CN117721167A

CN117721167A - Deer bone active polypeptide for promoting healing of key bones and preparation method thereof

Info

Publication number: CN117721167A
Application number: CN202410128110.XA
Authority: CN
Inventors: 王大麟; 韩梅; 王哲; 刘思译; 吴震宇; 冷冰; 王海涛; 李明成; 蒋维海
Original assignee: Beihua University
Current assignee: Beihua University
Priority date: 2024-01-30
Filing date: 2024-01-30
Publication date: 2024-03-19

Abstract

The invention provides deer bone active polypeptide for promoting healing of key bones and a preparation method thereof, wherein the preparation method comprises the following steps: (1) Degreasing deer bone powder, decalcifying, extracting with water, salting out, and dialyzing to obtain deer bone protein; (2) Performing primary enzymolysis and secondary enzymolysis on the deer bone protein to obtain crude deer bone polypeptide; (3) And (3) carrying out high performance liquid chromatography separation on the crude deer bone polypeptide to obtain deer bone polypeptide, carrying out alkylation reaction and neutralization reaction on the deer bone polypeptide, and desalting a reaction product to obtain the deer bone active polypeptide. The deer bone active polypeptide prepared by the invention can effectively promote healing of key bones and promote cell proliferation.

Description

Deer bone active polypeptide for promoting healing of key bones and preparation method thereof

Technical Field

The invention belongs to the technical field of peptide preparation, and in particular relates to deer bone active polypeptide for promoting healing of key bones and a preparation method thereof.

Background

The anterior cruciate ligament (Anterior Cruciate Ligaments, ACL) is a dense connective tissue band running from the femur to the tibia, which can effectively prevent excessive anterior-posterior translation of the knee joint, keeping the joint stable. ACL breaks are very common injuries in sports medicine, most commonly seen in teenagers who are loved to exercise. Currently, the classical surgical treatment of ACL fractures is arthroscopic anterior cruciate reconstruction, and tendon-bone interface healing strength has been considered an important factor in determining whether reconstruction was successful. In recent years, scholars at home and abroad have made a great deal of research to improve and optimize tendon-bone healing processes, including application of bioactive factors, mechanical stimulation, periosteum coverage, autologous platelet-rich plasma, bone marrow mesenchymal stem cell transplantation and the like, and have obtained good effects.

In recent years, research results of sika deer genome, transcriptome, proteomics and metabonomics show that sika deer genome is obviously superior to other deer and near-source species (cattle and sheep) which are released at present, and the total amount of the deer tissue protein in different periods and different sections can reach 4300 more species. It is well known that velvet antler has unique effects in promoting wound healing, inhibiting neuroinflammation, specific anti-inflammatory effects, promoting vascular and nerve regeneration, inhibiting osteoporosis, promoting rapid growth, etc. Development of industry technology, the magic place of the sika deer is revealed, the sika deer is taken as a traditional edible medicinal material, and along with technological progress, new product development of the sika deer can make a prominent contribution to human health, and future development of the sika deer industry is greatly considered. Jilin province has abundant deer bone resources, but its intensive research and product development are also relatively lagging. Most deer bones are used as common food materials and manufactured artworks and even discarded, so that resources are wasted greatly.

Deer bone is a traditional Chinese medicine with the efficacy of reinforcing deficiency and strengthening bones. Deer bone has effects of tonifying deficiency, strengthening tendons and bones, and is suitable for treating chronic diseases, asthenia, marrow deficiency, anemia, rheumatism, pain of limbs, etc. The 'materia medica outline mesh' records that deer bone is slightly hot, sweet and nontoxic in taste, has the effects of tonifying deficiency, strengthening bones and the like, is a rare and precious tonic, contains rich minerals such as calcium, phosphorus and the like, has the effect of setting bones and continuing tendons, and has remarkable effect on senile osteoporosis. Deer bone has very similar components with human bones, wherein calcium accounts for 30%, collagen accounts for 30%, other minerals, vitamins and the like account for about 30%, so that the deer bone five-collagen peptide is taken to supplement calcium and bone. The deer bone pentacollagen peptide comprehensively relieves symptoms such as waist soreness, back pain, leg cramp, numbness of hands and feet, humpback, hyperosteogeny, cervical vertebra pain, lumbar disc herniation, scapulohumeral periarthritis, arthritis, growth retardation of children, osteomalacia and the like caused by bone calcium deficiency through three in one bone formation, bone protection and bone nourishing, and builds healthier bones. The deer bone is rich in calcium, phosphorus, zinc, iron, a large amount of proteins, phospholipids, chondroitin, mucopolysaccharide, deer bone forming proteins, various trace elements and amino acids which cannot be synthesized by human body, and natural active vitamins.

CN107236775a discloses a deer bone protein polypeptide, and the preparation method of the deer bone protein polypeptide sequentially comprises the following steps: degreasing, decalcification, microwave-assisted alkaline extraction, microwave-assisted alkaline enzymolysis, microwave-assisted neutral enzymolysis, trypsin enzymolysis and concentration. The deer bone protein polypeptide has high oligopeptide ratio, and has high oxidation resistance, and refreshing and fatigue resisting effects. The deer bone protein polypeptide can be used for preparing health care products, and the health care wine prepared by the deer bone protein polypeptide has higher stability.

CN111235201a discloses a method for extracting deer bone peptide, which comprises the following steps: 1) Taking deer bone and taking out bone marrow for later use; pulverizing Os Cervi; 2) Dispersing bone marrow into purified warm water, adding zymophyte agent, and fermenting; after fermentation is completed, taking supernatant for later use; 3) The crushed deer bone is subjected to high-pressure steam blowing treatment to remove grease, the deer bone is softened by high-pressure steam treatment after cleaning, the deer bone is boiled in water after softening treatment, stirring is kept, deer bone soup is obtained after boiling, enzyme preparation is added, the enzyme preparation is deactivated by heating after hydrolysis is finished, deer bone hydrolysate is obtained, standing and precipitating are carried out, and a hydrolysate liquid part is reserved; circularly filtering the filtrate obtained after each filtering, and obtaining clear liquid after multiple times of filtering; 4) Mixing the supernatant obtained in 2) and the clarified liquid obtained in 3), stirring, and concentrating to obtain deer bone peptide concentrate.

Therefore, development of a deer bone active polypeptide with high purity, which can effectively promote bond bone healing and promote cell proliferation, is a research focus in the field.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide deer bone active polypeptide for promoting the healing of the key bones and a preparation method thereof, which can effectively promote the healing of the key bones and promote the proliferation of cells.

In order to achieve the aim of the invention, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a method for preparing a deer bone active polypeptide for promoting healing of a key bone, the method comprising:

(1) Degreasing deer bone powder, decalcifying, extracting with water, salting out, and dialyzing to obtain deer bone protein;

(2) Performing primary enzymolysis and secondary enzymolysis on the deer bone protein to obtain crude deer bone polypeptide;

(3) And (3) carrying out high performance liquid chromatography separation on the crude deer bone polypeptide to obtain deer bone polypeptide, carrying out alkylation reaction and neutralization reaction on the deer bone polypeptide, and desalting a reaction product to obtain the deer bone active polypeptide.

According to the invention, deer bone is salted out after water extraction, so that non-protein impurities can be effectively removed, deer bone proteins are subjected to stepwise enzymolysis, polypeptides with smaller molecular weight can be obtained, the obtained deer bone polypeptides are high in purity through high performance liquid chromatography separation, the activity of the polypeptides can be enhanced through alkylation of the deer bone polypeptides, the absorption by animals is more beneficial, and then neutralization reaction is carried out, so that impurities caused by alkylation can be removed, and further purification is carried out.

Preferably, the deer bone powder has a particle size of 30-50 mesh, for example, 35 mesh, 40 mesh, 45 mesh, etc.

Preferably, the degreasing agent is petroleum ether.

Preferably, the ratio of the liquid to the solid in degreasing is 1 (13-17), for example, 1:14, 1:15, 1:16, etc., and the degreasing time is 20-30h, for example, 22h, 24h, 26h, 28h, etc.

Preferably, the decalcification adopts a hydrochloric acid aqueous solution, and the concentration of hydrochloric acid in the hydrochloric acid aqueous solution is 0.3-0.7mol/L, for example, 0.4mol/L, 0.5mol/L, 0.6mol/L and the like.

Preferably, the feed liquid ratio in decalcification is 1 (8-12), for example, 1:9, 1:10, 1:11, etc.

Preferably, the decalcification temperature is 3-5deg.C, for example, 3.5deg.C, 4deg.C, 4.5deg.C, etc., and the time is 2-4 hr, for example, 2.5 hr, 3 hr, 3.5 hr, etc.

Preferably, the decalcification is repeated 7-9 times, for example, 7 times, 8 times or 9 times.

Preferably, the decalcification is followed by a drying step.

Preferably, the water extraction liquid ratio is 1 (8-12), for example, 1:9, 1:10, 1:11 and the like.

Preferably, the temperature of the water extraction is 90-100 ℃, for example, 92 ℃,95 ℃, 98 ℃ and the like, and the water extraction is repeatedly operated for 2-3 times.

Preferably, the water extraction further comprises the steps of filtering and concentrating.

Preferably, the filter screen pore size of the filter is 30-50 mesh, for example, 35 mesh, 40 mesh, 45 mesh, etc.

Preferably, the reagent used for salting out is an aqueous ammonium sulfate solution.

Preferably, the mass percentage of the ammonium sulfate in the ammonium sulfate aqueous solution is 75-85%, for example, 76%, 78%, 80%, 82%, 84% and the like.

Preferably, the mass ratio of the extract to the salting-out agent in salting-out is 1 (3-5), for example, 1:3.5, 1:4, 1:4.5, etc.

Preferably, the rotation speed at the salting-out is 4000-6000rpm, for example 4500rpm, 5000rpm, 5500rpm, etc., and the salting-out time is 8-12min, for example 9min, 10min, 11min, etc.

Preferably, the dialysis adopts a dialysis bag with a pore diameter of 30-40kDa, such as 32kDa, 35kDa, 38kDa, etc., and a dialysis time of 22-26h, such as 23h, 24h, 25h, etc.

Preferably, pepsin is adopted for the primary enzymolysis, and the dosage of the pepsin is 6000-6500U/g, for example, 6200U/g, 6300U/g, 6400U/g and the like.

Preferably, the pH value of the primary enzymolysis is 1.8-2.2, for example, 1.9, 2, 2.1 and the like, and the enzymolysis time is 3-4h, for example, 3.2h, 3.5h, 3.8h and the like.

Preferably, trypsin is used for the secondary enzymolysis, and the dosage of the trypsin is 6000-6500U/g, for example 6200U/g, 6300U/g, 6400U/g and the like.

Preferably, the pH value of the secondary enzymolysis is 7.8-8.2, for example, 7.9, 8, 8.1, etc., and the enzymolysis time is 3-5h, for example, 3.5h, 4h, 4.5h, etc.

Preferably, the detection conditions of the high performance liquid chromatography are as follows: the flow rate is 0.7mL/min, the sample injection amount is 50 mu L, and the chromatographic column is provided with: sepax BR-C18.6X1250 mm×5μm 120A, detection wavelength 214nm, analysis time 92min, mobile phase A98% water+2% ACN, mobile phase B98% ACN+2% water, wherein pH of mobile phase A is 10, mobile phase A adopts 25% ammonia water to adjust pH.

Preferably, the alkylation reaction dissolves deer bone polypeptide in NH ₄ HCO ₃ In the aqueous solution, the deer bone polypeptide concentration is set to 450-550. Mu.g/mL, for example, 480. Mu.g/mL, 500. Mu.g/mL, 530. Mu.g/mL, etc.

Preferably, the NH ₄ HCO ₃ NH in aqueous solution ₄ HCO ₃ The concentration of (C) is 45-55mmol/L, and may be, for example, 48mmol/L, 50mmol/L, 53mmol/L, etc.

Preferably, the reagent used in the alkylation reaction is DTT (dithiothreitol).

Preferably, the final concentration of DTT added during the alkylation reaction is 8-12mmol/L, for example, 9mmol/L, 10mmol/L, 11mmol/L, etc.

Preferably, the alkylation reaction is carried out at a temperature of 50 to 60℃and may be carried out at 52℃and 54℃and 56℃and 58℃for a period of 0.8 to 1.2 hours, for example, 0.9 hours, 1 hour, 1.1 hour, etc.

Preferably, the reagent used for the neutralization reaction is IAM (iodoacetamide).

Preferably, IAM is added at the time of the neutralization reaction at a final concentration of 45-55mmol/L, for example, 48mmol/L, 50mmol/L, 53mmol/L, etc.

Preferably, the neutralization reaction is carried out under a dark condition, and the reaction time is 35-45min, for example, 36min, 38min, 40min, 42min, 44min and the like.

Preferably, the desalination is preceded by the steps of activation, equilibration, loading.

Preferably, the reagent used for the activation is ACN (acetonitrile).

Preferably, the reagent used in the balancing is an aqueous solution of TFA, wherein the TFA in the aqueous solution has the same mass percentage of TFA, and the mass percentage of the TFA in the aqueous solution is 0.08-0.12%, for example, 0.09%, 0.1%, 0.11% and the like.

Preferably, the loading is repeated twice.

Preferably, the desalting solution used for the desalting is an aqueous TFA solution (aqueous trifluoroacetic acid solution).

Preferably, the desalting operation is performed 1 or 2 times.

Preferably, the desalting further comprises a step of eluting.

Preferably, the eluting reagent is an eluent obtained by mixing ACN and TFA water solution according to the volume ratio of (3.5-4.5): 1, for example, the eluent can be 3.8:1, 4:1, 4.2:1 and the like.

Preferably, the TFA aqueous solution in the desalting solution and the eluent has the same percentage by mass of TFA, and the percentage by mass of TFA is 0.08-0.12%, for example, 0.09%, 0.1%, 0.11%, etc.

Preferably, the preparation method comprises the following steps:

(1) Degreasing 30-50 mesh deer bone powder with 13-17 times of petroleum ether for 20-30h, decalcification with 8-12 times of 0.3-0.7mol/L hydrochloric acid aqueous solution at 3-5 ℃ for 2-4h, decalcification repeatedly for 7-9 times, drying, mixing with 8-12 times of water, extracting with 90-100 ℃ water for 2-3 times, filtering with 30-50 mesh, concentrating to obtain extract, salting out with 3-5 times of 75-85% ammonium sulfate aqueous solution at 4000-6000rpm for 8-12min, and dialyzing with 30-40kDa dialysis bag for 22-26h to obtain deer bone protein;

(2) Carrying out primary enzymolysis on the deer bone protein for 3-4 hours by adopting pepsin at the pH value of 1.8-2.2, and then carrying out secondary enzymolysis by adopting trypsin at the pH value of 7.8-8.2 for 3-5 hours to obtain crude deer bone polypeptide;

the dosage of pepsin is 6000-6500U/g, and the dosage of trypsin is 6000-6500U/g;

(3) Separating the crude deer bone polypeptide by high performance liquid chromatography to obtain deer bone polypeptide, dissolving the deer bone polypeptide in NH with concentration of 45-55mmol/L ₄ HCO ₃ In water solution, making the concentration of deer bone polypeptide be 450-550 mug/mL, adding DTT to make its final concentration be 8-12mmol/L, making alkylation reaction for 0.8-1.2 hr at 50-60 deg.C, adding IAM to make its final concentration be 45-55mmol/L, making neutralization reaction for 35-45min under the condition of light-proofing, adopting TFA water solution to make desalination of reaction product, adopting eluent obtained by mixing ACN and TFA water solution according to the volume ratio (3.5-4.5): 1 to make elution, the mass percentage of TFA in the above-mentioned TFA water solution used for desalination and elution is identical and is 0.08-0.12%Obtaining the deer bone active polypeptide.

The numerical ranges recited herein include not only the recited point values, but also any point values between the recited numerical ranges that are not recited, and are limited to, and for the sake of brevity, the invention is not intended to be exhaustive of the specific point values that the recited range includes.

In a second aspect, the present invention provides a deer bone active polypeptide for promoting healing of key bones, wherein the deer bone active polypeptide is prepared by the preparation method according to the first aspect.

Compared with the prior art, the invention has the following beneficial effects:

1. according to the invention, deer bone is salted out after water extraction, so that non-protein impurities can be effectively removed, deer bone proteins are subjected to step-by-step enzymolysis, polypeptides with smaller molecular weight can be obtained, the obtained deer bone polypeptides are separated by high performance liquid chromatography, the purity of the deer bone polypeptides is higher, and the alkylation of the deer bone polypeptides can enhance the activity of the polypeptides, so that the deer bone polypeptides are more beneficial to animal absorption;

2. the TDSCs cell test shows that the deer bone active polypeptide prepared by the invention can effectively promote bond bone healing and promote cell migration and proliferation.

Drawings

FIG. 1 is a spectrum of the high performance liquid chromatography test performed in example 1;

FIG. 2 is a spectrum of the standard in example 1 obtained by HPLC;

FIG. 3 is a graph showing the effect of deer bone active polypeptides of different concentrations on TDSCs cell proliferation in test example 2;

FIG. 4 is a diagram 10 ^-3 A microscopic image of TDSCs treated with the deer bone active polypeptide prepared in example 1 at mol/L;

FIG. 5 is 10 ^-5 A microscopic image of TDSCs treated with the deer bone active polypeptide prepared in example 1 at mol/L;

FIG. 6 is a graph showing the results of a scratch test of deer bone active polypeptide solution prepared in example 1 on TDSCs cells;

FIG. 7 is a graph showing the results of the TDSCs cell scratch test of the control group in test example 3.

Detailed Description

The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.

The terms "comprising," "including," "having," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, step, method, article, or apparatus.

"optional" or "any" means that the subsequently described event or event may or may not occur, and that the description includes both cases where the event occurs and cases where the event does not.

The indefinite articles "a" and "an" preceding an element or component of the invention are not limited to the requirement (i.e. the number of occurrences) of the element or component. Thus, the use of "a" or "an" should be interpreted as including one or at least one, and the singular reference of an element or component includes the plural reference unless the amount clearly dictates otherwise.

The description of the terms "one embodiment," "some embodiments," "exemplarily," "specific examples," or "some examples," etc., herein described means that a specific feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this document, the schematic representations of the above terms are not necessarily for the same embodiment or example.

The reagents or instrument sources in the following examples are as follows:

experimental example 1

High performance liquid chromatography separation and analysis

Column temperature: room temperature;

flow rate: 0.7mL/min;

the sample injection amount is 50 mu L;

chromatographic column: sepax BR-C18.6X100 mm. Times.5 μm 120A;

detection wavelength: 214nm;

the analysis time of each component is 92min;

mobile phase a was 98% water+2% ACN, mobile phase B was 98% acn+2% water, where mobile phase a had a pH of 10 and mobile phase a was pH adjusted with 25% aqueous ammonia.

Experimental example 2

Desalination process

a. Activating: the desalting cartridge was activated twice with 300 μl of 100% acn;

b. balance: the desalting cartridge was equilibrated 2 times with 200 μl of 0.1% tfa;

c. loading: loading the sample for 2 times;

d. desalting: desalting with 200. Mu.L of 0.1% TFA 2 times;

e. eluting: elution was performed 1 time with 300. Mu.L of CAN (80%) -0.1% TFA (20%).

Example 1

The embodiment provides a deer bone active polypeptide, which is prepared by the following steps:

(1) Crushing deer bone powder to 40 meshes, degreasing the obtained deer bone powder for 25 hours by using 15 times of petroleum ether, decalcification for 3 hours by using 10 times of 0.5mol/L hydrochloric acid aqueous solution at 4 ℃, repeatedly carrying out decalcification for 8 times, drying, mixing with 10 times of pure water, extracting with water for 2 times at 95 ℃, filtering, concentrating the filtrate to 1/3 of the original volume by adopting rotary evaporation, and obtaining deer bone extract;

(2) Salting out deer bone extract with 4 times of 80% ammonium sulfate aqueous solution at 5000rpm for 10min, re-dissolving the precipitate, dialyzing with 35kDa dialysis bag for 24 hr, and freeze drying to obtain deer bone protein;

(3) Redissolving deer bone protein in pure water to enable the concentration of a substrate to be 4%, carrying out primary enzymolysis for 3.5 hours by adopting 6200U/g pepsin at a pH value of 2, freeze-drying to obtain protein powder, redissolving the protein powder in pure water to enable the concentration of the substrate to be 4%, carrying out secondary enzymolysis for 4 hours by adopting 6300U/g trypsin at a pH value of 8, boiling for 15 minutes at 100 ℃ after the enzymolysis is finished, terminating enzymolysis, and freeze-drying to obtain crude deer bone polypeptide;

(4) Dissolving the crude deer bone polypeptide obtained in the step (3) in water to prepare a 10mg/mL test solution, performing high performance liquid chromatography analysis and separation by adopting the process of experimental example 1, detecting a sample solution to obtain a spectrogram shown in figure 1, and collecting components with retention time of 9.366min to obtain deer bone polypeptide;

the spectrogram obtained by detecting the standard substance solution is shown in figure 2;

(5) Dissolving deer bone polypeptide in NH with concentration of 50mmol/L ₄ HCO ₃ In the aqueous solution, the concentration of the deer bone polypeptide is 500 mug/mL, DTT is added to the concentration of 10mmol/L, reductive alkylation reaction is carried out for 1h at 56 ℃, IAM is added to the concentration of 50mmol/L, and neutralization reaction is carried out for 40min under the dark condition, thus obtaining a reaction product;

(6) Desalting the reaction product according to the method of experimental example 2, and vacuum drying the obtained solution at 45 ℃ to obtain deer bone active polypeptide.

Example 2

(1) Crushing deer bone powder to 30 meshes, degreasing the obtained deer bone powder for 30 hours by using 17 times of petroleum ether, decalcification for 4 hours at 3 ℃ by using 12 times of 0.3mol/L hydrochloric acid aqueous solution, repeating decalcification for 7 times, drying, mixing with 8 times of pure water, extracting with water for 3 times at 100 ℃, filtering, concentrating the filtrate to 1/3 of the original volume by adopting rotary evaporation, and obtaining deer bone extract;

(2) Salting out deer bone extract with 4 times of 80% ammonium sulfate aqueous solution at 5000rpm for 10min, re-dissolving the precipitate, dialyzing with 30kDa dialysis bag for 26 hr, and freeze drying to obtain deer bone protein;

(3) Redissolving deer bone protein in pure water to make the substrate concentration 4%, carrying out primary enzymolysis for 3h by using 6500U/g pepsin at pH value of 2, freeze-drying to obtain protein powder, redissolving the protein powder in pure water to make the substrate concentration 4%, carrying out secondary enzymolysis for 5h by using 6000U/g trypsin at pH value of 8, boiling at 100 ℃ for 15min after enzymolysis is finished, terminating enzymolysis, and freeze-drying to obtain crude deer bone polypeptide;

(4) Dissolving the crude deer bone polypeptide obtained in the step (4) in water to prepare a 10mg/mL test solution, performing high performance liquid chromatography analysis and separation by adopting the process of experimental example 1, and collecting components with retention time of 9.366min to obtain deer bone polypeptide;

(5) Dissolving deer bone polypeptide in NH with concentration of 45mmol/L ₄ HCO ₃ In the aqueous solution, the concentration of the deer bone polypeptide is 450 mug/mL, DTT is added to reach a final concentration of 8mmol/L, reductive alkylation reaction is carried out for 0.8h at 60 ℃, IAM is added to reach a final concentration of 45mmol/L, and neutralization reaction is carried out for 45min under the dark condition, so that a reaction product is obtained;

Example 3

(1) Crushing deer bone powder to 50 meshes, degreasing the obtained deer bone powder for 20 hours by using 13 times of petroleum ether, decalcification for 2 hours by using 8 times of 0.7mol/L hydrochloric acid aqueous solution at 5 ℃, repeatedly performing decalcification for 9 times, drying, mixing with 12 times of pure water, extracting with water for 3 times at 90 ℃, filtering, and concentrating the filtrate to 1/3 of the original volume by adopting rotary evaporation to obtain deer bone extract;

(3) Redissolving deer bone protein in pure water to make the substrate concentration 4%, performing primary enzymolysis for 4 hours at pH value of 2 by using 6000U/g pepsin, freeze-drying to obtain protein powder, redissolving the protein powder in pure water to make the substrate concentration 4%, performing secondary enzymolysis for 3 hours at pH value of 8 by using 6500U/g trypsin, boiling at 100 ℃ for 15 minutes after enzymolysis is completed, terminating enzymolysis, and freeze-drying to obtain crude deer bone polypeptide;

(4) Dissolving the crude deer bone polypeptide obtained in the step (3) in water to prepare a 10mg/mL test solution, performing high performance liquid chromatography analysis and separation by adopting the process of experimental example 1, and collecting components with retention time of 9.366min to obtain deer bone polypeptide;

(5) Dissolving deer bone polypeptide in NH with concentration of 55mmol/L ₄ HCO ₃ In the aqueous solution, the concentration of the deer bone polypeptide is 550 mu g/mL, DTT is added to a final concentration of 12mmol/L, reductive alkylation reaction is carried out for 1.2h at 50 ℃, IAM is added to a final concentration of 55mmol/L, and neutralization reaction is carried out for 35min under the condition of light shielding, so that a reaction product is obtained;

Example 4

The present embodiment provides a deer bone active polypeptide, which is different from embodiment 1 only in that the step (3) is:

redissolving deer bone protein in pure water to make the substrate concentration 4%, carrying out primary enzymolysis for 4 hours at pH value of 8 by using 6300U/g trypsin, freeze-drying to obtain protein powder, redissolving the protein powder in pure water to make the substrate concentration 4%, carrying out secondary enzymolysis for 3.5 hours at pH value of 2 by using 6200U/g pepsin, boiling for 15 minutes at 100 ℃ after enzymolysis is finished, terminating enzymolysis, and freeze-drying to obtain crude deer bone polypeptide; other raw materials, amounts and preparation methods were the same as in example 1.

Comparative example 1

The present comparative example provides a deer bone active polypeptide differing from example 1 only in the step (3), the step (3) of the present comparative example being:

redissolving deer bone protein in pure water to make the substrate concentration be 4%, carrying out enzymolysis for 3.5h under the condition that the pH value is 2 by using 6200U/g pepsin, boiling for 15min at 100 ℃ after the enzymolysis is finished, stopping enzymolysis, and freeze-drying to obtain crude deer bone polypeptide; other raw materials, amounts and preparation methods were the same as in example 1.

Comparative example 2

redissolving deer bone protein in pure water to make the substrate concentration be 4%, carrying out enzymolysis for 4 hours under the condition that the pH value is 8 by using 6300U/g trypsin, boiling for 15 minutes at 100 ℃ after the enzymolysis is finished, stopping enzymolysis, and freeze-drying to obtain crude deer bone polypeptide; other raw materials, amounts and preparation methods were the same as in example 1.

Comparative example 3

The comparative example provides a deer bone polypeptide which is different from the example 1 only in that the salting-out process of the step (2) is not performed, and the deer bone extract obtained in the step (1) is directly subjected to the enzymolysis of the step (3); other raw materials, amounts and preparation methods were the same as in example 1.

Comparative example 4

The present comparative example provides a deer bone polypeptide differing from example 1 only in that the high performance liquid chromatography separation of step (4) was not performed, and the product obtained in step (3) was directly subjected to the alkylation reaction of step (5); other raw materials, amounts and preparation methods were the same as in example 1.

Comparative example 5

This comparative example provides a deer bone polypeptide which differs from example 1 only in that the alkylation and neutralization reaction of step (5) is not performed, and the deer bone polypeptide obtained in step (4) is directly desalted in step (6); other raw materials, amounts and preparation methods were the same as in example 1.

Test example 1

1.1 main reagents: ca (Ca) ²⁺ ALP (alkaline phosphatase) kit is provided by North control Biotech Co.Ltd;

1.2 method:

1.2.1. grouping animals

60 Wistar male rats were selected. The randomization was divided into 6 groups: the control group, the model group, the deer bone polypeptide high, medium and low dose (250, 125, 62.5 mg/kg) group, the positive medicine compound deer antler bone-invigorating capsule group, 8 rats each. In addition to the control group, the other 5 groups of rats were 2.5mg/kg of im dexamethasone, 2 times per week, with an equal volume of physiological saline in the control group im.

The model set chooses to sever the anterior cruciate ligament. Deer bone polypeptide 250, 125 and 62.5mg/kg are respectively given to each component of deer bone polypeptide, 540mg/kg of compound deer antler bone-invigorating capsules (capsule content is dissolved by double distilled water), equivalent physiological saline is given to each component of control group and model group ig for 1 time per day, and 56 days (8 weeks) are continuously given. After the last administration, rats were anesthetized, abdominal aorta was bled, and Ca was detected in serum by ELISA ²⁺ ALP levels; cage-separated feeding (standard pellet feed is provided by experimental animal centers of Jilin university) and free drinking water;

1.2.2. ca in serum ²⁺ ALP level detection

Collecting 1mL of venous blood in the early morning on an empty stomach, centrifuging a blood sample at 3000r/min for 5min, separating serum for measurement, and adopting a kit provided by Zhongsheng North control biotechnology Co., ltd, wherein the detection method is an immunoturbidimetry;

1.2.3. statistical method

Statistical analysis is carried out by adopting SPSS13.0 statistical software, average standard deviation is adopted for representation, and p <0.05 is judged to have statistical significance by applying t test;

the results of the concentration test of each component in serum are shown in Table 1.

TABLE 1

As can be seen from the table data in examples 1 to 3, the results of animal experiments show that the effect is best by performing pepsin enzymolysis and then trypsin enzymolysis at firstInternal Ca ²⁺ ALP content was significantly increased compared to the blank.

As is clear from examples 1 to 3 and example 4, ca in serum was obtained when the order of primary and secondary enzymolysis was changed ²⁺ ALP concentration decreases; as is clear from examples 1 to 3 and comparative example 1, ca in serum was obtained when the stepwise enzymolysis was replaced by single pepsin enzymolysis ²⁺ The ALP concentration is greatly reduced; as is clear from examples 1-3 and comparative example 2, ca in serum was obtained when the stepwise enzymolysis was replaced by single trypsin enzymolysis ²⁺ The ALP concentration is obviously reduced; as is clear from examples 1 to 3 and comparative examples 3 to 5, ca in serum is present when no salting-out, ion exchange chromatography or liquid chromatography separation step is performed ²⁺ The ALP concentration was significantly reduced.

Test example 2

Test for proliferation potency of TDSCs

The proliferation of cells was examined by CCK-8 method, and the deer bone active polypeptides prepared in example 1 were prepared to 10 ^-8 mol/L、10 ^-7 mol/L、10 ^-6 mol/L、10 ^-5 mol/L、10 ^-3 As shown in FIG. 3, the results of the test on the concentration of mol/L for 24h, 48h and 72h respectively show that the OD value is gradually increased and then decreased with the increase of the concentration of the deer bone active polypeptide, and the concentration of the deer bone active polypeptide is 10 ^-5 In the case of mol/L, the cell proliferation ability was the strongest, and it was found from the figure that the proliferation of TDSCs cells was promoted and the time was in direct proportion.

Illustratively, use 10 ^-3 mol/L tendon stem cells treated with deer bone active polypeptide prepared in example 1 24h, and the proliferation of TDSCs is shown in FIG. 4; by 10 ^-5 mol/L tendon stem cells treated with deer bone active polypeptide prepared in example 1 24h, and the proliferation of TDSCs is shown in FIG. 5; as can be seen, use 10 ^-5 The deer bone active polypeptide of mol/L has obvious enhancement effect on the proliferation of TDSCs cells, 10 ^-3 The deer bone active polypeptide of mol/L has remarkable inhibition effect on TDSCs cells.

Test example 3

TDSCs migration Capacity test

By concentrationDegree of 10 ^-5 The deer bone active polypeptide solution prepared in the mol/L embodiment 1 acts on TDSCs cells for 12h and 24h, the scratch experiment result is shown in figure 6, the control group is a blank control without adding a drug, the control group experiment result is shown in figure 7, and the graph shows that the migration capability of the deer bone active polypeptide group TDSCs is obviously enhanced compared with that of the control group, and the migration capability of the deer bone active polypeptide group TDSCs is stronger than that of the deer bone active polypeptide group TDSCs when the deer bone active polypeptide solution is administrated for 24h, so that the migration capability of the deer bone active polypeptide can be effectively promoted.

The applicant states that the process of the invention is illustrated by the above examples, but the invention is not limited to, i.e. does not mean that the invention must be carried out in dependence on the above process steps. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of selected raw materials, addition of auxiliary components, selection of specific modes, etc. fall within the scope of the present invention and the scope of disclosure.

Claims

1. A method for preparing deer bone active polypeptide for promoting healing of key bones, which is characterized by comprising the following steps:

2. The method according to claim 1, wherein the deer bone powder has a particle size of 30-50 mesh;

preferably, the degreasing agent is petroleum ether;

preferably, the ratio of the feed liquid in degreasing is 1 (13-17), and the degreasing time is 20-30h.

3. The preparation method according to claim 1 or 2, wherein the decalcification agent is aqueous hydrochloric acid, and the concentration of hydrochloric acid in the aqueous hydrochloric acid is 0.3-0.7mol/L;

preferably, the ratio of feed liquid in decalcification is 1 (8-12);

preferably, the decalcification temperature is 3-5 ℃ and the decalcification time is 2-4h;

preferably, the decalcification is repeated 7-9 times;

preferably, the decalcification is followed by a drying step.

4. A process according to any one of claims 1 to 3, wherein the water extract has a feed to liquid ratio of 1 (8-12);

preferably, the temperature of the water extraction is 90-100 ℃, and the water extraction is repeatedly operated for 2-3 times;

preferably, the water extraction further comprises the steps of filtering and concentrating;

preferably, the filter screen pore size of the filter screen is 30-50 meshes.

5. The method according to any one of claims 1 to 4, wherein the reagent used for salting out is an aqueous ammonium sulfate solution;

preferably, the mass percentage of ammonium sulfate in the ammonium sulfate aqueous solution is 75-85%;

preferably, the mass ratio of the extract to the salting-out agent in salting-out is 1 (3-5);

preferably, the rotating speed during salting-out is 4000-6000rpm, and the salting-out time is 8-12min;

preferably, the dialysis adopts a dialysis bag with the aperture of 30-40kDa and the dialysis time of 22-26h.

6. The method according to any one of claims 1 to 5, wherein pepsin is used for the primary enzymolysis, and the amount of pepsin is 6000 to 6500U/g;

preferably, the pH value of the primary enzymolysis is 1.8-2.2, and the enzymolysis time is 3-4 hours;

preferably, trypsin is adopted for the secondary enzymolysis, and the dosage of the trypsin is 6000-6500U/g;

preferably, the pH value of the secondary enzymolysis is 7.8-8.2, and the enzymolysis time is 3-5h.

7. The method of any one of claims 1-6, wherein the alkylating reaction is performed by dissolving deer bone polypeptide in NH ₄ HCO ₃ In the aqueous solution, the concentration of the deer bone polypeptide is 450-550 mug/mL;

preferably, the NH ₄ HCO ₃ NH in aqueous solution ₄ HCO ₃ The concentration of (2) is 45-55mmol/L;

preferably, the reagent used in the alkylation reaction is DTT;

preferably, the final concentration of DTT added in the alkylation reaction is 8-12mmol/L;

preferably, the alkylation reaction is carried out at a temperature of 50-60 ℃ for a time of 0.8-1.2h;

preferably, the reagent used in the neutralization reaction is IAM;

preferably, IAM is added in the neutralization reaction at a final concentration of 45-55mmol/L;

preferably, the neutralization reaction is carried out in the dark for 35-45min.

8. The method according to any one of claims 1 to 7, wherein the desalting solution used for desalting is an aqueous TFA solution;

preferably, the desalting further comprises a step of eluting;

preferably, the eluting reagent is an eluent obtained by mixing ACN and TFA water solution according to the volume ratio of (3.5-4.5): 1;

preferably, the mass percentage of TFA in the aqueous solution of TFA in the desalting solution and the eluent is the same and is 0.08-0.12%.

9. The method of any one of claims 1-8, wherein the method of preparation comprises:

(3) Separating the crude deer bone polypeptide by high performance liquid chromatography to obtain deer bone polypeptide, dissolving the deer bone polypeptide in NH with concentration of 45-55mmol/L ₄ HCO ₃ In the aqueous solution, the concentration of the deer bone polypeptide is 450-550 mug/mL, DTT is added to a final concentration of 8-12mmol/L, alkylation reaction is carried out for 0.8-1.2h at 50-60 ℃, IAM is added to a final concentration of 45-55mmol/L, neutralization reaction is carried out for 35-45min under the condition of light shielding, the reaction product is desalted by adopting a TFA aqueous solution, and elution is carried out by adopting eluent obtained by mixing ACN and the TFA aqueous solution according to the volume ratio (3.5-4.5): 1, wherein the mass percentage of the TFA in the aqueous solution used in the desalting and elution is the same and is 0.08-0.12%, so that the deer bone active polypeptide is obtained.

10. A deer bone active polypeptide for promoting healing of key bones, wherein the deer bone active polypeptide is prepared by the preparation method of any one of claims 1-9.