Preparation method and application of bovine bone marrow protein peptide

Abstract

The invention relates to a preparation method and application of bovine bone marrow protein peptide. According to the bovine bone marrow antioxidant peptide BPP-I-C-2 and the preparation method thereof, the bovine bone marrow antioxidant peptide BPP-I-C-2 with the good antioxidant characteristic is separated from bovine bone marrow protein, the polypeptide can effectively eliminate oxygen free radicals generated by H2O2 stimulated organisms, reduce the content of GSSG and keep the stability of the GSH / GSSG ratio, and then the good protection effect on HEK293 cells damaged by H2O2 oxidation is achieved. The antioxidant peptide has a good application prospect after being prepared into a pharmaceutical composition.

Description

Technical Field

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

[0002] During the metabolism process of the body, reactive oxygen species (ROS) are generated by the oxidation of the mitochondrial respiratory chain, peroxidase, and oxidase. The level of ROS is controlled by the enzymatic and non-enzymatic antioxidant defense systems and maintained within a certain range. However, if the body produces excessive ROS due to factors such as aging, radiation, alcohol, or drugs, it will lead to oxidative stress in the body, thereby causing many non-communicable chronic diseases, such as diabetes, atherosclerosis, inflammation, and cancer. In addition, lipid peroxidation caused by ROS is one of the main reasons for lipid degradation in food and cosmetic matrices. Therefore, seeking various exogenous antioxidants has increasingly become the focus of people. Currently, widely used chemical synthetic antioxidants in the market, such as tert-butylhydroquinone (TBHQ), butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate (PG), etc., are subject to certain restrictions in the food and cosmetic industries due to their food safety issues. Finding safer antioxidants is an important research direction.

[0003] Antioxidant peptides are a class of natural bioactive peptides that have been widely studied in recent years. As a natural antioxidant, antioxidant peptides have a relatively simple structure, are easy to absorb, have good stability, and no immunoreactivity. They not only have strong antioxidant activity but also have other health care effects such as blood pressure lowering and anti-cancer, and are increasingly valued in the fields of food and healthcare products. The antioxidant activity of antioxidant peptides is closely related to their molecular weight, hydrophobicity, and amino acid composition and sequence. Peptides with a smaller molecular weight usually have stronger antioxidant activity than those with a larger molecular weight because they are more likely to interact with target free radicals and terminate the chain reaction. Most antioxidant peptides have a molecular weight of 500 - 1800 Da. Hydrophobicity is one of the key factors affecting the antioxidant activity of peptides. Hydrophobic amino acid residues such as tryptophan, proline, valine, phenylalanine, leucine, alanine, and methionine can promote the dissolution of peptides at the lipid-water interface, thereby better playing the role of scavenging free radicals. Especially when leucine or valine is located at the N-terminus, the antioxidant activity of the peptide is usually stronger. Aromatic amino acids also have an important impact 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 shows 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, and the charges carried by these amino acid residues directly determine the ability of the peptide to chelate metal ions (such as Fe 2+ and Cu 2+) chelating ability. In summary, lower molecular weight, hydrophobicity of specific amino acid residues, hydrogen donor effect, and chelating metal ion effect may be important reasons for the strong antioxidant activity of antioxidant peptides.

[0004] Studies have shown that antioxidant peptides can directly scavenge ROS through hydrogen donation or electron donation. The hydrogen donation or electron donation of antioxidant peptides depends on their amino acid residues. Aromatic amino acids tyrosine (Tyr), tryptophan (Trp), and phenylalanine (Phe) can provide 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 contribute hydrogen protons. Hydrophobic amino acids glycine (Gly), alanine (Ala), leucine (Leu), and proline (Pro) show strong antioxidant properties by transferring electrons to free radicals. On the other hand, their fatty side chains can enhance the solubility of antioxidant peptides in oils, thereby delaying oil oxidation. Some amino acid residues can both donate electrons and hydrogen. For example, the sulfhydryl group of cysteine (Cys) residues not only has a strong electron-donating ability but also a strong hydrogen-donating ability. Due to the presence of a hydroxyl group on its aromatic ring, Tyr can also donate electrons to free radicals to quench them. Not only does the type of amino acid residue have a significant impact on the free radical scavenging ability of antioxidant peptides, but the structure-activity relationships such as the peptide sequence, molecular weight, and secondary structure of antioxidant peptides also cannot be ignored. The binding of antioxidant peptides to ROS-producing oxidases mainly depends on the interactions between their amino acid residues, including van der Waals forces, hydrogen bonds, salt bonds, hydrophobic, and electrostatic interactions. Twenty-nine antioxidant peptides were isolated, purified, and identified from corn silk tryptic hydrolysates. Molecular docking found that 14 of them could successfully dock with XO. Among them, NDGPSR had the lowest binding energy and the strongest affinity when docking with XO. Molecular docking showed that NDGPSR had hydrophobic interactions with the catalytic residues (Glu802 and Arg880), substrate-binding residues (Phe914, Phe1009, and Thr1010) of XO, and residues related to the extended solvent channel leading to the molybdenum active center: formed hydrogen bonds with serine (Ser) 876, Thr1010, and valine (Val) 1011, and formed salt bonds with His875 and Glu1261, thus occupying the catalytic center of XO, hindering the entry of the substrate, and inhibiting XO activity and the generation of ROS. In addition, the number of Trp in antioxidant peptides was positively correlated with XO inhibitory activity. Similar to allopurinol (an XO inhibitor), Trp interacted with the key residues of XO and molybdopterin MOS3004, thereby inhibiting XO activity. There are also studies on two antioxidant peptides identified from hemp seed protein hydrolysates, YGRDEISV and LDLVKPQ, which can block the entrance of the MPO active cavity, thereby playing an antioxidant role. 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 scavenge ROS; inhibit the production of ROS by chelating pro-oxidant metal ions and reducing the levels of ROS-producing oxidases; but also play a cytoprotective role by anti-inflammatory, regulating apoptosis, activating autophagy and enhancing stress resistance, and rapidly restore the normal redox level of the body.

[0005] At present, the preparation methods of antioxidant peptides mainly include directly isolating and extracting endogenous antioxidant peptides from organisms, or preparing antioxidant peptides by proteolytic enzyme hydrolysis, acid hydrolysis or fermentation of edible proteins, or artificially synthesizing antioxidant peptides by chemical reactions or DNA recombination methods. Among these methods, enzymatic hydrolysis has been studied the most because of its mild conditions, easy control, strong specificity, few by-products, high safety and easy promotion. It has been found that animal and plant proteins are important raw material sources for the production of antioxidant peptides by enzymatic hydrolysis. Currently, the main raw materials of plant proteins are soybeans, corn, wheat, peanuts, rice and rapeseed, etc. Among them, soybeans, corn and wheat as grains and their processed by-products have been studied more and more deeply. Animal protein raw materials are mostly milk proteins, followed by meat proteins. Milk proteins include whole milk, whey protein and casein, etc., while meat proteins are mostly by-products of fish processing. In addition to animal and plant proteins, in recent years, more and more studies have been conducted on the preparation of antioxidant peptides from edible mushroom isolated proteins. For example, antioxidant peptides derived from edible mushroom proteins such as Pleurotus eryngii, Pleurotus ostreatus and Agaricus bisporus have been reported. A number of studies have confirmed that animal-derived bone collagen and peptides have good physicochemical properties and antioxidant activities, and have good application potential in food industrial processing. However, at present, the types of antioxidant peptides with high activity are not enough, and further research needs to be strengthened. Summary of the Invention

[0006] On the one hand, the present invention isolates and identifies bovine bone marrow antioxidant peptide BPP-I-C-2 with good antioxidant properties from bovine bone marrow protein, and its amino acid sequence is shown as SEQ ID NO: 1.

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

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

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

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

[0011] The use as described above is characterized in that a second therapeutic agent with antioxidant efficacy is further added to the drug.

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

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

[0014] According to the present invention, the "additional pharmaceutically acceptable excipients" can vary according to solid or liquid pharmaceutical preparations (intermediates or finished products). As used herein, a "liquid pharmaceutical preparation" is prepared by mixing the lipophilic basic or neutral low-molecular-weight active pharmaceutical ingredient of the required purity with one or more optional "pharmaceutically acceptable carriers" in the form of an aqueous solution. The "pharmaceutically acceptable carriers" used in combination with the "liquid pharmaceutical preparation" are generally non-toxic to the subject at the doses 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 octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl esters of p-hydroxybenzoic acid such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol); low molecular weight (less than about 10 residues) polypeptides; 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 dextrin; chelating agents such as EDTA; and polyols such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (such as zinc protein complexes); and / or ionic and non-ionic surfactants such as sodium dodecyl sulfate or poloxamer.

[0015] The medicaments obtained according to the present invention can be used in various forms for the administration of the polypeptide component, particularly for 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, milled or granulated with the dispersions (molecular or particulate dispersions) as described herein to form materials suitable for the above-mentioned dosage forms. Potentially beneficial additives can generally be classified into the following categories: other matrix materials or diluents, surfactants, drug complexing agents or solubilizers, fillers, disintegrants, binders, lubricants and pH regulators (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 solubilizers include polyethylene glycol, caffeine, xanthene, gentisic acid and cyclodextrin. Examples of disintegrants include sodium starch glycolate, sodium alginate, sodium carboxymethyl cellulose, methyl cellulose and cross-linked sodium carboxymethyl cellulose. Examples of binders include methyl cellulose, microcrystalline cellulose, starch and gums (such as guar gum and tragacanth gum). Examples of lubricants include magnesium stearate and calcium stearate. Examples of pH regulators 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 generally contain a mixture of an acid and a salt of the acid. In addition to the above 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 methods of operation known to those skilled in the art.

[0016] For enteral, e.g., oral, administration, the pharmaceutical composition can be formulated into various dosage forms. The pharmaceutically acceptable carrier can be solid, semi-solid or liquid. Solid forms of the preparation include powders, tablets, pills, lozenges, capsules, cachets, suppositories and dispersible granules. The solid carrier can be one or more substances, which can also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrants or encapsulating materials. In a powder, the carrier is usually a finely divided solid, which is a mixture with the finely divided active ingredient. In a tablet, the active ingredient is usually 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, methyl cellulose, sodium carboxymethyl cellulose, low melting wax, cocoa butter, etc. The preparation can 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 intended to be converted to a liquid - form preparation shortly before use. Emulsions can be prepared in solution, for example, in an aqueous propylene glycol solution, or can contain emulsifying agents such as lecithin, sorbitan monooleate, or gum arabic. Aqueous solutions can be prepared by dissolving the active ingredient in water and adding suitable coloring agents, flavoring agents, stabilizers, and thickening agents. 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 well - known suspending agents. Solid - form preparations include solutions, suspensions, and emulsions and, in addition to the active ingredient, can contain coloring agents, flavoring agents, stabilizers, buffering agents, artificial and natural sweeteners, dispersing agents, thickening agents, solubilizing agents, etc.

[0018] The pharmaceutical composition can also be administered parenterally, for example, by injection or infusion, such as by intravenous, intra - arterial, intra - osseous, intramuscular, intracerebral, intraventricular, intrasynovial, intrasternal, intrathecal, intralesional, intracranial, intratumoral, intradermal, and subcutaneous injection or infusion.

[0019] Thus, for parenteral administration, the pharmaceutical composition of the present invention can be in the form of a sterile injectable or infusible preparation, such as a sterile aqueous or oily suspension. This suspension can be formulated using suitable dispersing or wetting agents (such as Tween 80) and suspending agents according to techniques known in the art. The sterile injectable or infusible preparation can also be a sterile injectable or infusible solution or suspension in a non - toxic parenterally acceptable diluent or solvent. For example, the pharmaceutical composition can be a 1,3 - butanediol solution. Other examples of acceptable carriers and solvents that can be used in the compositions of the present invention include, but are not limited to, mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, a sterile fixed oil is commonly used as a solvent or suspending medium. For this purpose, any mild fixed oil can be used, including synthetic mono - or di - glycerides. Fatty acids, such as oleic acid and its glyceride derivatives, and natural pharmaceutically acceptable oils (such as olive oil or castor oil, especially their polyoxyethylated forms), can be used in the preparation of injectables. These oil solutions or suspensions can also contain long - chain alcohol diluents or dispersing agents.

[0020] Typically, the pharmaceutical composition will be administered in a therapeutically effective amount by any acceptable mode of administration of agents for similar uses. Suitable daily doses are generally 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 status of the patient, the route and form of administration, and the indication for which the administration is targeted, etc. 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 has isolated bovine bone marrow antioxidant peptide BPP-I-C-2 with good antioxidant properties from bovine bone marrow protein. The polypeptide can effectively eliminate the oxygen free radicals generated by H2O2-stimulated organisms, reduce the content of GSSG, maintain the stability of the GSH / GSSG ratio, and thus has a good protective effect on HEK293 cells damaged by H2O2 oxidation. The antioxidant peptide has good application prospects after being prepared into a pharmaceutical composition. Brief description of the drawings

[0023] Figure 1 Influence result diagram of TBARS value of the influence of polypeptide on lipid oxidation degree

[0024] Figure 2 Influence result diagram of the influence of polypeptide on the content of GSSG in cells Detailed implementation manners

[0025] Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant personnel can obviously make changes or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention. The methods, equipment, and materials in the following implementation cases, if not specifically stated, are all 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 bone, cut the bovine bone, wash it 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 mortar 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 reflux extract it 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 separatory funnel, collect the water layer, defat it 3 times with petroleum ether. Appropriately concentrate the aqueous solution, dialyze (3500 Da, 48 h), and vacuum freeze-dry 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, inactivate the enzyme in boiling water for 15 min, then add papain, under the conditions of pH 6 and 60 °C, the enzyme addition amount is 2.5%, hydrolyze for 2 h, 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 determine that the degree of hydrolysis reaches 38.3%. Subsequently, use 3 and 5 kDa molecular weight (MW) cut-off 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) 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 of it 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 (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 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 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), and a total of 6 fractions were obtained. Among them, the BPP-I-C fraction was found to have the highest scavenging ability by detecting the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging ability. The BPP-I-C was further separated on a Sephadex G-25 column (2.6 × 160 cm), and the fraction BPP-I-C-2 with the strongest antioxidant ability was obtained. The BPP-I-C-2 with high DPPH scavenging activity was separated 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% - 40% within 0 - 40 minutes) at a flow rate of 1.0 ml / min, and the small peptides were separated based on the absorbance at 214 nm and freeze-dried. The amino acid sequence of the separated peptide was measured on an Applied Biosystems 494 protein sequencer and a quadrupole time-of-flight mass spectrometer (Q-TOF MS) coupled with an electrospray ionization source (ESI) as shown in SEQ ID NO: 1, with a molecular weight of 2.08 kilodaltons. The polypeptide sequence was synthesized artificially and reserved for later use.

[0033] Example 2 Identification of the Antioxidant Characteristics of Bovine Marrow Antioxidant Peptide BPP-I-C-2

[0034] Weigh a certain amount of soybean lecithin and dissolve it in 0.12 mol·L -1 KCl, 5 mmol·L -1 histidine buffer solution (pH 6.8) to prepare a lecithin solution containing 0.2 mg·ml -1 lecithin. Homogenize it and sonicate it at 4°C for 45 min using ultrasonic waves. Then take 5 ml of the lecithin liposomes, add 1 ml of bovine marrow antioxidant peptide BPP-I-C-2 (concentrations are 10 mg / L, 50 mg / L, 100 mg / L respectively; set the positive control as 50 mg / L Vc; the blank control group is an equal amount of ultrapure water), and add 0.1 ml of 50 mmol·L -1 FeCl3 and 0.1 ml of 10 mmol·L -1Sodium ascorbate induces lipid oxidation, and then the sample is incubated in a 37 °C water bath for 1 h. The lipid oxidation is studied by measuring the thiobarbituric acid reactive substances (TBARS). Determination of TBARS: Take 1 ml of the sample, add 3 ml of thiobarbituric acid solution and 17 ml of trichloroacetic acid-hydrochloric acid solution. After mixing, react in a boiling water bath for 30 min, cool, take 5 ml of the sample, add an equal volume of chloroform, centrifuge at 3000 r / min for 10 min, and measure the absorbance at 532 nm. The TBARS value is expressed as the milligrams of malondialdehyde in each liter of the lipid oxidation sample solution. Calculation formula: TBARS (mg·L -1 ) = (A 532 / Vs) × 9.48. In the formula, A532 is the absorbance of the solution; Vs is the volume of the sample; 9.48 is a constant. The results are as Figure 1 shown.

[0035] It can be seen from Figure 1 that the bovine bone marrow antioxidant peptide BPP-I-C-2 has good antioxidant ability with dose-dependence, and there is a significant difference compared with the blank control group (P < 0.01); under the same concentration condition, the polypeptide has stronger antioxidant ability compared with the positive control group. When the concentration is 100 mg / L, the TBARS value is as low as (0.14 ± 0.03) mg / L.

[0036] Example 3 Protective effect of bovine bone marrow antioxidant peptide BPP-I-C-2 on HEK293 cells damaged by H2O2

[0037] Inoculate HEK293 cells with a density of 6.0×10 4 cells / mL into a 96-well plate, and inoculate 80 μL in each well. Set up a blank group, a control group, a damage group, a protection group, and a positive control group respectively. Add 100 μL of DMEM medium to the blank group; add 80 μL of cell suspension and 20 μL of medium to the control group; add 80 μL of cell suspension to the damage group, the protection group, and the positive control group. Set 6 replicates for each experimental concentration. After incubating in a 37 °C, 5% CO2 incubator for 24 h, add 10 μL of peptide solution with a final concentration of 10, 50, 100 mg / L to the protection group, add 10 μL of Vc with a final concentration of 50 mg / L to the positive control group, and at the same time add 10 μL of medium to the damage group. After continuing to incubate for 24 h, add 10 μL of H2O2 solution with a final concentration of 400 μmol / L to the damage group, the protection group, and the positive control group at the same time. After continuing to incubate for 12 h, measure the cell viability by the MTS method, and calculate the relative viability of other groups based on the 100% viability of the blank group. The results are shown in Table 2.

[0038] Table 2 Effects of each group on cell viability

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

[0040] As can be seen from the results in Table 2, the relative cell survival rate of the H2O2-treated injury group was (49.93 ± 0.35)%. After treating the oxidatively damaged HEK293 cells with peptides at different concentrations and the positive control group, the relative cell survival rate was significantly increased. Moreover, the improvement effect of the same concentration of peptide was more obvious than that of the positive control group. And with the increase of the peptide concentration, the relative cell survival rate increased accordingly, which fully demonstrated that the peptide of the present invention has a good effect on cell oxidative damage.

[0041] The cells in each of the above groups 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 Beyotime Biotechnology Co., Ltd. The results are as Figure 2 shown.

[0042] Since GSH is a natural antioxidant in cells, mainly responsible for scavenging oxygen free radicals, it is oxidized into GSSG by itself under oxidative stress. H2O2 stimulates cells to produce reactive oxygen species, thereby causing oxidative stress in cells. The presence of antioxidant components can effectively eliminate the oxygen free radicals produced by H2O2 stimulation of the body, reduce the GSSG content, and maintain the stability of the GSH / GSSG ratio. As Figure 2 shown, the GSSG concentration in the cells of the blank control group was 0.92 μmol / L, while that in the injury group increased significantly to 33.41 μmol / L. After treating the cells with peptides at different concentrations, the intracellular GSSG concentration decreased significantly. Among them, the GSSG concentration under the action of 100 mg / L peptide was 0.93 μmol / L, close to the GSSG concentration in the control group cells. This fully demonstrated that the polypeptide of the present invention has good antioxidant properties and a good antioxidant effect.

[0043] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention, not to limit it; although the present invention has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

[0044] In the description of this specification, the descriptions referring to terms such as "one embodiment", "some embodiments", "examples", "specific examples", or "some examples", etc., mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and purposes of the present invention, and the scope of the present invention is defined by the claims and their equivalents.

Claims

1. An antioxidant peptide BPP-I-C-2 from bovine bone marrow, characterized in that The amino acid sequence is shown as SEQ ID NO:

1.

2. A drug with antioxidant efficacy, characterized in that It contains bovine bone marrow antioxidant peptide BPP-I-C-2, and its amino acid sequence is shown as SEQ ID NO:

1.

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

1.

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

5. The use according to claim 3, characterized in that A second therapeutic agent with antioxidant efficacy is also added to the described drug.

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