A high-efficiency donkey-hide gelatin peptide with blood supplementing and nourishing functions and a preparation method thereof

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a method for preparing bioactive polypeptides and their applications. This invention prepares bioactive polypeptides from donkey-hide gelatin protein through an enzymatic hydrolysis reaction. The bioactive polypeptides possess iron-chelating ability, enhancing Hb and RBC production in a mouse anemia model. Therefore, the polypeptides of this invention have blood-nourishing and blood-tonifying functions, are non-toxic, have no side effects, and also have advantages such as simple preparation, easy absorption, and ease of widespread application.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an active polypeptide derived from donkey skin, its application in the treatment of anemia, and its preparation method. Background Technology

[0002] Anemia is frequently detected in clinical settings and can occur along with other diseases. When the hemoglobin content, hematocrit, and red blood cell count per unit volume of peripheral blood are below the standard, anemia can be preliminarily diagnosed

[50] . According to statistics, nearly one-third of the global population currently suffers from anemia. The proportion of people with anemia in my country is even higher, with a prevalence of 64% in women and about 30-40% in infants and young children.

[0003] Colla Corii Asini, a traditional Chinese medicine, is revered as a "holy medicine" for replenishing blood. It is a solid gelatin made from the dried or fresh hide of the donkey (Equus equina), after the hair has been removed. The hide is then boiled and concentrated with appropriate amounts of rice wine, soybeans, and rock sugar until it reaches a thick paste. The main components of Colla Corii Asini include proteins, amino acids, trace elements, polysaccharides, chondroitin sulfate, and hyaluronic acid. The proteins include donkey serum albumin, donkey collagen α1(I) type, and donkey collagen α2(I) type, with approximately 60%–85% being protein, with serum albumin having the highest content. Studies have shown that Colla Corii Asini collagen plays an important role in repair and regeneration in the human body, particularly in promoting skin health, enhancing bone toughness, repairing soft tissues, and improving joint function. Furthermore, Colla Corii Asini is rich in various amino acids, such as glycine, proline, lysine, and glutamic acid. These amino acids are not only crucial for maintaining human health but also enhance the body's antioxidant capacity and alleviate oxidative stress-induced cell damage. The carbohydrate components in donkey-hide gelatin include polysaccharides such as hyaluronic acid and dermatan sulfate. These components significantly promote skin health, effectively moisturize, and have anti-aging effects. Donkey-hide gelatin also contains polypeptides, one of its important active ingredients. These smaller molecular weight active ingredients exhibit significant biological activities in areas such as immune regulation, antioxidation, and anti-inflammation. Especially in studies focusing on donkey-hide gelatin polypeptides with a molecular weight below 3000 Da, these peptides have demonstrated remarkable health benefits in areas such as immune regulation, promoting blood replenishment and white blood cell production, antioxidation, and alleviating the progression of Alzheimer's disease. These unique bioactive functions indicate that donkey-hide gelatin polypeptides have broad potential for health applications. Summary of the Invention

[0004] In view of the current state of the technology, the purpose of this invention is to provide a bioactive polypeptide, specifically a donkey-hide gelatin-based active polypeptide, which has iron-chelating ability and enhances Hb and RBC production in a mouse anemia model, and can be used to treat anemia. The protein extract of this invention not only has the function of nourishing and replenishing blood, but is also non-toxic, has no side effects, and has the advantages of simple preparation and easy absorption.

[0005] The present invention first provides a bioactive polypeptide, characterized in that the polypeptide is an active polypeptide of donkey-hide gelatin, and its amino acid sequence is shown in SEQ ID NO: 1.

[0006] Another aspect of the present invention provides a nucleic acid molecule encoding the bioactive polypeptide described herein.

[0007] Another aspect of the present invention provides a carrier comprising the nucleic acid molecule described herein.

[0008] Another aspect of the present invention provides an isolated host cell comprising the bioactive polypeptide, the nucleic acid molecule, or the carrier described in the present invention.

[0009] Another aspect of the present invention provides the use of the bioactive polypeptide in the preparation of a medicament for treating anemia.

[0010] In some embodiments, the anemia is hemorrhagic anemia.

[0011] In some embodiments, the anemia is iron deficiency anemia.

[0012] In some embodiments, the anemia is nutritional anemia.

[0013] Another aspect of the present invention provides a pharmaceutical composition comprising an effective amount of the bioactive polypeptide of the present invention, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0014] The bioactive polypeptides of the present invention, or pharmaceutical compositions containing the bioactive polypeptides of the present invention, are administered in the form of lyophilized powder.

[0015] In some embodiments, the bioactive polypeptides of the present invention or pharmaceutical compositions containing the bioactive polypeptides of the present invention may be used in combination with other drugs for treating anemia.

[0016] In some embodiments, the anemia treatment drug may be an iron supplement, donkey-hide gelatin, and / or donkey-hide gelatin extract.

[0017] In some embodiments, the iron agent may be ferrous sulfate, ferrous fumarate, ferrous lactate, ferrous succinate, polysaccharide iron complex, dextran iron, or ferrous gluconate.

[0018] Beneficial effects This invention extracts and prepares an active polypeptide from donkey-hide gelatin. This active polypeptide possesses iron-chelating ability and enhances Hb and RBC production in a mouse anemia model. Therefore, the polypeptide of this invention has excellent therapeutic effects on anemia and has certain clinical application value. The polypeptide AG61 provided by this invention is easy to synthesize, low in cost, and easy to promote and apply. Attached Figure Description

[0019] Figure 1 The results shown are the Hb levels in the blood of mice in each group after different treatments.

[0020] Figure 2 The results shown are the RBC content in the blood of mice in each group after different treatments. Detailed Implementation

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0022] Except as provided in the operational examples or otherwise indicated, all figures for the amount of expressed components or reaction conditions used herein should be understood to be modified by the term "about" in all cases. When used in conjunction with percentages, the term "about" may mean ±1%.

[0023] In some embodiments, the pharmaceutical compositions provided herein comprise about 1 pg to about 2000 mg of the active polypeptide described herein (e.g., a polypeptide component, which may be a single polypeptide in some embodiments), optionally wherein the pharmaceutical composition comprises about 1 pg to about 1000 mg, about 1 pg to about 500 mg, about 1 pg to about 400 mg, about 1 pg to about 300 mg, about 1 pg to about 200 mg, about 1 pg to about 100 mg, about 1 pg to about 50 mg, about 1 pg to about 25 mg, about 1 pg to about 20 mg, about 1 pg to about 15 mg, about 1 pg to about 10 mg, about 1 pg to about The active polypeptides described herein (e.g., polypeptide components, which in some embodiments may be a single polypeptide) of 5 mg, about 1 pg to about 1 mg, about 1 pg to about 500 pg, about 1 pg to about 250 pg, about 1 pg to about 200 pg, about 1 pg to about 150 pg, about 1 pg to about 100 pg, about 1 pg to about 50 pg, about 1 mg to about 1000 mg, about 1 mg to about 500 mg, about 1 mg to about 400 mg, about 1 mg to about 300 mg, about 1 mg to about 200 mg, about 1 mg to about 100 mg, about 1 mg to about 50 mg, about 1 mg to about 25 mg.

[0024] As used herein, the terms “treatment” or “improvement” are used interchangeably. These terms refer to the means by which a beneficial or desired outcome is achieved, including but not limited to therapeutic and / or preventative benefits.

[0025] In this application, unless otherwise specifically stated, the use of the singular includes the plural. In this application, unless otherwise stated, the use of “or” means “and / or”. Furthermore, the use of the term “including” and other forms such as “includes” and “included” is not restrictive. Additionally, unless otherwise specifically stated, terms such as “element” or “component” cover elements and components that include one unit as well as elements and components that include more than one subunit. Additionally, the use of the term “part” can include a portion of a part or an entire portion. Throughout this specification, unless the context requires otherwise, the word “comprise” or variations such as “comprises” or “comprising” will be understood to imply inclusion of the specified integer or group of integers, but not to exclude any other integer or group of integers.

[0026] The term "therapeuticly effective amount" refers to the amount that produces the desired effect of its administration. In some embodiments, the term refers to an amount sufficient to treat a disease, condition, and / or ailment when administered according to a therapeutic dosing regimen to a population suffering from or susceptible to such a disease, condition, and / or ailment. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and / or severity of one or more symptoms of a disease, condition, and / or ailment, and / or delays its onset. Those skilled in the art will understand that a therapeutically effective amount does not necessarily achieve successful treatment in every particular individual. Rather, a therapeutically effective amount can be an amount that provides a specific desired pharmacological response in a large number of subjects when administered to patients who require such treatment. In some embodiments, references to a therapeutically effective amount can be to an amount measured, such as in one or more specific tissues (e.g., tissues affected by a disease, condition, or ailment) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those skilled in the art will understand that in some embodiments, a specific agent or therapy can be formulated and / or administered in a single dose. In some implementations, the therapeutic agent may be formulated and / or administered in multiple doses, for example, as part of a dosing regimen.

[0027] The pharmaceutical composition of the present invention further contains a pharmaceutically acceptable carrier.

[0028] Furthermore, the pharmaceutical compositions of the present invention can be oral dosage forms. Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active polypeptide is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following components: (a) fillers or solubilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin wax; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. In capsules, tablets, and pills, the dosage form may also contain a buffer. Solid dosage forms, such as tablets, sugar pills, capsules, pellets, and granules, can be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opaque agents, and the release of the active peptide or peptide in such compositions may be delayed at a site in the digestive tract. Examples of encapsulating components that may be used are polymeric substances and waxes. If necessary, the active peptide may also be formed into microcapsules with one or more of the excipients described above. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active peptide, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances.

[0029] The provided formulation may include lyophilization protectants, such as those selected from sucrose, lactose, trehalose, dextran, erythritol, arabinitol, xylitol, sorbitol, maltose, lactulose, maltulose, glucol, maltitol, lactitol, isomaltulose, and mannitol; amino acids, such as arginine, histidine, proline, or glycine; lyotropic salts, such as magnesium sulfate; propylene glycol, glycerol, poly(ethylene glycol), or poly(propylene glycol); gelatin, dextrin, modified starch, carboxymethyl cellulose, and combinations thereof.

[0030] Alternatively or additionally, in some embodiments, the provided formulation may comprise a penetration enhancer, such as one selected from bile salts, such as sodium trihydroxycholate, sodium glycocholate, sodium taurocholate and dihydroxycholate, sodium deoxycholate, sodium glycodeoxycholate, sodium taurodeoxycholate; fatty acids, their salts and esters, such as oleic acid, lauric acid, cod liver oil extract, sodium lauryl laurate, sodium decanoate, glyceryl monostearate, diethylene glycol monoethyl ether and various sucrose fatty acid esters, medium-chain fatty acid glycerides, polycaprolactone eoma-3 fatty acids, lecithin (phosphatidylcholine), lysophosphatidylcholine; surfactants, such as sodium lauryl sulfate, polysorbate (polysorbate 80), lauryl ether, Brijs and benzalkonium chloride; complexing agents, such as cyclodextrin, dextran sulfate, ethylenediaminetetraacetic acid sodium salt; complexing agents, such as cyclodextrin, dextran sulfate, ethylenediaminetetraacetic acid sodium salt; Dextran, sodium EDTA, cosolvents such as ethanol and propylene glycol, a combination of 1% oleic acid and 5% / 10% polyethylene glycol 200, a combination of 2% glyceryl monolaurate and 40% alcohol, sodium decanoate and alcohol or propylene glycol, a combination of 10% lauric acid in propylene glycol, polyoxyethylene, 2,3-lauryl ether, menthol, sodium decanoate, sodium octanoate, sodium glycinate, ethylene glycol; polysaccharides such as chitosan and chitosan glutamate; and others It includes, for example, aprotinin, benzalkonium chloride, hexadecylpyridinium chloride, hexadecyltrimethylammonium bromide, sodium salicylate, lysophosphatidylcholine, methoxysalicylate, hydroxymethyl oleate, sodium EDTA, sulfoxide, various alkyl glycosides, ethylenediaminetetraacetic acid (EDTA), tartaric acid; lyotropic salts, such as magnesium sulfate; propylene glycol, glycerol, poly(ethylene glycol) or poly(propylene glycol); gelatin, dextrin, modified starch, carboxymethyl cellulose and combinations thereof.

[0031] Optionally or additionally, in some embodiments, the provided formulation may include an absorption enhancer, such as one selected from surfactants, cholesterol, glycerides, salicylates, bile salts, chelating agents, sodium decanoate, salts of decanoic acid, and others including N-(5-chlorosalicylic acid)-8-aminooctanoic acid (5-CNAC), 4-((4-chloro-2-hydroxybenzoyl))-amino)butyric acid (4-CNAB), and N-(8-(2-hydroxybenzoyl))-amino)octanoic acid, also known as sodium salicylate (SNAC, octanoic acid, C8, castor oil, medium chain, acylcarnitine, EDTA, glyceryl monolaurate, bovine P-casein, tocopheryl succinate glycol chitosan conjugate, lecithin, glyceryl monostearate (GMS), chitosan, and alginate. PLGA, silica, stearic acid, oleic acid, hydrogenated castor oil and trimyridine glyceryl, etoposide phosphate, enalapril maleate, ramipril, olmesartan medoxomil, valacyclovir, midodrine, gabapentin enalacarbide, sulfasalazine, or alternatively or additionally, in some embodiments, the provided formulation may contain a mucosal bioadhesive, such as selected from sucrose, lactose, trehalose, dextran, erythritol, arabinitol, xylitol, sorbitol, maltose, lactulose, maltulose, glucosyl alcohol, maltitol, lactitol, isomaltulose and mannitol; amino acids, such as arginine or histidine or proline or glycine; lyotropic salts, such as magnesium sulfate; propylene glycol, glycerol, poly(ethylene glycol) or poly(propylene glycol);Gelatin, dextrin, modified starch, carboxymethyl cellulose and combinations thereof; mucosal adhesion systems, such as those derived from natural sources, such as gelatin, agarose, chitosan, hyaluronic acid, and synthetic polymers, such as polyvinylpyrrolidone (PVP), polyacrylates, polyvinyl alcohol, sodium carboxymethyl cellulose (SCMC), and pectin; all anionic polymers, chitosan (cationic), and hydroxypropyl methyl cellulose (HPMC) as a nonionic polymer; polyacrylic acid (PAA) derivatives (CP934, CP940, PCP), 15% CMC and 35% CP; copolymers of acrylic acid and poly(ethylene glycol) monomethyl ether monomethacrylate (PEGMM); eudragitlNE40D is a neutral poly(ethyl acrylate methacrylate); hydrophilic polymers, such as methocel K4M, methocel K15M, SCMC 400, Cekol 700, Cekol 10000, CP934P, CP971P and CP974P, carboxyvinyl polymers and triethanolamine, HPC (hydroxypropyl cellulose), CP (Carbopol 934P), Carbopol (CP) Ex-55CMC (sodium carboxymethyl cellulose), HPMC (hydroxypropyl methyl cellulose), HEC (hydroxyethyl cellulose), PIP [poly(isoprene)], PIB [poly(isobutylene)], xanthan gum, locust bean gum, pectin, polycarbofil, benzyl ester, hydroxyethyl cellulose Formulations comprising: poly(acrylic acid), poly(acrylic acid-co-acrylamide), poly(acrylic acid-co-methyl methacrylate), poly(acrylic acid-co-butyl acrylate), (bioadhesive polymer blends of CP and PIB), composed of PVP, hexadecylpyridinium chloride (as stabilizer), chlorinated chitosan, polyethylene oxide, polymethyl vinyl ether / maleic anhydride (PME / MA) and tragacanth gum, polyethylene glycol monomethyl ether monomethyl acrylate, drum-dried waxy corn starch (DDWM), carbopol 974P and stearyl fumarate sodium, and cellulose derivatives; hydrogels of acrylic acid (polar) and butyl acrylate (non-polar) and combinations thereof.

[0032] In addition to these inert diluents, the composition may also contain auxiliaries such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and fragrances.

[0033] In addition to active peptides, suspensions may contain suspending agents such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0034] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0035] Dosage forms of the polypeptides of the present invention for topical administration include ointments, powders, patches, sprays, and inhalers. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or, if necessary, propellants.

[0036] The pharmaceutical compositions of the present invention are formulated to a pH of 5.5 to 7.5. In one embodiment, the pH of the aqueous medium can be adjusted by low concentrations of suitable biocompatible buffering agents, non-limiting examples of which are glycerol, sodium carbonate and sodium bicarbonate, and sodium dihydrogen phosphate and disodium hydrogen phosphate.

[0037] The compositions of the present invention can be administered daily or intermittently, with a frequency of once daily or two to three times daily. If each of the two active ingredients is a single formulation, their administration frequencies can be the same or different. Furthermore, the compositions of the present invention can be used alone or in combination with other drugs for treating anemia. Considering all the foregoing factors, it is important to administer the lowest possible dose to achieve optimal efficacy without side effects, which can be readily determined by those skilled in the art. In some embodiments, the dosing regimen is repeated, for example, once, twice, three times, or more; for example, repeated over the remaining lifespan of the individual in need.

[0038] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0039] Example 1: Screening of active peptides from donkey-hide gelatin 1.1 Preparation of donkey-hide gelatin peptides Take an appropriate amount of donkey-hide gelatin powder that has passed through a 60-mesh sieve, add 20 times the amount of purified water and mix well. Melt the mixture at 80℃ for 30 minutes to obtain a donkey-hide gelatin solution. Take an appropriate amount of the solution and adjust the pH to 7.0 with 10% sodium hydroxide solution. Add bromelain at a ratio of 5% of the raw donkey-hide gelatin and hydrolyze at 50℃ and a rotation speed of 45-50 rpm for 2 hours. After hydrolysis, adjust the pH to 7.5 again with 10% sodium hydroxide solution. Add trypsin at a ratio of 2% of the raw donkey-hide gelatin and continue hydrolysis at 40℃ and a rotation speed of 45-50 rpm for 3 hours. After the reaction is complete, boil for 15 minutes to inactivate the enzyme, cool to room temperature, and refrigerate at 0-4℃ for 6 hours. Then centrifuge at 4500 rpm for 10 minutes and collect the supernatant, which is the donkey-hide gelatin hydrolysate.

[0040] The donkey-hide gelatin enzymatic hydrolysate was extracted using a 3kDa ultrafiltration tube, and the filtrate was collected to obtain a solution of donkey-hide gelatin active peptides. The active peptides were then desalted. First, the desalting column was activated with an aqueous solution containing 0.1% TFA and 80% acetonitrile, and then equilibrated with an aqueous solution containing 0.1% TFA and 1% acetonitrile. The donkey-hide gelatin active peptide solution was slowly passed through the equilibrated desalting column to collect the peptides; salts and other non-hydrophobic small molecules elute. The desalting column was washed with an aqueous solution containing 0.1% TFA and 0.5% acetonitrile to remove residual salts. Finally, the peptides in the desalting column were eluted with an aqueous solution containing 0.1% TFA and 80% acetonitrile, and the eluent was collected and freeze-dried.

[0041] 1.2 LC-MS / MS analysis of donkey-hide gelatin peptides LC conditions: Mobile phase: A is 0.1% formic acid solution, B is 0.1% formic acid-acetonitrile solution. Injection volume: 2µL, loading flow rate: 500nL / min, separation flow rate: 300nL / min. Gradient elution conditions: 0–105 min, 95%–70% A, 5%–30% B; 105–110 min, 70%–10% A, 30%–90% B; 110–112 min, 10% A, 90% B; 112–113 min, 10%–95% A, 90%–5% B; 95% A, 5% B held for 7 min.

[0042] MS conditions: Ion source spray voltage 2.2kV, capillary temperature 320℃, switching between MS and MS / MS acquisition. Full-scan MS: scan range m / z 400–1600, scan resolution 120,000 (at m / z 200), maximum ion introduction time 50ms, automatic gain control (AGC) set to 1.0 × 10⁻⁶. 6MS / MS: Scan resolution 15000, scan range 110–2000 m / z, minimum ion intensity 50000, maximum ion introduction time 100 ms, AGC set to 1.0 × 10⁻⁶. 5 The precursor ion selection was set to 1.6 Da.

[0043] 1.3 Activity Screening of Donkey-hide Gelatin-derived Active Peptides X-ray crystal structures of transferrin and / or erythropoietin receptors were obtained from the RCSB PDB database. Using Discovery Studio (DS) 2017 R2 (Dassault Systemes Biovia, San Diego, CA, USA) software, water molecules were removed from the receptor targets and hydrogen atoms were added. The active sites were then defined in the "Receptor-Ligand Interactions" module. The active site (x, y, z) and docking radius (r) information are shown in Table 1. The peptide structures were plotted using the "Macromolecules" module of DS software. The "Minimize Ligands" and "Prepare Ligands" tools were used for energy optimization of the peptide structures under the CHARMm force field. Molecular docking of the receptor and peptide was performed using the "CDOCKER" tool in the "Receptor-Ligand Interactions" module. Peptides with potential hematopoietic effects were screened based on the CDOCKER_Energy value. The 20 peptides with the highest CDOCKER_Energy values ​​were selected for subsequent screening experiments.

[0044] Example 2. Iron chelating ability of donkey-hide gelatin peptides The iron chelating ability of donkey-hide gelatin peptides was determined using the o-phenanthroline colorimetric method. A standard working solution containing 200 μL of 20 mg / mL ascorbic acid in a concentration range of 0.00–2.00 μg / mL was prepared, with a total volume of 10 mL. The pH of the system was adjusted to 5.0. After adding 400 μL of 5 mg / mL o-phenanthroline solution, the reaction was allowed to proceed for 15 min. Using ultrapure water as a reference blank, the absorbance was measured at 510 nm. A standard curve of Fe²⁺ chelating ability was plotted: y = 0.1145x + 0.0039, R² = 0.999.

[0045] Mix the donkey-hide gelatin peptide solution with 0.2 mol / L acetate-sodium acetate buffer at a ratio of 2:1 (v / v), then add 0.1 times the volume of the donkey-hide gelatin peptide solution in 20 mg / mL ascorbic acid solution and mix well. Take 2.5 mL of the mixed solution and add 1.0 mL of 100 μg / mL FeSO4 solution, then mix well. Adjust the pH to 5.0 for each group, with three replicates per group. Place the treated solution in a constant temperature shaker at 37℃ and 200 rpm for 30 min. After the chelation reaction is complete, centrifuge the solution at 6000 rpm for 20 min, collect the supernatant, and determine the absorbance value according to the o-phenanthroline colorimetric method. Substitute the absorbance value into the standard curve to calculate the iron content of the supernatant after chelation. Calculate the chelated iron content of the donkey-hide gelatin peptide according to the formula.

[0046] Formula: Iron chelation amount (μg / mg) = (Added iron content - Iron content in the supernatant after chelation) / Peptide mass The results showed that the polypeptide with the highest iron chelation was AG61, with an iron chelation of 57.97 μg / mg, and its amino acid sequence is shown in SEQ ID NO: 1.

[0047] Example 3. Effect of polypeptide AG61 on a mouse model of anemia induced by tail bleeding. Forty BALB / c mice were randomly divided into four groups. Three groups (AG61 group, positive control group, and model group) were used to induce a blood loss anemia model, while the other group served as a blank control group. Blood was collected from the tail of all four groups before the experiment to measure normal hemoglobin (Hb) and red blood cell (RBC) levels. In the AG61 group, positive control group, and model group, 0.5 mL of blood was expelled from the tail. Blood was collected again from the tail 24 hours after blood loss to measure Hb and RBC levels. The mice were then administered 12 g / kg of AG61 polypeptide, 0.1 g / kg of ferrous succinate, and the same volume of physiological saline (0.6 mL), respectively. The blank control group was administered only the same volume of physiological saline. The medication was administered once daily for 15 consecutive days. Blood was collected from the abdominal vein on the day after the last administration to measure Hb and RBC levels.

[0048] The results are as follows Figure 1-2 As shown, donkey-hide gelatin peptides can increase the levels of Hb and RBC in a mouse model of blood loss-induced anemia, and the blood-tonifying effect of donkey-hide gelatin peptides is better than that of ferrous succinate.

Claims

1. A bioactive polypeptide, characterized in that, The polypeptide is an active polypeptide of donkey-hide gelatin, and its amino acid sequence is shown in SEQ ID NO:

1.

2. A nucleic acid molecule encoding the bioactive polypeptide as described in claim 1.

3. A vector comprising the nucleic acid molecule as described in claim 2.

4. An isolated host cell comprising the bioactive polypeptide of claim 1, the nucleic acid molecule of claim 2, or the carrier of claim 3.

5. Use of the bioactive polypeptide according to claim 1 in the preparation of a medicament for treating anemia.

6. The use according to claim 5, wherein the anemia is iron deficiency anemia, hemorrhagic anemia, or nutritional anemia.

7. A pharmaceutical composition comprising an effective amount of the bioactive polypeptide as claimed in claim 1.

8. The pharmaceutical composition according to claim 7 may further be used in combination with other drugs for treating anemia.

9. The pharmaceutical composition according to claim 8, wherein the anemia treatment drug may be an iron supplement, donkey-hide gelatin, and / or donkey-hide gelatin extract.

10. The pharmaceutical composition according to claim 9, wherein the iron agent may be ferrous sulfate, ferrous fumarate, ferrous lactate, ferrous succinate, polysaccharide iron complex, dextran iron, or ferrous gluconate.

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