CN115197296A - Antioxidant peptide, egg shell membrane hydrolysate containing antioxidant peptide and application of antioxidant peptide and egg shell membrane hydrolysate - Google Patents
Antioxidant peptide, egg shell membrane hydrolysate containing antioxidant peptide and application of antioxidant peptide and egg shell membrane hydrolysate Download PDFInfo
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- CN115197296A CN115197296A CN202111667534.6A CN202111667534A CN115197296A CN 115197296 A CN115197296 A CN 115197296A CN 202111667534 A CN202111667534 A CN 202111667534A CN 115197296 A CN115197296 A CN 115197296A
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention discloses an antioxidant peptide, an eggshell membrane hydrolysate containing the antioxidant peptide and application thereof. The amino acid sequence of the antioxidant peptide is any one of the following: NDADYPWPH, DFGPGPM, FPMRT, KPLCPP, DKDGPFRL, CRPPSLGR. The antioxidant peptide has strong antioxidant activity, and has one or more of ABTS free radical scavenging activity, ORAC activity, DPPH free radical scavenging activity, total antioxidant activity TAA and cell antioxidant activity. Wherein 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 have high ABTS, ORAC and cell antioxidant activity. The antioxidant peptide is obtained by hydrolyzing, separating and purifying eggshell membrane, and screening peptide groups and bioinformatics, is a novel unreported antioxidant peptide, has the characteristics of small molecular weight, direct absorption by human bodies, targeting and regulation of cell antioxidant pathways, and can be widely applied to the fields of foods, medicines, health care products, cosmetics and the like.
Description
Technical Field
The invention relates to the technical field of biological small molecule active peptides, in particular to antioxidant peptides, an egg shell membrane hydrolysate containing the antioxidant peptides and application thereof.
Background
Dysregulation of cellular oxidative stress and defense can cause various cellular dysfunctions and inflammatory diseases, such as osteoarthritis, cardiovascular diseases, cancer, and the like. Among them, osteoarthritis is a chronic disease with an increasing incidence rate, and 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) present certain safety risks to human health, may cause attention deficit, allergy and dermatitis, and even cause cancer. The natural antioxidant has high efficiency and no toxic and side effect, and is urgently needed. Antioxidant peptides are one of natural and highly effective antioxidant sources.
The eggshells are used as main byproducts of the poultry egg industry, about 800 ten thousand tons of eggshells are discarded every year around the world, and serious environmental pollution and resource waste are caused. The eggshell membrane is adhered to the inner surface of the eggshell, and the waste amount is huge. The research shows that the egg shell membrane hydrolysate has antioxidant and anti-inflammatory activity. Studies on eggshell membranes have been reported: for example, chinese patent CN 103275205B discloses a production method of a water-soluble egg membrane active peptide; chinese patent CN 108323764B discloses a preparation method of eggshell membrane polypeptide with bone joint health care function, but all the bioactive peptides with determined structure and function can not be accurately identified, the product components are not clear, and a single purified bioactive peptide product is not available. At present, no bioactive eggshell membrane antioxidant peptide sequence is reported, and various problems can be encountered in the development process of later-stage medicaments and health-care products.
Therefore, it is necessary to identify the structure and sequence of the highly active eggshell membrane antioxidant peptide.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides antioxidant peptide, an eggshell membrane hydrolysate containing the antioxidant peptide and application thereof. The invention hydrolyzes egg shell membrane by proper hydrolysis technology to obtain egg shell membrane hydrolysate, separates and purifies the hydrolysate by ultrafiltration, gel chromatography and reversed-phase high performance liquid chromatography, measures the chemical and cell antioxidant activity of each component, performs peptide group identification by LC-MS/MS, screens peptide sequence by bioinformatics means, synthesizes solid phase, and verifies the activity, thereby completing the following invention.
In order to achieve the purpose, the invention adopts the following technical scheme:
an antioxidant peptide, the amino acid sequence of which is any one of the following sequences:
NDADYPWPH(Asn-Asp-Ala-Asp-Tyr-Pro-Trp-Pro-His)；
DFGPGPM(Asp-Phe-Gly-Pro-Gly-Pro-Met)；
FPMRT(Phe-Pro-Met-Arg-Thr)；
KPLCPP(Lys-Pro-Leu-Cys-Pro-Pro)；
DKDGPFRL(Asp-Lys-Asp-Gly-Pro-Phe-Arg-Leu)；
CRPPSLGR(Cys-Arg-Pro-Pro-Ser-Leu-Gly-Arg)。
preferably, the antioxidant peptide is derived from eggshell membrane.
A polynucleotide encoding the above antioxidant peptide.
An egg shell membrane hydrolysate comprising one or more of the following antioxidant peptides;
an antioxidant peptide having an amino acid sequence of NDADYPWPH (Asn-Asp-Ala-Asp-Tyr-Pro-Trp-Pro-His);
an antioxidant peptide with the amino acid sequence of DFGPGPGPM (Asp-Phe-Gly-Pro-Gly-Pro-Met);
an antioxidant peptide with an amino acid sequence of FPMRT (Phe-Pro-Met-Arg-Thr);
antioxidant peptide with amino acid sequence KPLCPP (Lys-Pro-Leu-Cys-Pro-Pro);
an antioxidant peptide with an amino acid sequence of DKDGPFRL (Asp-Lys-Asp-Gly-Pro-Phe-Arg-Leu);
the amino acid sequence is CRPPSLGR (Cys-Arg-Pro-Pro-Ser-Leu-Gly-Arg).
The antioxidant peptide or eggshell membrane hydrolysate can be used for preparing bone joint medicine.
The antioxidant peptide or eggshell membrane hydrolysate can be used for preparing antioxidant drugs.
The antioxidant peptide or eggshell membrane hydrolysate can be used for preparing bone joint health products.
The antioxidant peptide or eggshell membrane hydrolysate can be used for preparing antioxidant health product.
The antioxidant peptide or egg shell membrane hydrolysate can be used in food.
The antioxidant peptide or egg shell membrane hydrolysate can be used in cosmetic.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention identifies 6 novel polypeptides with high antioxidant activity from the egg shell membrane hydrolysate, the molecular weight is mainly and intensively distributed between 600 Da and 1200Da, the molecular weight is small, and the novel polypeptides are beneficial to absorption and utilization by human bodies.
(2) The invention adopts a plurality of indexes to jointly verify the in vitro and cell antioxidant activity.
(1) Wherein the ORAC value of antioxidant peptides KPLCPP, FPMRT, DFGPGPM, NDADYPWPH is 1.58-8.64 times of that of reduced glutathione;
(2) the ABTS values of the antioxidant peptides KPLCPP and NDADYPWPH are respectively 2.00 times and 3.42 times of that of reduced glutathione;
(3) the antioxidant peptide can protect inflammatory cells from H 2 O 2 Damage by induced oxidative stress.
(3) The antioxidant peptide disclosed by the invention can interact with an antioxidant cell target Keap1 protein, and effectively regulates and controls a Keap1-Nrf2 signal path of an organism antioxidant path.
(4) The invention adopts bioinformatics and computer-aided technology to carry out virtual screening, and finally screens out the peptide with high antioxidant activity.
(5) The invention uses the egg shell membrane as the protein raw material, reasonably utilizes the poultry egg processing by-products, reduces the environmental pollution, reduces the production cost of the antioxidant peptide, improves the utilization rate of resources, and has the advantages of low cost, environmental protection and sustainable development.
Drawings
FIG. 1 is the in vitro antioxidant results of preferred eggshell membrane antioxidant peptides;
FIG. 2 is a mass spectrum of eggshell membrane antioxidant peptide NDADYPWPH of the present invention;
FIG. 3 is a mass spectrum of the eggshell membrane antioxidant peptide DFGPGPGPM of the present invention;
FIG. 4 is a mass spectrum of the eggshell membrane antioxidant peptide FPMRT of the present invention;
FIG. 5 is a mass spectrum of the eggshell membrane antioxidant peptide KPLCPP of the present invention;
FIG. 6 is a mass spectrum of the eggshell membrane antioxidant peptide DKDDGPFRL of the invention;
FIG. 7 is a mass spectrum of the eggshell membrane antioxidant peptide CRPPSLGR of the present invention;
FIG. 8 shows the eggshell membrane hydrolysate pair H of the present invention 2 O 2 Comparative schematic of the effect of induced ROS levels in RAW264.7 cells;
FIG. 9 shows the effect of antioxidant peptide KPLCPP on the expression of Keap1-Nrf2 signal pathway-related proteins of cellular antioxidant pathway (A is no H) 2 O 2 The result of the treatment, B, is H 2 O 2 The result of the processing);
FIG. 10 shows the molecular docking results of the antioxidant peptide KPLCPP and the cell antioxidant key protein Keap 1;
FIG. 11 shows the molecular docking results of the antioxidant peptide CRPPSLGR and the cell antioxidant key protein Keap 1.
Detailed Description
The present invention is further described with reference to the following examples, which should not be construed as limiting the scope of the invention as claimed.
1. Description of the specific antioxidant Activity of antioxidant peptides
The antioxidant peptide of the present invention can be used for determining the ORAC value by the method described in (Habinshuti I, mu T H, zhang M. Ultrasonic microwave-assisted enzymatic production and chromatography of antioxidant peptides from sweet potato protein [ J ]. Ultrason Sonochem,2020,69, 105262.).
ABTS, determination of DPPH free radical scavenging Rate methods are described by reference to (dying Wang, mengting Ma, zhipen Yu, shuang-kui Du. Preparation and identification of antioxidant peptides from chemically synthesized proteins [ J ]. Food Chemistry,2021,352, 129399.) and (Qiaozhi Zhang, xiaohong Tong, yang Li, human Wang, zhongjiang Wang, baokun Qi, xian Sun Sui, lianzhou J. Purification and characterization of antioxidant peptides from Alcala-purified genes [ J. ] -2. Additive J.: 36. Amino-2. Conversion and analysis.
Method for measuring total Antioxidant activity TAA the method described in (Qian-Cheng Zhao, je-Yuan Zhao, dong Uk Ahn, yong-Guo Jin, xi huang. Separation and Identification of high effective ingredients from egg Membrane [ J ]. Antioxidants, 2019).
Specific polypeptide sequences and activity results are as follows:
(1) The antioxidant peptide CRPPSLGR showed good DPPH, ABTS free radical scavenging activity, where DPPH free radical scavenging activity was higher than that of the positive control GSH.
(2) The antioxidant peptides NDADYPWPH and KPLCPP show high ORAC and ABTS values, and the determination results are higher than the results of a positive control GSH; the antioxidant peptides DFGPGPM and FPMRT show high ORAC value, the determination result is higher than the GSH result, and DFGPM and DKDGPCRL show high DPPH value.
(3) The eggshell membrane hydrolysate (preferably the component with the molecular weight less than 3 kDa) with the antioxidant peptide of the invention reacts with H when the concentration is 0.75mg/mL 2 O 2 Compared with a model group, the survival rate of inflammatory cells (RAW 264.7) of the eggshell membrane hydrolysate pretreatment group is improved by 48.36 percent, which shows that the eggshell membrane antioxidant peptide can protect the inflammatory cells and reduce the H-cell-mediated inflammatory response 2 O 2 Damage by induced oxidative stress.
(4) After egg shell membrane hydrolysate (0.75 mg/mL) with antioxidant peptide of the invention is pretreated, H is added 2 O 2 Compared with a model group, the MDA content of inflammatory cells (RAW 264.7) is obviously reduced, which shows that the eggshell membrane antioxidant peptide can inhibit cell lipid peroxidation, thereby preventing H 2 O 2 Induced oxidative stress damage of cells.
(5) The eggshell membrane antioxidant peptide NDADYPWPH, KPLCPP can protect inflammatory cells (RAW 264.7) from being damaged by oxidative stress and prevent inflammation-related diseases such as osteoarthritis and the like.
The antioxidant peptide of the present invention can be obtained according to methods commonly used in the art, such as enzymatic hydrolysis, chemical synthesis, and genetic engineering. Preferably, the novel antioxidant peptide obtained by the invention is obtained by using solid phase synthesis.
2. Application in food, health product, medicine and cosmetics
It should be noted that, as a typical example, the present invention can prepare eggshell membrane hydrolysate containing the antioxidant peptide of the present invention by hydrolyzing eggshell membrane protein material by an enzymatic hydrolysis method. The antioxidant useful as food, medicine and cosmetic is preferably at least one of antioxidant peptides NDADYPWPH, DFGPGPM, FPMRT, KPLCPP, DKDGPFRL, CRPPSLGR exhibiting high ORAC value, high ABTS scavenging activity and high DPPH scavenging activity from a hydrolysate of eggshell membrane.
The antioxidant peptide of the present invention and the eggshell membrane hydrolysate containing the same are also useful as pharmaceuticals, typically, as antioxidant pharmaceuticals. Examples of diseases caused by oxidation of a living body include osteoarthritis, arteriosclerosis, myocardial infarction, cancer, diabetes, alzheimer's disease, pollinosis, and the like. The antioxidant peptide of the present invention and the eggshell membrane hydrolysate containing the same can be used as a medicine for preventing and treating these diseases. When the drug is supplied in the form of a pharmaceutical product, it can be used in various forms such as liquid, powder, tablet, capsule, and the like.
The antioxidant peptide and the eggshell membrane hydrolysate containing the same of the present invention can be used as an effective antioxidant ingredient in various foods, including foods to be added for the purpose of preventing the self-oxidation of the foods, beverages, tablets, food blocks (briquetted finished foods), snacks, nutritional supplements, and the like, which exert physiological antioxidant functions on the ingesters.
The antioxidant peptide and the eggshell membrane hydrolysate containing the same can be used as effective antioxidant components for cosmetic products, including facial masks, lotions, creams and the like with antioxidant and anti-aging effects.
The present invention will be described more specifically with reference to examples.
3. Preparation, purification, identification and screening of egg shell membrane antioxidant peptide
Example 1
< preparation of egg shell Membrane hydrolysate 1 >
a. Obtaining the eggshell membrane:
is prepared from the wastes of egg processing factory by tearing off the eggshell membrane, washing with pure water, and oven drying.
b. Preparing the eggshell membrane hydrolysate:
refer to Chinese patent CN 108323764B (a method for extracting eggshell membrane polypeptide and preparing bone joint health product) to prepare eggshell membrane hydrolysate.
Example 2
< preparation of egg shell Membrane hydrolysate 2 >
a. Obtaining the eggshell membrane:
is prepared from the wastes of egg processing factory by tearing off the eggshell membrane, washing with pure water, and oven drying.
b. Preparing the eggshell membrane hydrolysate:
1) Crushing the dried eggshell membrane;
2) The ratio of the eggshell membrane powder to water is 1:30 to 1:50 (w/v);
3) The pH value of the hydrolysis system is 7.5-10.5;
4) Adding metalloprotease and serine protease in a hydrolysis system according to the proportion of 0.5-3%;
5) The temperature of the reaction system is set to be 55-65 ℃;
6) The hydrolysis time is 10 to 12 hours;
7) After the hydrolysis is finished, quickly putting the container filled with the hydrolysis liquid into boiling water for 10-20 min to inactivate the protease;
8) After the hydrolysate is cooled to the room temperature, the pH value of the hydrolysate is adjusted to 7.0-7.6, so that the influence of the pH value on the antioxidant activity is avoided;
9) Centrifuging the hydrolysate for 10-30 min under 8000-12000 r/min to obtain supernatant as egg shell membrane hydrolysate 2, and storing at-20 deg.c.
Example 3
< ultrafiltration of egg shell membrane hydrolysate >
Using ultrafiltration membranes with molecular weights of 30kDa, 10kDa and 3kDa to carry out primary separation on hydrolysates respectively to obtain ultrafiltration components with the peptide segment molecular weights of more than 30kDa, 30-10 kDa, 10-3 kDa and less than 3kDa, respectively determining the oxidative free Radical absorption Capacity (ORAC) of each component, and selecting the component with the largest ORAC value (less than 3 kDa) for amino acid sequence identification.
Example 4
< Mass Spectrometry identification of Eggshell Membrane antioxidant peptides >
And identifying the amino acid sequence of the polypeptide component with the highest antioxidant activity obtained by ultrafiltration by adopting a liquid chromatography-tandem mass spectrometry (LC-MS/MS). LC-MS/MS was coupled with a Thermo LTQ linear ion trap mass spectrometer (Thermo Fisher, san Jose, CA, USA) using an eks ignent nano LC instrument (eks Technologies, dublin, CA, USA), an LC Packins PepMap 300C 18 column (75 μm × 150mm pore size
particle size 5 μm; dionex, sunnyvale, CA, USA). Eluent A was 2% ACN aqueous solution (containing 0.1% formic acid), eluent B was 80% ACN aqueous solution (containing 0.1% formic acid), and flow rate was 300nL/min. The elution conditions were: 0 to 85min,5 percent of B; 85-95min, 5-50% of B; 95-125min, 50-95% of B. The eluted peptide amino acid sequence was evaluated using MS/MS data with a spray voltage of 2.2kV, a m/z scan range of 400-2000, a data acquisition time of 110min, a capillary temperature of 200 ℃ and a normalized collision energy of 35%. Finally, MS/MS data were analyzed using Mascot (version 2.1.0, matrix sciences, london, UK).
Example 5
< screening of antioxidant peptides based on bioinformatics >
From example 4, we can obtain the amino acid sequence of the antioxidant peptide, predict the potential of activity from bioinformatics website PeptideRanker (http:// distilldeep. Ucd. Ie/PeptideRanker /), and predict physicochemical indexes of the antioxidant peptide, such as molecular weight, hydrophilicity, hydrophobic amino acid content, isoelectric point, according to ProtParam tool (https:// web. Expasy. Org/ProtParam /) website.
The results of the molecular weight, hydrophilicity, hydrophobic amino acid content and isoelectric point of the polypeptides identified in eggshell membrane hydrolysates 1, 2 are shown in tables 1, 2 below, with polypeptides having a predicted activity greater than 0.80 being preferred.
TABLE 1 molecular weight, hydrophilicity, content of hydrophobic amino acids, isoelectric point of the polypeptides identified in eggshell membrane hydrolysate 1
Note: negative values of GRAVY indicate hydrophilicity, with smaller values being more hydrophilic.
TABLE 2 molecular weight, hydrophilicity, content of hydrophobic amino acids, isoelectric point of the polypeptides identified in eggshell membrane hydrolysate 2
Note: negative values of GRAVY indicate hydrophilicity, with smaller values being more hydrophilic.
4. Activity determination of egg shell membrane antioxidant peptide
(determination of antioxidant Activity in vitro)
Example 6
< measurement of antioxidant Activity in vitro >
The antioxidant peptide sequences contained in the eggshell membrane hydrolysates 1 and 2 were synthesized by solid phase synthesis and the antioxidant index thereof was measured, and the results are shown in tables 3 and 4 below.
TABLE 3 antioxidant peptides contained in egg shell membrane antioxidant peptide hydrolysate 1 and antioxidant activity thereof
Note: different letters represent significant differences (P < 0.05), table 4, 5, 6, 7, 8, 9, and so on.
TABLE 4 antioxidant peptides contained in egg shell membrane antioxidant peptide hydrolysate 2 and antioxidant activities thereof
From the results in table 3, it can be found that the antioxidant peptide CRPPSLGR has high antioxidant activity, and simultaneously shows the highest DPPH and ABTS free radical scavenging activity.
As can be seen from fig. 1 and table 4, antioxidant peptide NDADYPWPH, DFGPGPM, FPMRT, KPLCPP showed a high ORAC value, and compared with the positive control GSH, the results of the above three antioxidant peptides were all higher than the positive control GSH, and in particular, antioxidant peptide NDADYPWPH had an ORAC value 8.64 times higher than that of GSH. Antioxidant peptide NDADYPWPH showed the highest ABTS value, followed by KPLCPP, all higher than the ABTS value of the positive control GSH. The antioxidant peptide KPLCPP showed the highest DPPH value, higher than the results of the positive control GSH, followed by the antioxidant peptides dfgpgpgm, ndadyppgh, DKDGPFRL.
Wherein, fig. 2 is a mass spectrogram of the eggshell membrane antioxidant peptide NDADYPWPH of the invention;
FIG. 3 is a mass spectrum of the eggshell membrane antioxidant peptide DFGPGPGPM of the present invention;
FIG. 4 is a mass spectrum of the eggshell membrane antioxidant peptide FPMRT of the present invention;
FIG. 5 is a mass spectrum of the eggshell membrane antioxidant peptide KPLCPP of the present invention;
FIG. 6 is a mass spectrum of the eggshell membrane antioxidant peptide DKDDGPFRL of the invention;
FIG. 7 is a mass spectrum of the eggshell membrane antioxidant peptide CRPPSLGR of the present invention;
from the above results, it can be concluded that the antioxidant peptide of the present invention has high antioxidant activity and can be added to foods, pharmaceuticals and cosmetics as a novel antioxidant ingredient. Compared with the antioxidant synthesized chemically, the antioxidant peptide has the advantages of safety and high efficiency.
(determination of antioxidant Activity of egg Shell Membrane hydrolysate on RAW264.7 cells)
Example 7
<Antioxidant peptide pair H 2 O 2 Induced protection of RAW264.7 cells>
Macrophages are multifunctional immune cells that are widely distributed throughout all tissues and organs of the body. These cells are responsible for the elimination of pathogens or damaged cells, play an important role in homeostatic maintenance, inflammatory regulation and repair, and are associated with various human diseases.
The eggshell membrane hydrolysate containing the antioxidant peptide can obviously improve H 2 O 2 Cell viability in the model of induced oxidative damage to inflammatory cells (RAW 264.7) after pretreatment with 0.75mg/mL eggshell membrane hydrolysate, in combination with H 2 O 2 Compared with a model group, the survival rate of the RAW cells can be improved by 48.36 percent.
Preferably, the egg shell membrane antioxidant peptide pair H 2 O 2 The protective effects of the toxicity and oxidative damage model for inducing RAW cells can be determined as follows:
culture of raw cells: RAW cells were cultured in DMEM high-glucose medium (containing 10% fetal bovine serum, 100U/mL penicillin and 100. Mu.g/mL streptomycin) at 37 ℃ with 5% CO 2 A saturated humidity incubator. Cell monolayer culture and subculture by digestion with 0.25% trypsin solution.
2. Study of egg shell membrane hydrolysate on proliferation of RAW cells: RAW cells at 2X 10 4 Inoculating the egg shell membrane hydrolysate in a 96-well plate at the cell concentration of 0.25mg/mL and 0.75mg/mL for 24h, respectively, adding the egg shell membrane antioxidant peptide solution (dissolved in a cell culture solution, and subjected to filtration, impurity removal and sterilization by a 0.22-micrometer filter membrane), and continuously culturing for 24h to measure the cell survival rate, wherein the results are shown in the following table 5, and the preferred egg shell membrane hydrolysate has no toxicity to RAW cells at the concentration of 0.25mg/mL and 0.75mg/mL, which indicates that the egg shell membrane hydrolysate identified by the invention has the characteristics of safety and no toxicity.
TABLE 5 cytotoxicity of egg shell membrane hydrolysate containing antioxidant peptides of the present invention
| Group of | Blank group | 0.25mg/mL egg shell membrane antioxidant peptide | 0.75mg/mL egg shell membrane antioxidant peptide |
| Cell viability% | 100.00±2.00 b | 109.10±7.46 ab | 119.46±5.71 a |
RAW cell oxidative damage model (H) 2 O 2 Model group) establishment: RAW cells at 2X 10 4 The cells were seeded in 96-well plates at a concentration of 200. Mu. M H per well for 48h 2 O 2 Solution (in DMEM) and incubated for 1h.
Determination of raw cell viability: RAW cells at 2X 10 4 Inoculating each/mL cell in 96-well plate, culturing for 24 hr, adding 0.75mg/mL egg shell membrane antioxidant peptide solution (dissolved in cell culture solution, and filtered with 0.22 μm filter membrane to remove impurities and bacteria), pretreating for 24 hr, and adding 200 μ M H into each well 2 O 2 And culturing the solution for 1h. Removing the culture medium, adding 100. Mu.L of CCK-8 solution (10%) per well, standing at 37 deg.C, 5% CO 2 After the culture is continued for 1h in the saturated humidity incubator, the light absorption value A of each hole is measured by the microplate reader 450nm 。
5. Cell viability was calculated according to the following formula:
wherein A represents A in the experimental group 450nm ；A 0 A representing blank group 450nm ；A 1 A representing normal group 450nm ；
The results are shown in table 6 below:
TABLE 6 egg shell membrane hydrolysate pairs H containing the antioxidant peptides of the invention 2 O 2 Results of protective Effect of induced RAW cells
After pretreatment with 0.75mg/mL egg shell membrane hydrolysate, the mixture is mixed with H 2 O 2 Compared with a model group, the survival rate of the RAW264.7 cells can be improved by 48.36 percent.
Example 8
< Effect of eggshell membrane antioxidant peptide on lipid peroxidation of cells >
MDA as a product of lipid peroxidation can directly reflect the degree of lipid peroxidation and indirectly characterize the degree of oxidative damage of ROS to cells. Pretreating egg shell membrane hydrolysate for 24h, and then using 175 mu M H 2 O 2 Treatment for 1h, washing 2 times with PBS, followed by sonication for 30s in an ice bath. Subsequently, the protein content was determined with BCA kit. The activity of MDA in the cell suspension is detected by adopting the kit, and the detection result is expressed by nmol/mgprotein.
The experiment was divided into blank groups (no polypeptide and H) 2 O 2 Treatment), model group (only 175 μ M H) 2 O 2 Treating for 1 h) and polypeptide group (0.75 mg/mL egg shell membrane hydrolysate is pretreated for 24h, and then 175 mu M H 2 O 2 Treatment 1 h).
TABLE 7 Effect of eggshell membrane hydrolysate containing antioxidant peptides of the present invention on lipid peroxidation of cells
The results are shown in Table 7, comparison with the blank group, via H 2 O 2 The MDA content of the treated model group is obviously higher than that of the blank group, in the polypeptide-treated group, the MDA content is obviously reduced and is restored to a normal level, which indicates that the oxidation damage degree of cells is reduced after the eggshell membrane treatment, and further indicates that the eggshell membrane treatment can inhibit lipid peroxidation of RAW cells, thereby preventing H 2 O 2 Induced oxidative stress damage of cells.
Example 9
<Egg shell membrane hydrolysate pair H containing antioxidant peptide 2 O 2 Effect of induced ROS levels in RAW264.7 cells>
The DCFH-DA fluorescence probe method is selected to research the H pair of the eggshell membrane hydrolysate 2 O 2 Effect of induced ROS levels in RAW264.7 cells. Pretreating RAW264.7 cells with eggshell membrane hydrolysate, and adding H 2 O 2 Induction was carried out for 1h. DCFH-DA (10. Mu.M) probe was added immediately and incubated for 30 min. PBS washed cells three times and RAW264.7 cell images were obtained with fluorescence microscopy.
As shown in fig. 8, compared with the model group, the fluorescent signal was significantly reduced after the treatment of 0.25mg/mL and 0.5mg/mL egg shell membrane hydrolysate, which indicates that the intracellular ROS level was reduced after the treatment of egg shell membrane hydrolysate, and indicates that the egg shell membrane hydrolysate can effectively protect inflammatory cells from ROS-mediated oxidative stress.
(antioxidant action of preferred egg shell membrane antioxidant peptides on RAW264.7 cells)
Example 10
<Toxicity of antioxidant peptides KPLCPP and NDADYPWPH on RAW264.7 cells and H 2 O 2 Protective effects of induced cell damage>
Preferred are oxidation resistanceStudy of the effect of peptides on proliferation of RAW cells: RAW cells at 2X 10 4 Inoculating the cells per mL in a 96-well plate, culturing for 24h, adding 500 and 1000 μ M eggshell membrane antioxidant peptide solutions (KPLCPP and NDADYPWPH), and culturing for 24h to determine the cell survival rate. The results are shown in the following table 8, and the preferred antioxidant peptides KPLCPP and NDADYPWPH have no toxicity to RAW cells at the concentration of 500 and 1000. Mu.M, which indicates that the eggshell membrane antioxidant peptide identified by the invention has the characteristics of safety and no toxicity.
TABLE 8 cytotoxicity of antioxidant peptides KPLCPP and NDADYPWPH
Preferred antioxidant peptide pairs H 2 O 2 Study of protective effects of induced cellular injury: RAW cells at 2X 10 4 Inoculating each/mL cell in 96-well plate, culturing for 24 hr, adding 1000 μ M egg shell membrane antioxidant peptide solution (dissolved in cell culture solution, filtering with 0.22 μ M filter membrane to remove impurities and bacteria), culturing for 24 hr, adding 175 μ M H into the rest wells except blank wells 2 O 2 The cells were assayed for viability in each well after 1h incubation in solution (in DMEM). The results are shown in table 9 below, after pretreatment of antioxidant peptides KPLCPP and ndadyppwph, cell viability was increased from 50.97% to 62.13% and 78.21%, respectively.
TABLE 9 antioxidative peptides KPLCPP and NDADYPWPH vs H 2 O 2 Results of protective Effect of induced RAW264.7 cells
Example 11
< study on the influence of antioxidant peptide KPLCPP on intracellular antioxidant pathway based on western blot >
RAW264.7 cells (5.1X 10) 5 Cells/well) were seeded into 6-well plates and cultured for 24h. Preferred antioxidant peptides KPLCPP (500 and 1000. Mu.M) were pretreated for 24h, 175. Mu. M H 2 O 2 And treating for 1h. Collection RAW264.7Cells were lysed with RIPA buffer containing PMSF and phosphatase inhibitor. After analysis of the total protein content by BCA, the proteins were separated on a 12% SDS-PAGE gel (40. Mu.g) and transferred to a PVDF membrane. Five milliliters of blocking solution was added, one hour later blocking solution was removed, primary antibody diluted in blocking solution was added, and incubation was performed slowly overnight at 4 ℃. Washing with TBST three times, adding TBST diluted secondary antibody, incubating for one hour at 24 ℃, washing with TBST 3 times, adding ECL liquid to the washed PVDF membrane, allowing chemical reaction for one to two minutes, placing in dark environment, and developing and fixing to obtain the result of western blot.
As shown in fig. 9: in the meridian H 2 O 2 Treatment (B) and not with H 2 O 2 In the case of treatment (A), the antioxidant peptide KPLCPP can up-regulate the expression of Nrf2 and HO-1 protein after treatment, thereby protecting inflammatory cells (RAW 264.7) from H 2 O 2 Damage by induced oxidative stress.
5. Structure-activity relationship research of eggshell membrane antioxidant peptide
Example 12
< study on the influence of antioxidant peptides KPLCPP and CRPPSLGR on intracellular antioxidant pathways based on molecular docking >
Molecular docking is carried out through a CDOCKER program of Discovery Studio (DS) 2018 client software, eggshell membrane antioxidant peptide is used as a ligand, and Keap1 (PDB ID:2 FLU) is used as a receptor. The docking results are expressed as the-CDOCKER interaction energy (-CIE) score, with higher scores indicating higher binding affinity and tighter binding, and the specific values are shown in Table 10.
TABLE 10 molecular docking results of eggshell membrane antioxidant peptides with Keap1
| Peptide sequence | –CIE |
| NDADYPWPH | 53.0792 |
| DFGPGPM | 54.8485 |
| FPMRT | 55.3801 |
| DKDGPFRL | 69.3744 |
| KPLCPP | 52.0045 |
| CRPPSLGR | 57.2673 |
All the eggshell membrane antioxidant peptides related by the invention can be successfully docked with the active site of Keap 1. Among them, KPLCPP and CRPPSLGR are used as the preferred eggshell membrane antioxidant peptides to study the binding mechanism of eggshell membrane antioxidant peptides with Keap 1. The results are shown in fig. 10 and 11, KPLCPP forms 3 conventional hydrogen bonds with Arg380, arg415 and Gln530 of Keap1, 1 charge-attracting interaction with Arg380, 1 carbon hydrogen bond with Ser602, 2 alkyl bonds with Ala556 and Arg415, and 1 pi-alkyl bond with Tyr 525. CRPPSLGR forms 6 conventional hydrogen bonds with Arg415, ser508, arg483 and Ser602 of Keap1, wherein Arg380, arg415, ser602, gln530, tyr525, ser508 and Arg483 are key amino acid residues of the Keap1-Kelch structural domain on the Keap-Nrf 2 interaction binding site, which indicates that the eggshell membrane antioxidant peptide inhibits the ubiquitination degradation of Nrf2 by occupying the binding site of Nrf2 in the Keap1-Kelch structural domain, thereby protecting inflammatory cells from H2 2 O 2 Damage from induced oxidative stress.
The technical solutions provided by the present invention are described in detail in the embodiments, and the principles and embodiments of the present invention are described herein by using specific examples, and the description of the embodiments is only used to help understanding the principles of the embodiments of the present invention; meanwhile, for a person skilled in the art, according to the embodiments of the present invention, there may be variations in the specific implementation manners and application ranges, and in summary, the content of the present description should not be construed as a limitation to the present invention.
Claims (10)
1. An antioxidant peptide, characterized in that the amino acid sequence of the antioxidant peptide is any one of the following sequences:
NDADYPWPH(Asn-Asp-Ala-Asp-Tyr-Pro-Trp-Pro-His)；
DFGPGPM(Asp-Phe-Gly-Pro-Gly-Pro-Met)；
FPMRT(Phe-Pro-Met-Arg-Thr)；
KPLCPP(Lys-Pro-Leu-Cys-Pro-Pro)；
DKDGPFRL(Asp-Lys-Asp-Gly-Pro-Phe-Arg-Leu)；
CRPPSLGR(Cys-Arg-Pro-Pro-Ser-Leu-Gly-Arg)。
2. the antioxidant peptide as claimed in claim 1, wherein the antioxidant peptide is derived from eggshell membrane.
3. A polynucleotide encoding the antioxidant peptide of claim 1.
4. An egg shell membrane hydrolysate, comprising one or more of the following antioxidant peptides:
an antioxidant peptide having an amino acid sequence of NDADYPWPH (Asn-Asp-Ala-Asp-Tyr-Pro-Trp-Pro-His);
an antioxidant peptide with the amino acid sequence of DFGPGPGPM (Asp-Phe-Gly-Pro-Gly-Pro-Met);
an antioxidant peptide with an amino acid sequence of FPMRT (Phe-Pro-Met-Arg-Thr);
an antioxidant peptide with an amino acid sequence of KPLCPP (Lys-Pro-Leu-Cys-Pro-Pro);
an antioxidant peptide with an amino acid sequence of DKDGPFRL (Asp-Lys-Asp-Gly-Pro-Phe-Arg-Leu);
the amino acid sequence is CRPPSLGR (Cys-Arg-Pro-Pro-Ser-Leu-Gly-Arg).
5. Use of the antioxidant peptide of claim 1 or the eggshell membrane hydrolysate of claim 4 in the manufacture of a medicament for osteoarticular use.
6. Use of the antioxidant peptide of claim 1 or the eggshell membrane hydrolysate of claim 4 in the manufacture of an antioxidant medicament.
7. Use of the antioxidant peptide of claim 1 or the eggshell membrane hydrolysate of claim 4 in the manufacture of a bone joint health product.
8. Use of the antioxidant peptide of claim 1 or the eggshell membrane hydrolysate of claim 4 in the manufacture of an antioxidant health product.
9. Use of the antioxidant peptide of claim 1 or the eggshell membrane hydrolysate of claim 4 in a food product.
10. Use of the antioxidant peptide of claim 1 or the eggshell membrane hydrolysate of claim 4 in cosmetics.
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