bone and joint health care functions, but they have not been able to accurately identify active peptides with certain structures and functions, the product ingredients are unclear, and there is no single purified active peptide product. There is currently no report on the sequence of eggshell membrane antioxidant peptides with biological activity, and various problems will be encountered in the later development of drugs and health products.

[0004] Therefore, it is necessary to identify the structure and sequence of highly active eggshell membrane antioxidant peptides. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides antioxidant peptides, eggshell membrane hydrolysates containing antioxidant peptides and their applications. The present invention hydrolyzes eggshell membranes by appropriate hydrolysis technology to obtain eggshell membrane hydrolysates, separates and purifies the hydrolysates by ultrafiltration, gel chromatography and reversed-phase high performance liquid chromatography, measures the chemical and cellular antioxidant activity of each component, performs peptideomics identification by LC-MS / MS, screens peptide sequences by bioinformatics means, solid phase synthesizes, and verifies its activity, thereby completing the following invention.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] An antioxidant peptide, whose amino acid sequence is any of the following sequences:

[0008] NDADYPWPH(Asn-Asp-Ala-Asp-Tyr-Pro-Trp-Pro-His);

[0009] DFGPGPM(Asp-Phe-Gly-Pro-Gly-Pro-Met);

[0010] FPMRT (Phe-Pro-Met-Arg-Thr);

[0011] KPLCPP(Lys-Pro-Leu-Cys-Pro-Pro);

[0012] DKDGPFRL(Asp-Lys-Asp-Gly-Pro-Phe-Arg-Leu);

[0013] CRPPSLGR (Cys-Arg-Pro-Pro-Ser-Leu-Gly-Arg).

[0014] Preferably, the antioxidant peptide is derived from chicken egg shell membrane.

[0015] A polynucleotide, characterized in that it encodes the above-mentioned antioxidant peptide.

[0016] An hen's egg shell membrane hydrolyzate comprising one or more of the following antioxidant peptides;

[0017] an antioxidant peptide with an amino acid sequence of NDADYPWPH (Asn-Asp-Ala-Asp-Tyr-Pro-Trp-Pro-His);

[0018] An antioxidant peptide with an amino acid sequence of DFGPGPM (Asp-Phe-Gly-Pro-Gly-Pro-Met);

[0019] An antioxidant peptide with an amino acid sequence of FPMRT (Phe-Pro-Met-Arg-Thr);

[0020] An antioxidant peptide with the amino acid sequence of KPLCPP (Lys-Pro-Leu-Cys-Pro-Pro);

[0021] An antioxidant peptide with an amino acid sequence of DKDGPFRL (Asp-Lys-Asp-Gly-Pro-Phe-Arg-Leu);

[0022] An antioxidant peptide with the amino acid sequence of CRPPSLGR (Cys-Arg-Pro-Pro-Ser-Leu-Gly-Arg).

[0023] Application of the above antioxidant peptides or eggshell membrane hydrolysate in the manufacture of bone and joint drugs.

[0024] Application of the antioxidant peptide or eggshell membrane hydrolyzate in the manufacture of antioxidant drugs.

[0025] Application of the above antioxidant peptides or eggshell membrane hydrolysate in the manufacture of bone and joint health products.

[0026] Application of the antioxidant peptides or eggshell membrane hydrolysates in the manufacture of antioxidant health products.

[0027] Application of the above antioxidant peptides or eggshell membrane hydrolysate in food.

[0028] Application of the above antioxidant peptides or eggshell membrane hydrolysate in cosmetics.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The present invention identified 6 novel polypeptides with high antioxidant activity from eggshell membrane hydrolysate, whose molecular weights are mainly concentrated in the range of 600 to 1200 Da. The small molecular weight is conducive to human absorption and utilization.

[0031] (2) The present invention uses multiple indicators to jointly verify the in vitro and cellular antioxidant activities.

[0032] ① The ORAC values ​​of antioxidant peptides KPLCPP, FPMRT, DFGPGPM, and NDADYPWPH are 1.58-8.64 times that of reduced glutathione;

[0033] ② The ABTS values ​​of antioxidant peptides KPLCPP and NDADYPWPH were 2.00 and 3.42 times that of reduced glutathione, respectively;

[0034] ③ The antioxidant peptides of the present invention can protect inflammatory cells from damage by oxidative stress induced by H2O2.

[0035] (3) The antioxidant peptides of the present invention can interact with the antioxidant cell target Keap1 protein and effectively regulate the body's antioxidant pathway Keap1-Nrf2 signaling pathway.

[0036] (4) The present invention uses bioinformatics and computer-aided technology to perform virtual screening, and ultimately screens out peptides with high antioxidant activity.

[0037] (5) The present invention uses chicken egg shell membrane as the protein raw material, rationally utilizes the by-products of egg processing, reduces environmental pollution, reduces the production cost of antioxidant peptides, and improves resource utilization, thus having the advantages of low cost, green environmental protection, and sustainable development. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The in vitro antioxidant results of the preferred hen's egg shell membrane antioxidant peptides;

[0039] Figure 2 is the mass spectrum of the eggshell membrane antioxidant peptide NDADYPWPH of the present invention;

[0040] Figure 3 The mass spectrum of the hen's egg shell membrane antioxidant peptide DFGPGPM of the present invention;

[0041] Figure 4 The mass spectrum of the chicken egg shell membrane antioxidant peptide FPMRT of the present invention;

[0042] Figure 5 The mass spectrum of the chicken egg shell membrane antioxidant peptide KPLCPP of the present invention;

[0043] Figure 6 The mass spectrum of the eggshell membrane antioxidant peptide DKDGPFRL of the present invention;

[0044] Figure 7 is the mass spectrum of the eggshell membrane antioxidant peptide CRPPSLGR of the present invention;

[0045] Figure 8 Schematic diagram showing the effect of the chicken egg shell membrane hydrolyzate of the present invention on the ROS level in RAW264.7 cells induced by H2O2;

[0046] Fig. 9 The effect of the antioxidant peptide KPLCPP on the expression of proteins related to the Keap1-Nrf2 signaling pathway of the cell antioxidant pathway (A is the result without H2O2 treatment, B is the result after H2O2 treatment);

[0047] Fig.10 The molecular docking results of the antioxidant peptide KPLCPP and the key cellular antioxidant protein Keap1;

[0048] Fig.11 This is the molecular docking result of the antioxidant peptide CRPPSLGR and the key cellular antioxidant protein Keap1. DETAILED DESCRIPTION

[0049] The present invention is further described below in conjunction with examples, but it should be noted that the examples do not limit the scope of protection claimed in the present invention.

[0050] 1. Specific antioxidant activity of antioxidant peptides

[0051] The antioxidant peptides involved in the present invention are measured by the method described in (Habinshuti I, Mu TH, Zhang M. Ultrasound microwave-assisted enzymatic production and characterization of antioxidant peptides from sweet potato protein [J]. Ultrason Sonochem, 2020, 69: 105262.) to determine the ORAC value.

[0052] The method for determining the scavenging rate of ABTS and DPPH free radicals was determined by referring to the methods described in (Liying Wang, Mengting Ma, Zhipeng Yu, Shuang-kui Du. Preparation and identification of antioxidant peptides from cottonseed proteins [J]. Food Chemistry, 2021, 352: 129399.) and (Qiaozhi Zhang, Xiaohong Tong, Yang Li, Huan Wang, Zhongjiang Wang, Baokun Qi, Xiaonan Sui, Lianzhou Jiang. Purification and characterization of antioxidant peptides from Alcalase-hydrolyzed soybean (Glycine max L.) hydrolysate and their cytoprotective effects in human intestinal Caco-2 cells [J]. Agricultural and Food Chemistry. 2019: 1-36.).

[0053] The total antioxidant activity TAA was determined by referring to the method described in (Qian-Cheng Zhao, Jie-Yuan Zhao, Dong UkAhn, Yong-Guo Jin, Xi Huang. Separation and Identification of Highly Efficient Antioxidant Peptides from Eggshell Membrane[J]. Antioxidants, 2019: 1-16.).

[0054] The specific peptide sequences and activity results are as follows:

[0055] (1) The antioxidant peptide CRPPSLGR showed good scavenging activity for DPPH and ABTS free radicals, among which the DPPH free radical scavenging activity was higher than that of the positive control GSH.

[0056] (2) The antioxidant peptides NDADYPWPH and KPLCPP showed high ORAC and ABTS values, and the results were higher than those of the positive control GSH; the antioxidant peptides DFGPGPM and FPMRT showed high ORAC values, and the results were higher than those of GSH. At the same time, DFGPGPM and DKDGPFRL showed high DPPH values.

[0057] (3) When the concentration of the egg shell membrane hydrolyzate containing the antioxidant peptides of the present invention (preferably the component with a molecular weight less than 3 kDa) was 0.75 mg / mL, the survival rate of inflammatory cells (RAW264.7) in the egg shell membrane hydrolyzate pretreatment group was increased by 48.36% compared with the H2O2 model group, indicating that the egg shell membrane antioxidant peptides can protect inflammatory cells and reduce their damage to H2O2-induced oxidative stress.

[0058] (4) After pretreatment with the eggshell membrane hydrolyzate (0.75 mg / mL) containing the antioxidant peptides of the present invention, the MDA content of inflammatory cells (RAW264.7) was significantly reduced compared with the H2O2 model group, indicating that the eggshell membrane antioxidant peptides can inhibit cellular lipid peroxidation, thereby preventing H2O2-induced cellular oxidative stress damage.

[0059] (5) The eggshell membrane antioxidant peptides NDADYPWPH and KPLCPP involved in the present invention can protect inflammatory cells (RAW264.7) from damage by oxidative stress and prevent the occurrence of inflammation-related diseases, such as osteoarthritis.

[0060] The antioxidant peptides of the present invention can be obtained by common methods in the art, such as enzymatic hydrolysis, chemical synthesis and genetic engineering. Preferably, the novel antioxidant peptides obtained by the present invention are obtained by solid phase synthesis.

[0061] 2. Application in food, health products, medicines and cosmetics

[0062] It should be noted that, as a typical example, the present invention can prepare the eggshell membrane hydrolyzate containing the antioxidant peptide of the present invention by hydrolyzing the eggshell membrane protein raw material by enzymatic hydrolysis. The above antioxidant that can be used as food, medicine and cosmetics refers to at least one of the antioxidant peptides NDADYPWPH, DFGPGPM, FPMRT, KPLCPP, DKDGPFRL and CRPPSLGR that preferably show high ORAC value, high ABTS scavenging activity and high DPPH scavenging activity in the eggshell membrane hydrolyzate.

[0063] The antioxidant peptide of the present invention and the eggshell membrane hydrolyzate containing the same can also be used as a medicine, typically, as an antioxidant medicine. Diseases caused by oxidation of organisms include osteoarthritis, arteriosclerosis, myocardial infarction, cancer, diabetes, Alzheimer's disease, pollinosis, etc. The antioxidant peptide of the present invention and the eggshell membrane hydrolyzate containing the same can be used as a medicine for preventing and treating these diseases. When provided in the form of a medicine, it can be used in various forms such as liquid, powder, tablet and capsule.

[0064] The antioxidant peptides of the present invention and the eggshell membrane hydrolyzate containing the same can be used as effective antioxidant ingredients in various foods, including being added to foods for the purpose of preventing the oxidation of the food itself, and being added to beverages, tablets, food blocks (processed foods that are pressed into blocks), snacks, and nutritional supplements, etc., which can exert physiological antioxidant functions on the ingestors.

[0065] The antioxidant peptide of the present invention and the eggshell membrane hydrolyzate containing the same can be used as an effective antioxidant ingredient in cosmetic products, including facial masks, lotions, creams, etc. with antioxidant and anti-aging effects.

[0066] Hereinafter, the present invention will be described in more detail by way of examples.

[0067] 3. Preparation, purification, identification and screening of antioxidant peptides from eggshell membrane

[0068] Example 1

[0069] <Preparation of Chicken Egg Shell Membrane Hydrolyzate 1>

[0070] a. Obtaining eggshell membrane:

[0071] It comes from waste from egg processing plants. The egg shell membrane is torn off, washed with pure water, and dried for later use.

[0072] b. Preparation of hen egg shell membrane hydrolysate:

[0073] The egg shell membrane hydrolysate was prepared with reference to Chinese patent CN 108323764 B (a method for extracting egg shell membrane polypeptides and preparing bone and joint health products).

[0074] Example 2

[0075] <Preparation of Chicken Egg Shell Membrane Hydrolyzate 2>

[0076] a. Obtaining eggshell membrane:

[0077] It comes from waste from egg processing plants. The egg shell membrane is torn off, washed with pure water, and dried for later use.

[0078] b. Preparation of hen egg shell membrane hydrolysate:

[0079] 1) crushing the dried egg shell membrane;

[0080] 2) mixing eggshell membrane powder and water at a solid-liquid ratio of 1:30 to 1:50 (w / v);

[0081] 3) The pH value of the hydrolysis system is 7.5 to 10.5;

[0082] 4) adding metalloproteinase and serine protease in the hydrolysis system at a ratio of 0.5% to 3%;

[0083] 5) The reaction system temperature is set to 55-65°C;

[0084] 6) The hydrolysis time is 10 to 12 hours;

[0085] 7) After the hydrolysis is completed, quickly place the container containing the hydrolyzate in boiling water for 10 to 20 minutes to inactivate the protease;

[0086] 8) After the hydrolyzate is cooled to room temperature, the pH of the hydrolyzate is adjusted to between 7.0 and 7.6 to avoid the influence of pH on antioxidant activity;

[0087] 9) Centrifuge the hydrolyzate at 8000-12000 r / min for 10-30 min. The supernatant is the hen egg shell membrane hydrolyzate 2, which is stored at -20°C for future use.

[0088] Example 3

[0089] <Ultrafiltration of Chicken Egg Shell Membrane Hydrolyzate>

[0090] The hydrolyzate was initially separated using ultrafiltration membranes with molecular weights of 30 kDa, 10 kDa, and 3 kDa, respectively, to obtain ultrafiltration fractions with peptide molecular weights >30 kDa, 30-10 kDa, 10-3 kDa, and <3 kDa. The oxidative radical absorption capacity (ORAC) of each fraction was measured, and the fraction with the largest ORAC value (<3 kDa) was selected for amino acid sequence identification.

[0091] Example 4

[0092] <Mass spectrometry identification of antioxidant peptides from hen eggshell membrane>

[0093] The amino acid sequence of the peptide fraction with the highest antioxidant activity obtained by ultrafiltration was identified by liquid chromatography-tandem mass spectrometry (LC-MS / MS). LC-MS / MS was performed using an Eksignent nano-LC instrument (Eksigent Technologies, Dublin, CA, USA) coupled to a Thermo LTQ linear ion trap mass spectrometer (Thermo Fisher, San Jose, CA, USA) and an LC Packins PepMap300 C18 column (75 μm × 150 mm; pore size The particle size was 5 μm; Dionex, Sunnyvale, CA, USA). The eluent A was 2% ACN aqueous solution (containing 0.1% formic acid), and the eluent B was 80% ACN aqueous solution (containing 0.1% formic acid). The flow rate was 300 nL / min. The elution conditions were: 0-85 min, 5% B; 85-95 min, 5-50% B; 95-125 min, 50-95% B. The amino acid sequence of the eluted peptides was evaluated using MS / MS data, with a spray voltage of 2.2 kV, an m / z scan range of 400-2000, a data acquisition time of 110 min, a capillary temperature of 200 °C, and a normalized collision energy of 35%. Finally, the MS / MS data were analyzed using Mascot (version 2.1.0, Matrix Sciences, London, UK).

[0094] Example 5

[0095] <Screening of antioxidant peptides based on bioinformatics>

[0096] From Example 4, we can obtain the amino acid sequence of the antioxidant peptide, and predict the potential for activity by the bioinformatics website PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ), and predict the physicochemical indicators of the antioxidant peptide, such as molecular weight, hydrophilicity, hydrophobic amino acid content, and isoelectric point, according to the ProtParam tool (https: / / web.expasy.org / protparam / ) website.

[0097] The molecular weight, hydrophilicity, hydrophobic amino acid content and isoelectric point of the polypeptides identified in the hen egg shell membrane hydrolysates 1 and 2 are shown in Tables 1 and 2 below. Peptides with predicted activities greater than 0.80 are preferred.

[0098] Table 1 Molecular weight, hydrophilicity, hydrophobic amino acid content, and isoelectric point of the peptides identified in hen egg shell membrane hydrolyzate 1

[0099]

[0100] Note: Negative GRAVY value represents hydrophilicity, the smaller the value, the more hydrophilic it is.

[0101] Table 2 Molecular weight, hydrophilicity, hydrophobic amino acid content, and isoelectric point of the peptides identified in hen egg shell membrane hydrolyzate 2

[0102]

[0103]

[0104] Note: Negative GRAVY value represents hydrophilicity, the smaller the value, the more hydrophilic it is.

[0105] IV. Activity determination of antioxidant peptides from eggshell membrane

[0106] (In vitro antioxidant activity assay)

[0107] Example 6

[0108] <In vitro antioxidant activity assay>

[0109] The antioxidant peptide sequences contained in the hen egg shell membrane hydrolysates 1 and 2 were synthesized by solid phase synthesis, and their antioxidant indexes were measured. The results are listed in Tables 3 and 4 below.

[0110] Table 3 Antioxidant peptides contained in hen egg shell membrane antioxidant peptide hydrolysate 1 and their antioxidant activities

[0111]

[0112] Note: Different letters represent significant differences (P<0.05), the same as Tables 4, 5, 6, 7, 8, and 9.

[0113] Table 4 Antioxidant peptides contained in eggshell membrane antioxidant peptide hydrolysate 2 and their antioxidant activities

[0114]

[0115]

[0116] From the results in Table 3, it can be found that the antioxidant peptide CRPPSLGR has a high antioxidant activity and exhibits the highest DPPH and ABTS free radical scavenging activity.

[0117] Depend on Figure 1As shown in Table 4, the antioxidant peptides NDADYPWPH, DFGPGPM, FPMRT, and KPLCPP showed high ORAC values. Compared with the positive control GSH, the results of the above three antioxidant peptides were higher than those of the positive control GSH. In particular, the ORAC value of the antioxidant peptide NDADYPWPH was 8.64 times that of GSH. The antioxidant peptide NDADYPWPH showed the highest ABTS value, followed by KPLCPP, both of which were higher than the ABTS value of the positive control GSH. The antioxidant peptide KPLCPP showed the highest DPPH value, which was higher than the result of the positive control GSH, followed by the antioxidant peptides DFGPGPM, NDADYPWPH, and DKDGPFRL.

[0118] in, Figure 2 is the mass spectrum of the eggshell membrane antioxidant peptide NDADYPWPH of the present invention;

[0119] Figure 3 The mass spectrum of the hen's egg shell membrane antioxidant peptide DFGPGPM of the present invention;

[0120] Figure 4 The mass spectrum of the chicken egg shell membrane antioxidant peptide FPMRT of the present invention;

[0121] Figure 5 The mass spectrum of the chicken egg shell membrane antioxidant peptide KPLCPP of the present invention;

[0122] Figure 6 The mass spectrum of the eggshell membrane antioxidant peptide DKDGPFRL of the present invention;

[0123] Figure 7 is the mass spectrum of the eggshell membrane antioxidant peptide CRPPSLGR of the present invention;

[0124] The above results indicate that the antioxidant peptides of the present invention have high antioxidant activity and can be added to food, medicine and cosmetics as novel antioxidant ingredients. Compared with chemically synthesized antioxidants, the antioxidant peptides of the present invention have the advantages of safety and high efficiency.

[0125] (Determination of the antioxidant activity of hen egg shell membrane hydrolysate on RAW264.7 cells)

[0126] Example 7

[0127] <Protective effect of antioxidant peptides on H2O2-induced RAW264.7 cells>

[0128] Macrophages are multifunctional immune cells that are widely distributed in all tissues and organs of the body. These cells are responsible for clearing pathogens or damaged cells, play an important role in homeostasis maintenance, inflammation regulation and repair, and are associated with various human diseases.

[0129] The egg shell membrane hydrolyzate containing the antioxidant peptides of the present invention can significantly improve the cell survival rate of the H2O2-induced inflammatory cell (RAW264.7) oxidative damage model. When pretreated with 0.75 mg / mL egg shell membrane hydrolyzate, the survival rate of RAW cells can be increased by 48.36% compared with the H2O2 model group.

[0130] As a preferred solution, the protective effect of the aforementioned chicken egg shell membrane antioxidant peptide on the toxicity and oxidative damage model of RAW cells induced by H2O2 can be determined according to the following method:

[0131] 1. Cultivation of RAW cells: RAW cells were cultured in DMEM high-glucose medium (containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin) in a 37°C, 5% CO2 saturated humidity incubator. Cells were cultured in monolayers and digested with 0.25% trypsin solution for subculture.

[0132] 2. Study on the effect of eggshell membrane hydrolysate on RAW cell proliferation: RAW cells were cultured at 2×10 4 The cells were inoculated at a concentration of 100 / mL in a 96-well plate and cultured for 24 h. 0.25 and 0.75 mg / mL of egg shell membrane antioxidant peptide solution (dissolved in cell culture medium and filtered through a 0.22 μm filter membrane to remove impurities and sterilize) were added respectively. After continuing to culture for 24 h, the cell survival rate was measured. The results are shown in Table 5 below. The preferred egg shell membrane hydrolyzate had no toxicity to RAW cells at concentrations of 0.25 and 0.75 mg / mL, indicating that the egg shell membrane hydrolyzate identified by the present invention has the characteristics of safety and non-toxicity.

[0133] Table 5 Cytotoxicity of the hen egg shell membrane hydrolyzate containing the antioxidant peptides of the present invention

[0134] Group Blank Group 0.25mg / mL Eggshell membrane antioxidant peptide 0.75mg / mL Eggshell membrane antioxidant peptide Cell viability % <![CDATA[100.00±2.00 b ]]> <![CDATA[109.10±7.46 ab ]]> <![CDATA[119.46±5.71 a ]]>

[0135] 3. Establishment of RAW cell oxidative damage model (H2O2 model group): RAW cells were incubated at 2×10 4 The cells were seeded at a concentration of 100 / mL in a 96-well plate and cultured for 48 h. A 200 μM H2O2 solution (dissolved in DMEM) was added to each well and cultured for 1 h.

[0136] 4. Determination of RAW cell survival rate: RAW cells were cultured at 2×10 4The cells were inoculated into a 96-well plate at a concentration of 100 cells / mL and cultured for 24 hours. A 0.75 mg / mL eggshell membrane antioxidant peptide solution (dissolved in cell culture medium and filtered through a 0.22 μm filter to remove impurities and sterilize) was added. After pretreatment for 24 hours, 200 μM H2O2 solution was added to each well and cultured for 1 hour. The culture medium was removed, and 100 μL CCK-8 solution (10%) was added to each well. After continued culture in a 37°C, 5% CO2 saturated humidity incubator for 1 hour, the absorbance value A of each well was measured by an ELISA reader. 450nm .

[0137] 5. The cell survival rate was calculated according to the following formula:

[0138]

[0139] Where A represents the A of the experimental group 450nm ; A0 represents A of the blank group 450nm ; A1 represents A in the normal group 450nm ;

[0140] The results are shown in Table 6 below:

[0141] Table 6 Results of the protective effect of the chicken egg shell membrane hydrolyzate containing the antioxidant peptides of the present invention on H2O2-induced RAW cells

[0142]

[0143]

[0144] When pretreated with 0.75 mg / mL hen's egg shell membrane hydrolyzate, the survival rate of RAW264.7 cells could be increased by 48.36% compared with the H2O2 model group.

[0145] Example 8

[0146] <Effects of Chicken Egg Shell Membrane Antioxidant Peptides on Cellular Lipid Peroxidation>

[0147] MDA, as a product of lipid peroxidation, can directly reflect the degree of lipid peroxidation and indirectly characterize the degree of oxidative damage to cells by ROS. After pretreatment with eggshell membrane hydrolysate for 24 hours, the cells were treated with 175μM H2O2 for 1 hour, washed twice with PBS, and then lysed by ultrasound in an ice bath for 30 seconds. Subsequently, the protein content was determined using a BCA kit. The activity of MDA in the cell suspension was detected using a kit, and the results were expressed in nmol / mgprotein.

[0148] The experiment was divided into a blank group (no peptide and H2O2 treatment), a model group (only 175 μM H2O2 treatment for 1 h) and a peptide group (pretreated with 0.75 mg / mL chicken egg shell membrane hydrolyzate for 24 h, then treated with 175 μM H2O2 for 1 h).

[0149] Table 7 Effect of the chicken egg shell membrane hydrolyzate containing the antioxidant peptides of the present invention on cellular lipid peroxidation

[0150]

[0151] The results are shown in Table 7. Compared with the blank group, the MDA content in the model group treated with H2O2 was significantly higher than that in the blank group. In the peptide-treated group, the MDA content was significantly reduced and returned to normal levels, indicating that the degree of cell oxidative damage was reduced after eggshell membrane treatment, further indicating that eggshell membrane treatment can inhibit lipid peroxidation in RAW cells, thereby preventing H2O2-induced cell oxidative stress damage.

[0152] Example 9

[0153] <Effect of the chicken egg shell membrane hydrolyzate containing the antioxidant peptide of the present invention on the ROS level in RAW264.7 cells induced by H2O2>

[0154] The DCFH-DA fluorescent probe method was selected to study the effect of hen egg shell membrane hydrolysate on ROS levels in H2O2-induced RAW264.7 cells. RAW264.7 cells were pretreated with hen egg shell membrane hydrolysate and induced with H2O2 for 1 h. DCFH-DA (10 μM) probe was immediately added and incubated for 30 min. The cells were washed three times with PBS and RAW264.7 cell images were obtained using a fluorescence microscope.

[0155] like Figure 8 As shown, compared with the model group, the fluorescence signal was significantly weakened after the cells were treated with 0.25 and 0.5 mg / mL of egg shell membrane hydrolysate, indicating that the ROS level in the cells was reduced after treatment with egg shell membrane hydrolysate, indicating that egg shell membrane hydrolysate can effectively protect inflammatory cells from ROS-mediated oxidative stress damage.

[0156] (Antioxidant effect of the preferred chicken egg shell membrane antioxidant peptide on RAW264.7 cells)

[0157] Example 10

[0158] <The protective effects of antioxidant peptides KPLCPP and NDADYPWPH on the toxicity of RAW264.7 cells and H2O2-induced cell damage>

[0159] Study on the effect of selected antioxidant peptides on the proliferation of RAW cells: RAW cells were cultured at 2×10 4The cells were inoculated in a 96-well plate at a concentration of 100 / mL and cultured for 24 hours. 500 and 1000 μM eggshell membrane antioxidant peptide solutions (KPLCPP and NDADYPWPH) were added respectively, and the cell survival rate was measured after continuing to culture for 24 hours. The results are shown in Table 8 below. The preferred antioxidant peptides KPLCPP and NDADYPWPH were not toxic to RAW cells at concentrations of 500 and 1000 μM, indicating that the eggshell membrane antioxidant peptides identified by the present invention have safe and non-toxic characteristics.

[0160] Table 8 Cytotoxicity of antioxidant peptides KPLCPP and NDADYPWPH

[0161]

[0162] Study on the protective effect of selected antioxidant peptides on H2O2-induced cell damage: RAW cells were cultured at 2×10 4 The cells were inoculated in a 96-well plate at a concentration of 1000 μM eggshell membrane antioxidant peptide solution (dissolved in cell culture medium, filtered through a 0.22 μm filter membrane to remove impurities and sterilize) after culturing for 24 hours. After culturing for another 24 hours, 175 μM H2O2 solution (dissolved in DMEM) was added to all wells except the blank well. After culturing for 1 hour, the cell viability of each well was measured. The results are shown in Table 9 below. After pretreatment with antioxidant peptides KPLCPP and NDADYPWPH, the cell viability increased from 50.97% to 62.13% and 78.21%, respectively.

[0163] Table 9 Results of the protective effects of antioxidant peptides KPLCPP and NDADYPWPH on H2O2-induced RAW264.7 cells

[0164]

[0165] Embodiment 11

[0166] <Study the effect of antioxidant peptide KPLCPP on intracellular antioxidant pathways based on western blot>

[0167] RAW264.7 cells (5.1×10 5Cells / well) were inoculated into 6-well plates and cultured for 24 hours. The preferred antioxidant peptide KPLCPP (500 and 1000 μM) was pretreated for 24 hours and then treated with 175 μM H2O2 for 1 hour. RAW264.7 cells were collected and lysed with RIPA buffer containing PMSF and phosphatase inhibitors. After analyzing the total protein content with BCA, the protein (40 μg) was separated with 12% SDS-PAGE gel and transferred to a PVDF membrane. Five milliliters of blocking solution were added, and the blocking solution was removed after one hour. The primary antibody diluted with the blocking solution was added and slowly incubated at 4°C overnight. Rinse three times with TBST, add the secondary antibody diluted with TBST, incubate at 24°C for one hour, rinse three times with TBST, add ECL liquid to the washed PVDF membrane, let it react chemically for one to two minutes, place it in a dark environment, and obtain the results of western blot after development and fixation.

[0168] like Fig. 9 As shown: Under the conditions of H2O2 treatment (B) and without H2O2 treatment (A), the antioxidant peptide KPLCPP can upregulate the expression of Nrf2 and HO-1 proteins after treatment, thereby protecting inflammatory cells (RAW264.7) from damage by H2O2-induced oxidative stress.

[0169] 5. Study on the structure-activity relationship of eggshell membrane antioxidant peptides

[0170] Example 12

[0171] <Study on the effects of antioxidant peptides KPLCPP and CRPPSLGR on intracellular antioxidant pathways based on molecular docking>

[0172] Molecular docking was performed using the CDOCKER program of Discovery Studio (DS) 2018 client software, with the eggshell membrane antioxidant peptide of the present invention as a ligand and Keap1 (PDB ID: 2FLU) as a receptor. The docking results were expressed by the –CDOCKERinteraction energy (–CIE) score, where a higher score indicates a higher binding affinity and a tighter binding, and the specific values ​​are shown in Table 10.

[0173] Table 10 Molecular docking results of eggshell membrane antioxidant peptides and Keap1

[0174] Peptide sequence –CIE NDA D Y P WPH 53.0792 DFGPGPM 54.8485 FPMRT 55.3801 DKDGPFRL 69.3744 KPLCPP 52.0045 CRPPSLGR 57.2673

[0175] All the eggshell membrane antioxidant peptides involved in the present invention can successfully dock with the active site of Keap1. Among them, KPLCPP and CRPPSLGR are preferred eggshell membrane antioxidant peptides, which are used to study the binding mechanism of eggshell membrane antioxidant peptides and Keap1. The results are as follows Fig.10 , 11As shown, KPLCPP forms three conventional hydrogen bonds with Arg380, Arg415 and Gln530 of Keap1, one attractive charge interaction with Arg380, one carbon-hydrogen bond with Ser602, two alkyl bonds with Ala556 and Arg415, and one π-alkyl bond with Tyr525. CRPPSLGR formed six conventional hydrogen bonds with Arg415, Ser508, Arg483 and Ser602 of Keap1, among which Arg380, Arg415, Ser602, Gln530, Tyr525, Ser508 and Arg483 were the key amino acid residues of the Keap1-Kelch domain at the Keapl-Nrf2 interaction binding site, indicating that the eggshell membrane antioxidant peptides occupy the Nrf2 binding site in the Keap1-Kelch domain, inhibit the ubiquitination degradation of Nrf2, and thus protect inflammatory cells from damage induced by H2O2-induced oxidative stress.

[0176] The above embodiments have provided a detailed introduction to the technical solutions provided by the present invention. Specific examples are used herein to illustrate the principles and implementation methods of the embodiments of the present invention. The description of the above embodiments is only applicable to help understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, according to the embodiments of the present invention, there may be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. An antioxidant peptide, characterized in that Its amino acid sequence is as follows: NDADYPWPH(Asn-Asp-Ala-Asp-Tyr-Pro-Trp-Pro-His).

2. A polynucleotide, characterized in that Encodes the antioxidant peptide according to claim 1.

3. An hen egg shell membrane hydrolyzate, characterized in that It contains the following antioxidant peptides: An antioxidant peptide with the amino acid sequence of NDADYPWPH (Asn-Asp-Ala-Asp-Tyr-Pro-Trp-Pro-His).

4. Use of the antioxidant peptide according to claim 1 or the eggshell membrane hydrolyzate according to claim 3 in the manufacture of antioxidant drugs.

5. Use of the antioxidant peptide according to claim 1 or the eggshell membrane hydrolyzate according to claim 3 in the manufacture of antioxidant health products.

6. Use of the antioxidant peptide according to claim 1 or the eggshell membrane hydrolyzate according to claim 3 in food.

7. Use of the antioxidant peptide according to claim 1 or the eggshell membrane hydrolyzate according to claim 3 in cosmetics.

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