Preparation method and application of bovine bone marrow protein peptide

Abstract

The present invention relates to a method for preparing and applying bovine bone marrow protein peptides. The present invention isolates bovine bone marrow antioxidant peptide BPP-I-C-2 from bovine bone marrow protein, which exhibits excellent antioxidant properties. The peptide effectively eliminates oxygen free radicals generated by H2O2 stimulation, reduces GSSG content, and maintains a stable GSH / GSSG ratio, thereby providing excellent protection against HEK293 cells damaged by H2O2 oxidation. The antioxidant peptide has promising application prospects when prepared as a pharmaceutical composition.

Description

Technical Field

[0001] The present application relates to the biological field, and specifically to a preparation method and application of bovine bone marrow protein peptides. Background Art

[0002] During metabolic processes, reactive oxygen species (ROS) are generated by the mitochondrial respiratory chain, peroxidases, and oxidases. ROS levels are controlled and maintained within a certain range by enzymatic and non-enzymatic antioxidant defense systems. However, excessive ROS production due to factors such as aging, radiation, alcohol, or medications can lead to oxidative stress, which in turn contributes to many non-communicable chronic diseases such as diabetes, atherosclerosis, inflammation, and cancer. Furthermore, ROS-induced lipid peroxidation is one of the primary causes of lipid degradation in food and cosmetic matrices. Therefore, the search for various exogenous antioxidants has become a growing focus. Currently, widely used chemically synthesized antioxidants, such as tertiary butylhydroquinone (TBHQ), butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and propyl gallate (PG), are subject to certain restrictions in the food and cosmetics industries due to food safety concerns. Therefore, the search for safer antioxidants is an important research direction.

[0003] Antioxidant peptides are a class of natural bioactive peptides that have been extensively studied in recent years. As natural antioxidants, antioxidant peptides possess relatively simple structures, are easily absorbed, have good stability, and are non-immunoreactive. They not only possess strong antioxidant activity but also possess other health benefits, such as lowering blood pressure and preventing cancer. They are gaining increasing attention in the food and healthcare sectors. The antioxidant activity of antioxidant peptides is closely related to their molecular weight, hydrophobicity, and amino acid composition and sequence. Peptides with smaller molecular weights generally exhibit stronger antioxidant activity than those with larger molecular weights because they are more likely to interact with target free radicals and terminate chain reactions. Most antioxidant peptides have molecular masses between 500 and 1800 Da. Hydrophobicity is a key factor influencing the antioxidant activity of peptides. Hydrophobic amino acid residues such as tryptophan, proline, valine, phenylalanine, leucine, alanine, and methionine promote the solubility of peptides at the lipid-water interface, thereby enhancing their free radical scavenging activity. In particular, peptides with leucine or valine at the N-terminus exhibit stronger antioxidant activity. Aromatic amino acids also have an important influence on the antioxidant activity of peptides, because the aromatic groups in tryptophan, phenylalanine and tyrosine residues can scavenge free radicals by providing hydrogen protons. In addition, data show that acidic or basic amino acids such as aspartic acid, glutamic acid, arginine and histidine play an important role in the antioxidant activity of peptides. The charge carried by these amino acid residues directly determines the peptide's resistance to metal ions (such as Fe 2+ and Cu 2+In conclusion, the lower molecular weight, the hydrophobicity of specific amino acid residues, the hydrogen donor function and the chelation of metal ions may be the important reasons why antioxidant peptides have strong antioxidant activity.

[0004] Studies have shown that antioxidant peptides can directly scavenge ROS by donating either hydrogen or electrons. The hydrogen or electron-donating activity of antioxidant peptides depends on the amino acid residues. The aromatic amino acids tyrosine (Tyr), tryptophan (Trp), and phenylalanine (Phe) can donate hydrogen protons to electron-deficient free radicals and maintain stability through resonance structures. The ionized side chain of glutamic acid (Glu) produced by partial deamidation of glutamine (Gln) residues can act as a reducing agent to donate hydrogen protons. The hydrophobic amino acids glycine (Gly), alanine (Ala), leucine (Leu), and proline (Pro) exhibit strong antioxidant properties by transferring electrons to free radicals. Furthermore, their aliphatic side chains enhance the solubility of antioxidant peptides in oils and fats, thereby delaying oil oxidation. Some amino acid residues, such as cysteine ​​(Cys), have both electron and hydrogen donating abilities. The sulfhydryl group of cysteine ​​(Cys) possesses both strong electron and hydrogen donating abilities. Tyr, due to the presence of a hydroxyl group on its aromatic ring, can also donate electrons to free radicals, thereby quenching them. Not only does the type of amino acid residue significantly influence the free radical scavenging ability of antioxidant peptides, but structure-activity relationships, such as peptide sequence, molecular weight, and secondary structure, also play a significant role in their antioxidant activity. The binding of antioxidant peptides to ROS-producing oxidases primarily relies on interactions between their amino acid residues, including van der Waals forces, hydrogen bonds, salt bonds, hydrophobic interactions, and electrostatic interactions. Twenty-nine antioxidant peptides were isolated, purified, and identified from corn silk pancreatic protein hydrolysate. Molecular docking revealed that 14 of these peptides could successfully dock with XO. Among these, NDGPSR exhibited the lowest binding energy and the strongest affinity for XO. Molecular docking revealed that NDGPSR interacts hydrophobically with XO catalytic residues (Glu802 and Arg880), substrate-binding residues (Phe914, Phe1009, and Thr1010), and residues associated with the extended solvent channel leading to the molybdenum active center. It forms hydrogen bonds with Ser876, Thr1010, and Val1011, and salt bonds with His875 and Glu1261. This interaction occupies the XO catalytic center, hindering substrate access and inhibiting XO activity and ROS generation. Furthermore, the amount of Trp in the antioxidant peptides is positively correlated with XO inhibitory activity. Similar to allopurinol (a XO inhibitor), Trp interacts with key XO residues and molybdenum pterin MOS3004, thereby inhibiting XO activity. Two antioxidant peptides, YGRDEISV and LDLVKPQ, identified from hemp seed protein hydrolysate, have also been shown to block the entrance to the MPO active cavity, thereby exerting antioxidant activity. An organism is a complex organic whole. Oxidative stress caused by excessive ROS can cause cell damage and induce diseases such as inflammation, tumors, and aging.Antioxidant peptides can not only directly eliminate ROS; they can also inhibit the production of ROS by chelating pro-oxidant metal ions and reducing the level of ROS-producing oxidases; they can also play a cellular protective role by anti-inflammation, regulating cell apoptosis, activating cell autophagy and enhancing stress resistance, thereby quickly restoring the body's normal redox level.

[0005] Currently, antioxidant peptides are primarily produced by directly isolating and extracting endogenous antioxidant peptides from organisms, degrading edible proteins through enzymatic hydrolysis, acid hydrolysis, or fermentation, or synthesizing them artificially through chemical reactions or DNA recombination. Of these methods, enzymatic hydrolysis has received the most research due to its advantages, including mild conditions, ease of control, high specificity, few byproducts, high safety, and ease of scalability. Research has found that animal and plant proteins are important sources of antioxidant peptides for enzymatic hydrolysis. Currently, the main raw materials for plant protein include soybeans, corn, wheat, peanuts, rice, and rapeseed. Of these, soybeans, corn, and wheat, as grains, and their processed by-products have received the most extensive and in-depth research. Dairy protein is the most common animal protein source, followed by meat protein. Dairy proteins include whole milk, whey protein, and casein, while meat protein is primarily derived from by-products of fish processing. In addition to animal and plant proteins, research on the preparation of antioxidant peptides using edible fungus protein isolates has also been increasing in recent years. For example, antioxidant peptides derived from proteins from edible fungi such as Pleurotus ferulae, Trichoderma lucidum, and Agaricus bisporus have all been reported. Numerous studies have confirmed that animal-derived collagen and peptides possess excellent physicochemical properties and antioxidant activity, demonstrating their potential for application in the food industry. However, the current availability of highly active antioxidant peptides is limited, necessitating further research. Summary of the Invention

[0006] In one aspect of the present invention, bovine bone marrow antioxidant peptide BPP-IC-2 with good antioxidant properties is isolated and identified from bovine bone marrow protein, and its amino acid sequence is shown in SEQ ID NO: 1.

[0007] The abundant hydrophobic amino acids in the peptide sequence of the low molecular weight antioxidant peptide isolated by the present invention can enhance the antioxidant activity by increasing the solubility of the peptide in lipids and interacting with free radical species. The peptide can easily interact with the target free radical to terminate the free radical chain reaction, thereby exerting antioxidant ability.

[0008] More specifically, the present invention also provides a drug with antioxidant efficacy, characterized by containing bovine bone marrow antioxidant peptide BPP-IC-2, whose amino acid sequence is shown in SEQ ID NO: 1.

[0009] More specifically, the present invention also provides a health functional food with antioxidant efficacy, characterized by containing bovine bone marrow antioxidant peptide BPP-IC-2, whose amino acid sequence is shown in SEQ ID NO: 1.

[0010] Specifically, the drug contains a pharmaceutically acceptable carrier or excipient or other active agent.

[0011] The use as described above is characterized in that a second therapeutic agent with antioxidant effect is also added to the medicine.

[0012] Furthermore, the present invention also provides the use of bovine bone marrow antioxidant peptide BPP-IC-2 in the preparation of a drug with antioxidant efficacy. The amino acid sequence of the antioxidant peptide BPP-IC-2 is shown in SEQ ID NO: 1.

[0013] Specifically, the drug contains a pharmaceutically acceptable carrier or excipient or other active agent.

[0014] According to the present invention, the "additional pharmaceutically acceptable excipients" may vary depending on whether the pharmaceutical formulation is solid or liquid (intermediate or finished product). As used herein, a "liquid pharmaceutical formulation" is prepared by mixing the lipophilic, basic or neutral low molecular weight active pharmaceutical ingredient of the desired purity with one or more optional "pharmaceutically acceptable carriers" in the form of an aqueous solution. The "pharmaceutically acceptable carriers" used in conjunction with the "liquid pharmaceutical formulation" are generally non-toxic to the subject at the dosages and concentrations used, and include, but are not limited to: buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol); low molecular weight (less than about 10 residues) polyols. peptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyethylene glycol (PEG) or polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; and polyols such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc protein complexes); and / or ionic and nonionic surfactants, such as sodium lauryl sulfate or poloxamers, respectively.

[0015] The medicaments obtained according to the present invention can be used in a variety of forms for administering the polypeptide components, particularly in oral or topical dosage forms. Exemplary dosage forms include powders or granules that can be taken dry or reconstituted with water to form a paste, slurry, suspension, or solution for oral administration; tablets, capsules, or pills. Various additives can be mixed, ground, or granulated with the dispersions described herein (either molecularly or partically) to form materials suitable for the above dosage forms. Potentially beneficial additives can generally be divided into the following categories: other matrix materials or diluents, surfactants, drug complexing agents or solubilizing agents, fillers, disintegrants, binders, lubricants, and pH adjusters (e.g., acids, bases, or buffers). Examples of other matrix materials, fillers, or diluents include lactose, mannitol, xylitol, microcrystalline cellulose, calcium diphosphate, and starch. Examples of surfactants include sodium lauryl sulfate and polysorbate 80. Examples of drug complexing agents or solubilizing agents include polyethylene glycol, caffeine, xanthenes, gentisic acid, and cyclodextrins. Examples of disintegrants include sodium starch glycolate, sodium alginate, sodium carboxymethylcellulose, methylcellulose, and cross-linked sodium carboxymethylcellulose. Examples of binders include methylcellulose, microcrystalline cellulose, starch, and gums (such as guar gum and tragacanth). Examples of lubricants include magnesium stearate and calcium stearate. Examples of pH adjusters include acids such as citric acid, acetic acid, ascorbic acid, lactic acid, aspartic acid, succinic acid, phosphoric acid, etc.; bases such as sodium acetate, potassium acetate, calcium oxide, magnesium oxide, trisodium phosphate, sodium hydroxide, calcium hydroxide, aluminum hydroxide, etc.; and buffers that typically contain a mixture of an acid and a salt of the acid. In addition to the above-mentioned additives or excipients, it may be useful to formulate and prepare oral or topical dosage forms with the compositions disclosed herein using any conventional materials and procedures known to those skilled in the art.

[0016] For enteral administration, such as oral administration, pharmaceutical compositions can be formulated into various dosage forms. Pharmaceutically acceptable carriers can be solid, semisolid, or liquid. Solid form preparations include powders, tablets, pills, lozenges, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances that can also serve as a diluent, flavoring agent, solubilizer, lubricant, suspending agent, binder, preservative, tablet disintegrant, or encapsulating material. In powders, the carrier is typically a finely divided solid that is mixed with the finely divided active ingredient. In tablets, the active ingredient is typically mixed with a carrier having the necessary binding capacity in a suitable ratio and compressed into the desired shape and size. Suitable carriers include, but are not limited to, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth gum, methylcellulose, sodium carboxymethylcellulose, low-melting wax, cocoa butter, and the like. The preparation may include an encapsulating material as a carrier.

[0017] Other forms suitable for oral administration include liquid form preparations, including emulsions, syrups, elixirs, aqueous solutions, aqueous suspensions, or solid form preparations, which are intended to be converted into liquid form preparations shortly before use. Emulsions can be prepared in solutions, for example, in aqueous propylene glycol solutions, or can contain emulsifiers, such as lecithin, sorbitan monooleate, or gum arabic. Aqueous solutions can be prepared by dissolving the active ingredient in water and adding suitable colorants, flavorings, stabilizers, and thickeners. Aqueous suspensions can be prepared by dispersing the finely divided active ingredient in water with a viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other known suspending agents. Solid form preparations include solutions, suspensions, and emulsions and, in addition to the active ingredient, can also contain colorants, flavorings, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.

[0018] The pharmaceutical compositions can also be administered parenterally, e.g., by injection or infusion, such as by intravenous, intraarterial, intraosseous, intramuscular, intracerebral, intraventricular, intrasynovial, intrasternal, intrathecal, intralesional, intracranial, intratumoral, intradermal, and subcutaneous injection or infusion.

[0019] Therefore, for parenteral administration, the pharmaceutical compositions of the present invention may be in the form of sterile injectable or infusible preparations, for example, as sterile aqueous or oily suspensions. Such suspensions can be formulated using suitable dispersants or wetting agents (e.g., Tween 80) and suspending agents according to techniques known in the art. Sterile injectable or infusible preparations may also be sterile injectable or infusible solutions or suspensions in nontoxic parenterally acceptable diluents or solvents. For example, the pharmaceutical composition may be a 1,3-butanediol solution. Other examples of acceptable carriers and solvents that can be used for the compositions of the present invention include, but are not limited to, mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are commonly used as solvents or suspending media. For this purpose, any bland fixed oil may be used, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives, as well as natural pharmaceutically acceptable oils (e.g., olive oil or castor oil, particularly their polyoxyethylated forms), may be used in the preparation of injectables. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants.

[0020] Typically, the pharmaceutical composition will be administered in a therapeutically effective amount by any acceptable mode of administration for medicaments of similar use. Suitable daily doses are typically in the range of 1-1500 mg, such as 1-500 mg / day, or 1-50 mg / day, but will also depend on many factors, such as the severity of the disease to be treated, the age and relative health of the patient, the route and form of administration, and the indication for which the administration is intended. Those of ordinary skill in the art of treating these diseases will be able to determine the therapeutically effective amount of the pharmaceutical composition proposed herein for a given disease without undue experimentation and relying on personal knowledge and the disclosure of this application.

[0021] Beneficial effects

[0022] The present invention isolated bovine bone marrow antioxidant peptide BPP-IC-2 from bovine bone marrow protein, which exhibits excellent antioxidant properties. The peptide can effectively eliminate oxygen free radicals generated by H₂O₂, reduce GSSG content, and maintain a stable GSH / GSSG ratio, thereby providing excellent protection against HEK293 cells damaged by H₂O₂. The antioxidant peptide has promising application prospects when formulated into a pharmaceutical composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The effect of peptides on TBARS values ​​of lipid oxidation

[0024] Figure 2 Effect of peptides on GSSG content in cells DETAILED DESCRIPTION

[0025] Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and apply the technology of the present invention. The methods, equipment, and materials in the following implementation cases, if not otherwise specified, are conventional methods, equipment, and materials in the art and can be purchased from the market.

[0026] Example 1 Preparation of bovine bone marrow protein peptide

[0027] Take out the frozen bovine bones, cut the bones, wash them with ultrapure water, remove the residual debris and excess meat residues in the bone marrow and wash away the excess blood stains. Place the obtained bovine bone marrow in a medicine-pounding pot and add liquid nitrogen for cryogenic grinding according to a solid-liquid ratio of 1:6 (g / mL). Dissolve the powder with distilled water, and perform reflux extraction 3 times with a magnetic stirrer at 45 °C. The liquid-solid ratios and extraction times for the three extractions are 1:10, 1:7, 1:5 g / mL and 2, 1, 0.5 h respectively. Combine the extraction solutions. Separate the oil and water layers with a separating funnel, collect the water layer, degrease it with petroleum ether 3 times, appropriately concentrate the aqueous solution, dialyze (3500 Da, 48 h), and freeze-dry under vacuum to obtain bovine bone marrow protein powder. Weigh 300 mg of bovine bone marrow protein powder, add 150 mL of distilled water, add alkaline protease, under the conditions of pH 9 and 55 °C, the enzyme addition amount is 2%, hydrolyze for 2 h, then inactivate the enzyme in boiling water for 15 min. Subsequently, add papain, under the conditions of pH 6 and 60 °C, the enzyme addition amount is 2.5%, hydrolyze for 2 h, then inactivate the enzyme in boiling water for 15 min, cool, adjust the pH to 7, centrifuge at 4000 r / min for 20 min, take the supernatant, and measure that the degree of hydrolysis reaches 38.3%. Subsequently, use 3 and 5 kDa molecular weight (MW) retention membranes to separate the supernatant, collect and freeze-dry the three components, named BPP-I (MW < 3 kDa), BPP-II (3 kDa < MW < 5 kDa) and BPP-III (MW > 5 kDa).

[0028] Detect the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging ability of the above three components. Specifically, prepare a test sample solution with a mass concentration of 0.5 mg / mL, accurately pipette 2 mL into a stoppered test tube respectively, add 2 mL of DPPH solution (0.2 mmol / L) to each, place it in a constant temperature incubator (at 37 °C) for 30 min at a constant temperature, and measure the absorbance at 517 nm. The blank control group uses distilled water, and the positive control group uses 2 mL of 0.5 mg / mL ascorbic acid. The scavenging rate of DPPH· is calculated according to the following formula: Scavenging rate = (1 - [A1 - A2] / A0) × 100%, where: A0 - blank group; A1 - sample group; A2 - reference group. The results are shown in Table 1.

[0029] Table 1 Free radical scavenging ability of each component

[0030] Each component Free radical scavenging ability (%) BPP-I 86.34±0.76 BPP-II 56.67±0.48 BPP-III 38.37±0.26 supernatant 58.43±0.42 Positive control 97.86±1.03

[0031] As can be seen from Table 1, the BPP-I (MW < 3 kDa) component has the strongest free radical scavenging ability, indicating that small molecular weight polypeptides are the main components with antioxidant ability.

[0032] The BPP-I (MW <3 kDa) fraction was further separated using a DEAE-52 cellulose column (1.6 × 80 cm), yielding six fractions. Fraction BPP-IC demonstrated the highest scavenging activity against 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) free radicals. This fraction was further separated on a Sephadex G-25 column (2.6 × 160 cm), yielding BPP-IC-2, which exhibited the strongest antioxidant activity. BPP-IC-2, exhibiting high DPPH scavenging activity, was isolated using a C-18 column in an Agilent 1260 HPLC system. The sample was eluted with a linear gradient of acetonitrile containing 0.1% TFA (0% to 40% over 0-40 minutes) at a flow rate of 1.0 mL / min. The peptides were separated based on their absorbance at 214 nm and lyophilized. The amino acid sequence of the isolated peptide was measured on an Applied Biosystems 494 protein sequencer and a quadrupole time-of-flight mass spectrometer (Q-TOF MS) coupled to an electrospray ionization source (ESI), and was shown in SEQ ID NO: 1, with a molecular weight of 2.08 kilodaltons. The polypeptide sequence was artificially synthesized for later use.

[0033] Example 2 Identification of the Antioxidant Properties of Bovine Bone Marrow Antioxidant Peptide BPP-IC-2

[0034] Weigh a certain amount of soybean lecithin and dissolve it in 0.12 mol·L -1 KCl, 5mmol·L -1 Histidine buffer solution (pH 6.8) was prepared to contain 0.2 mg ml -1 The lecithin solution was homogenized and ultrasonicated at 4°C for 45 min. Then, 5 ml of lecithin liposomes were added with 1 ml of bovine bone marrow antioxidant peptide BPP-IC-2 (concentrations were 10 mg / L, 50 mg / L, and 100 mg / L; the positive control was 50 mg / L Vc; the blank control was an equal amount of ultrapure water). 0.1 ml of 50 mmol·L was added to the mixture of liposomes and bovine bone marrow antioxidant peptide BPP-IC-2. -1 FeCl3 and 0.1ml 10mmol·L -1Sodium ascorbate triggers lipid oxidation, and then the sample is placed in a 37°C water bath for 1 hour. The fat oxidation status is studied by measuring thiobarbituric acid reactive substances (TBARS). TBARS determination: Take 1 ml of sample, add 3 ml of thiobarbituric acid solution and 17 ml of trichloroacetic acid-hydrochloric acid solution, mix well, react in a boiling water bath for 30 minutes, cool, take 5 ml of sample and add an equal volume of chloroform, centrifuge at 3000 r / min for 10 minutes, and measure the absorbance at 532 nm. The TBARS value is expressed as milligrams of malondialdehyde per liter of lipid oxidation sample solution. Calculation formula: TBARS (mg·L -1 )=(A 532 / Vs)×9.48. Where A532 is the absorbance of the solution; Vs is the volume of the sample; and 9.48 is a constant. Figure 1 shown.

[0035] from Figure 1 As can be seen from the results, bovine bone marrow antioxidant peptide BPP-IC-2 exhibited strong antioxidant capacity in a dose-dependent manner, with a significant difference compared to the blank control group (P<0.01). At the same concentration, the peptide exhibited stronger antioxidant capacity than the positive control group. At a concentration of 100 mg / L, the TBARS value was as low as (0.14±0.03) mg / L.

[0036] Example 3 Protective Effect of Bovine Bone Marrow Antioxidant Peptide BPP-IC-2 on HEK293 Cells Injured by H2O2

[0037] The density is 6.0×10 4 HEK293 cells were seeded at a concentration of 100 μL / well in a 96-well plate. A blank group, control group, injury group, protection group, and positive control group were set up. The blank group was supplemented with 100 μL of DMEM medium; the control group was supplemented with 80 μL of cell suspension and 20 μL of culture medium; the injury group, protection group, and positive control group were supplemented with 80 μL of cell suspension. Six replicates were set up for each experimental concentration. After incubation for 24 hours at 37°C and 5% CO₂, the protection group was supplemented with 10 μL of peptide solution at a final concentration of 10, 50, or 100 mg / L; the positive control group was supplemented with 10 μL of Vc at a final concentration of 50 mg / L; and the injury group was supplemented with 10 μL of culture medium. After an additional 24 hours of incubation, 10 μL of H₂O₂ solution at a final concentration of 400 μmol / L was added to the injury group, protection group, and positive control group. After an additional 12 hours of incubation, cell viability was determined by MTS assay. The relative viability of the other groups was calculated based on the blank group as 100%. The results are shown in Table 2.

[0038] Table 2 Effects of each group on cell survival rate

[0039] Group Relative cell survival rate (%) 10mg / L peptide protection group 92.14±0.37* 50mg / L peptide protection group 95.13±0.48* 100mg / L peptide protection group 98.76±0.52* Positive control group 93.52±0.47* Injury group 49.93±0.35

[0040] As shown in Table 2, the relative cell survival rate in the H2O2-treated damaged group was (49.93±0.35)%. Treatment of oxidatively damaged HEK293 cells with different peptide concentrations, as well as the positive control group, significantly improved the relative cell survival rate. Furthermore, the improvement was even more pronounced at the same peptide concentration than in the positive control group. Furthermore, the relative cell survival rate increased with increasing peptide concentration, demonstrating the effective efficacy of the peptides presented herein in protecting cells from oxidative damage.

[0041] The above-mentioned groups of cells were operated according to the instructions of the GSSG detection kit to detect the GSSG content in the cells. Specifically, the reduced glutathione (glutathione, GSH) and oxidized glutathione (oxidized GSH, GSSH) detection kits were purchased from Shanghai Biyuntian Biotechnology Co., Ltd. The results are as follows Figure 2 shown.

[0042] Since GSH is a natural antioxidant in cells, it mainly removes oxygen free radicals. Under oxidative stress, it is oxidized to GSSG. H2O2 stimulates cells to produce reactive oxygen species, thereby causing oxidative stress in cells. The presence of antioxidant ingredients can effectively eliminate the oxygen free radicals produced by the body under the stimulation of H2O2, reduce the GSSG content, and maintain the stability of the GSH / GSSG ratio. Figure 2 As shown, the GSSG concentration in the cells of the blank control group was 0.92 μmol / L, while that in the damaged group was significantly increased to 33.41 μmol / L. After the cells were treated with different concentrations of the polypeptide, the intracellular GSSG concentration was significantly reduced. Among them, the GSSG concentration under the action of 100 mg / L polypeptide was 0.93 μmol / L, which was close to the intracellular GSSG concentration of the control group. This fully demonstrates that the polypeptide of the present invention has good antioxidant properties and has a good antioxidant effect.

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

[0044] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in an appropriate manner in any one or more embodiments or examples. Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and purpose of the present invention, and the scope of the present invention is defined by the claims and their equivalents.

Claims

1. A bovine bone marrow antioxidant peptide BPP-IC-2, characterized in that The amino acid sequence is shown in SEQ ID NO:

1.

2. A drug with antioxidant effect, characterized in that The invention contains bovine bone marrow antioxidant peptide BPP-IC-2, the amino acid sequence of which is shown in SEQ ID NO:

1.

3. Use of bovine bone marrow antioxidant peptide BPP-IC-2 in the preparation of a drug with antioxidant efficacy, wherein: The amino acid sequence of the antioxidant peptide BPP-IC-2 is shown in SEQ ID NO:

1.

4. The use according to claim 3, characterized in that The medicine contains a pharmaceutically acceptable carrier.

5. The use according to claim 3, characterized in that The medicine further comprises a second therapeutic agent having an antioxidant effect.

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