A deer antler bone-strengthening peptide, its screening method and application

Abstract

This invention discloses a deer antler bone-strengthening peptide, its screening method, and its application, belonging to the field of traditional Chinese medicine peptide technology. The deer antler bone-strengthening peptide is one of G35, G40, G44, and G45. Deer antler is ground and pulverized, the lysate is homogenized, ultrasonically centrifuged, and the protein is precipitated with methanol. The supernatant is discarded after centrifugation. Extraction and desalting are performed, followed by freeze-drying to obtain the deer antler peptide extract. Liquid chromatography-mass spectrometry (LC-MS), Peaks 8 search software, and the De Novo method are used for analysis. Free peptides are obtained based on ALC≥95%, local confidence≥95%, Area≥1000000, and comparison with the NCBI database. The bioactivity probability, toxicity, and sensitization of peptides are predicted to obtain candidate peptides. Candidate peptides are synthesized using a solid-phase synthesis method. The effect of candidate peptides on osteoblast proliferation is evaluated, and the deer antler bone-strengthening peptide is screened. The deer antler bone-strengthening peptide promotes osteoblast proliferation, revealing its potential bone repair mechanism.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide technology of traditional Chinese medicine, specifically relating to a deer antler bone-strengthening peptide, its screening method, and its application. Background Technology

[0002] Deer antler bone-strengthening peptides are active peptides extracted from deer antlers, possessing significant effects in promoting bone growth and repair. Their main components include various amino acids, polypeptides, and trace elements, which can enhance bone strength by regulating bone metabolism and promoting osteoblast proliferation and differentiation. In traditional Chinese medicine, deer antler is considered a precious tonic, primarily used for strengthening tendons and bones and tonifying kidney yang. Modern medical research indicates that the active ingredients in deer antler have significant therapeutic effects on osteoporosis, fracture healing, and other diseases.

[0003] Polypeptides are a class of small, bioactive molecules with significant value in drug development and biomedical research. Deer antler, a traditional Chinese medicine, is believed to tonify kidney yang, replenish essence and blood, strengthen tendons and bones, and regulate the Chong and Ren meridians. Its structural diversity and specific functions suggest broad application prospects in anti-inflammatory, antibacterial, and cell proliferation-promoting fields. Modern research also indicates that deer antler extract is rich in proteins and peptides, potentially participating in bone metabolism by regulating osteoblast activity. However, systematic screening of deer antler peptides and their mechanisms of action remain relatively scarce, particularly regarding the structure and function of individual peptides. This limits their clinical application and exploration of molecular mechanisms. Summary of the Invention

[0004] In view of the problems existing in the prior art, the present invention provides a deer antler bone-strengthening peptide, its screening method and application, providing a theoretical basis for the pharmaceutical development of deer antler polypeptides and revealing its potential bone repair mechanism.

[0005] This invention is achieved through the following technical solution:

[0006] In a first aspect, the present invention discloses a deer antler bone-strengthening peptide, wherein the deer antler bone-strengthening peptide is one of G35, G40, G44, and G45;

[0007] The amino acid sequence of G35 is shown in SEQ ID NO.10;

[0008] The amino acid sequence of G40 is shown in SEQ ID NO.15;

[0009] The amino acid sequence of G44 is shown in SEQ ID NO.19;

[0010] The amino acid sequence of G45 is shown in SEQ ID NO.20.

[0011] In a second aspect, the present invention discloses a method for screening the aforementioned deer antler bone-strengthening peptide, comprising the following steps:

[0012] (1) Extraction of antler polypeptide: The antler was ground and pulverized under liquid nitrogen conditions, added to the lysis buffer for homogenization, ultrasonic centrifugation, methanol was added to the supernatant to precipitate the protein, and the supernatant was discarded after centrifugation; the solid was extracted with the extract and then freeze-dried, dissolved with formic acid solution, desalted by C18 desalting column, and freeze-dried to obtain antler polypeptide extract.

[0013] (2) The antler peptide extract was analyzed by liquid chromatography-mass spectrometry, Peaks 8 search software and De Novo analysis method. Peptides were further screened according to ALC≥95%, local confidence≥95% and Area≥1,000,000. Free peptides were obtained by comparison with the NCBI database. The bioactivity probability of peptides was predicted by the online website PeptideRanker, the toxicity of peptides was predicted by ToxIBTL and the sensitization was predicted by Algpred 2.0 to obtain candidate peptides.

[0014] (3) The candidate peptides obtained in step (2) were synthesized using solid-phase synthesis.

[0015] (4) The effect of the candidate peptides synthesized in step (3) on osteoblast proliferation was evaluated by the CCK-8 method, and antler peptides that promote osteoblast proliferation were screened out.

[0016] Further, the lysis buffer in step (1) contains 1 wt% SDC, 10 mM TCEP, 40 mM CAA and 100 mMTEAB, and the pH of the lysis buffer is 8.5; the extract is an acetonitrile solution containing 3 wt% hydrochloric acid; and the formic acid solution is a 0.1 wt% aqueous formic acid solution.

[0017] Further, the desalting treatment method in step (1) is as follows: activate the desalting column with 100% acetonitrile, balance the desalting column with 0.1wt% formic acid aqueous solution, load the formic acid aqueous solution containing peptides onto the desalting column, then wash the desalting column with 0.1wt% formic acid aqueous solution to remove impurities, and finally elute with 40vt% acetonitrile, collect the eluent, and complete the desalting treatment.

[0018] Further, in step (2), the antler polypeptide extract was dissolved in 0.1 wt% formic acid aqueous solution, and the supernatant was collected by centrifugation for liquid chromatography-mass spectrometry (LC-MS) analysis.

[0019] The liquid chromatography conditions for liquid chromatography-mass spectrometry (LC-MS) detection were as follows: mobile phase A: 0.1 wt% formic acid aqueous solution; mobile phase B: 0.1 wt% formic acid aqueous solution containing 80 vt% acetonitrile; the elution program was as follows: 0~8 min 6% mobile phase B, 8~15 min 6~12% mobile phase B, 15~63 min 12~30% mobile phase B, 63~73 min 30~40% mobile phase B, 73~74 min 40~95% mobile phase B, 74~84 min 95% mobile phase B, 84~85 min 95~6% mobile phase B.

[0020] The mass spectrometry conditions for LC-MS were as follows: FAIMS Pro™ Interface mass spectrometer, compensation voltage (CV) switched every 1 second between -45 and 65 kV, Nanospray Flex™ (NSI) ion source, ion spray voltage set to 2.0 kV, ion transfer tube temperature set to 320°C, data-dependent acquisition mode, full scan range of m / z 350-1500, primary mass spectrometry resolution set to 120,000 m / z, and AGC set to 4 × 10⁻⁶ m / z. 5 The maximum injection time for C-trap is 50 ms; the secondary mass spectrometry detection uses Top Speed ​​mode, with a resolution of 15000 (200 m / z) and an AGC of 5 × 10⁻⁶. 4 The maximum injection time is 22ms, and the peptide fragmentation collision energy is set to 33%.

[0021] Furthermore, the osteoblasts mentioned in step (4) are the MC3T3-E1 cell line.

[0022] Furthermore, the antlers mentioned in step (1) are those of a sika deer.

[0023] In a third aspect, the present invention discloses the application of the aforementioned antler-strengthening peptide in the preparation of bone repair drugs, wherein the antler-strengthening peptide is one of G35, G40, G44, and G45.

[0024] Furthermore, the antler-strengthening peptide is G35.

[0025] The beneficial effects achieved by this invention are as follows:

[0026] The antler-derived osteogenic peptides G35, G40, G44, and G45 obtained by the method of this invention, especially G35, can significantly promote osteoblast proliferation in a concentration-dependent manner, providing a theoretical basis for the pharmaceutical development of antler peptides and revealing their potential bone repair mechanism. Attached Figure Description

[0027] Figure 1 The graph shows the cell proliferation rate of osteoblasts induced by 25 different peptides.

[0028] Figure 2 The graph shows the cell proliferation rate of osteoblasts at four different concentrations of antler-derived osteopeptide.

[0029] Figure 3 This is a graph showing the growth curves of osteoblasts after intervention with antler-derived osteogenic peptides G35 and G45. Detailed Implementation

[0030] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. This example is merely a detailed implementation scheme and specific operation process based on the present invention, and is not intended to limit the scope of protection of the present invention. Any modifications or improvements made within the principles of the present invention should be included within the scope of protection.

[0031] Example 1

[0032] Extracting antler polypeptides from sika deer antlers:

[0033] Take 30 mg of sika deer antler, grind it into powder under liquid nitrogen conditions, add lysis buffer (lysate containing 1 wt% SDC, 10 mM TCEP, 40 mM CAA, 100 mM TEAB, pH 8.5) and homogenize, sonicate and centrifuge to collect the supernatant, add methanol to precipitate the protein, centrifuge and discard the supernatant; add 1 mL of acetonitrile solution containing 3% hydrochloric acid (970 μL acetonitrile + 30 μL dilute hydrochloric acid) for extraction, freeze-dry the extract and dissolve it in 0.1 wt% formic acid aqueous solution, desalt it using a C18 desalting column, freeze-dry to obtain antler polypeptide extract;

[0034] The desalting process is as follows: the desalting column is activated with 100% acetonitrile, the column is equilibrated with 0.1 wt% formic acid, an aqueous solution containing peptides is loaded onto the desalting column, the desalting column is then washed with 0.1 wt% formic acid to remove impurities, and finally eluted with 40 wt% acetonitrile solution. The eluent is collected to complete the desalting process.

[0035] Example 2

[0036] The antler polypeptide extract from Example 1 was dissolved in a 0.1 wt% formic acid aqueous solution. The supernatant was collected by centrifugation and analyzed by liquid chromatography-mass spectrometry (LC-MS), Peaks 8 search software, and De Novo analysis method. Peptides were screened based on ALC≥95%, local confidence≥95%, and Area≥1,000,000. Free polypeptides were obtained by comparison with the NCBI database. Furthermore, the online websites PeptideRanker were used to predict the bioactivity probability of peptides, ToxIBTL to predict peptide toxicity, and Algpred2.0 to predict sensitization, to obtain candidate polypeptides.

[0037] The liquid chromatography conditions were as follows: mobile phase A: 0.1 wt% formic acid aqueous solution; mobile phase B: 0.1 wt% formic acid aqueous solution containing 80 wt% acetonitrile; the elution program was as follows: 0~8 min 6% mobile phase B, 8~15 min 6~12% mobile phase B, 15~63 min 12~30% mobile phase B, 63~73 min 30~40% mobile phase B, 73~74 min 40~95% mobile phase B, 74~84 min 95% mobile phase B, 84~85 min 95~6% mobile phase B.

[0038] Mass spectrometry conditions were as follows: FAIMS Pro™ Interface mass spectrometer, compensation voltage (CV) switched every 1 second between -45 and -65, Nanospray Flex™ (NSI) ion source, ion spray voltage set to 2.0 kV, ion transfer tube temperature set to 320 °C, data-dependent acquisition mode, full scan range of m / z 350–1500, primary mass spectrometry resolution set to 120,000 m / z, and AGC set to 4 × 10⁻⁶ m / z. 5 The maximum C-trap injection time is 50 ms; the secondary mass spectrometry detection uses Top Speed ​​mode, with a secondary mass spectrometry resolution of 15000 (200 m / z) and an AGC of 5×10⁻⁶. 4 The maximum injection time was 22 ms, the peptide fragmentation collision energy was set to 33%, and raw mass spectrometry detection data was generated.

[0039] The parameters for Peaks 8 software were set as follows: Enzyme, no; Precursor ion mass tolerance, ±15ppm; Fragment ion mass tolerance, ±0.02 Da; ALC (%), ≥50.

[0040] Based on ALC≥95%, local confidence≥95%, and Area≥1,000,000, a total of 246 candidate peptides were obtained. Comparison with the NCBI database identified 53 free peptides. Further screening using online platforms (PeptideRanker predicting peptide bioactivity probability, ToxIBTL predicting peptide toxicity, and Algpred2.0 predicting sensitization) identified 25 candidate peptides. The amino acid sequences of these 25 candidate peptides are shown in Table 1 below, where amino acid sequences numbered G26~G45 correspond to SEQ ID NO.1~25, respectively.

[0041] Table 1. Information on candidate peptides obtained from screening in Example 2

[0042] .

[0043] Example 3

[0044] The 25 candidate peptides screened in Example 2 were synthesized using a solid-phase synthesis method:

[0045] The solid-phase synthesis method described above is as follows:

[0046] (1) Solvent treatment: DMF and methanol are soaked overnight in G3 pore molecular sieves to remove impurities and water before use;

[0047] (2) Full swelling of resin: Weigh 2.0 g blank Wang resin into a clean and dry reaction tube, add 15 mL DMF, and activate at room temperature for about 30 min;

[0048] (3) Adding the first amino acid: At room temperature, filter out the solvent from step (2) through a sand core filter, add 1 mmol of 5 times the molar excess of the first C-terminal amino acid, 5 times the molar excess of DMAP, 5 times the molar excess of DIC, and DMF as the solvent. React at room temperature for 3 hours. After the reaction is complete, wash with DMF 5 times, 5-6 mL each time. Then add a pyridine and acetic anhydride mixture with a volume ratio of 1:1 and react for 30 minutes. After the reaction is complete, wash with DMF 5 times, 5-6 mL each time.

[0049] (4) Removal of Fmoc protecting group: Remove the solvent in step (3) by filtration, add 10 mL of 20% piperidine DMF solution to the resin, stir with N2 for 10 min and filter out the solution, add another 10 mL of 20% piperidine DMF solution, stir with N2 for 5 min and filter out the solution again. Repeat this operation twice, then stir with DMF 4 times and wash with methanol 2 times, 5-6 mL each time.

[0050] (5) Detection of ninhydrin removal effect: Take 15 mg of resin, wash it three times with methanol, add one drop each of ninhydrin, KCN and phenol solution, heat at 105-110 ℃ for 5 min, and the reaction is positive when it turns dark blue, indicating that the removal is complete and the next step can be carried out; if it is colorless, it means that the protecting group has not been completely removed and the above deprotection operation needs to be repeated.

[0051] (6) Removal of the second amino acid and Fmoc protecting group: Weigh 3 times the molar excess of the second C-terminal amino acid, 3 times the molar excess of HBTU, and 3 times the molar excess of HOBT into a reaction tube. Add an appropriate amount of DMF solution to completely dissolve them, then add 10 times the molar excess of DIEA. React at room temperature for 40 min, and wash with DMF 5 times, 5-6 mL each time. Take a small amount of resin and test it with ninhydrin reagent. If it turns colorless, add 10 mL of 20% piperidine DMF solution to remove Fmoc. Repeat this process twice, for 10 min and 5 min respectively. Then wash with DMF 4 times and methanol 2 times, 5-6 mL each time. Take a small amount of resin and test it with ninhydrin reagent. If it turns blue, proceed to the next step of the reaction.

[0052] (7) Repeat step (6) in this manner until the last amino acid at the N-terminus is synthesized, remove the Fmoc protecting group, and then dry the mixture.

[0053] (8) Resin shedding and pure product separation detection: The peptide was cut with trifluoroacetic acid cutting solution (95% TFA: 2% TIS: 2% EDT: 1% H2O) for 2 h. The reaction solution was filtered to obtain a trifluoroacetic acid solution of the peptide. The lysis solution was dried as much as possible with nitrogen gas, then precipitated with ether, centrifuged, and then washed with ether 4 times to obtain a white solid. After being dissolved in pure water, it was desalted and purified by HPLC, and then lyophilized to precipitate crystals to obtain the target peptide.

[0054] The 25 candidate peptides listed in Table 1 were all synthesized using the methods described in steps (1) to (7) above.

[0055] Example 4

[0056] Effects of different antler peptides on osteoblast activity

[0057] The culture medium described below is α-MEM medium containing 10% fetal bovine serum and 1 wt% penicillin and antibiotics.

[0058] (1) Culture and passage of osteoblasts (MC3T3-E1): Osteoblasts (MC3T3-E1) are a good model for in vitro study of osteoblasts. Osteoblasts MC3T3-E1 were placed in culture medium and cultured in a 37 ℃, 5% CO2 cell culture incubator. The culture medium was changed every 3 days. When the cells reached about 80% confluence, they were passaged and the logarithmic growth phase cells were used for subsequent experiments.

[0059] (2) Take cells in the logarithmic growth phase, digest them, and then use 2×10⁻⁶ cells in the experimental group. 3100 μL (candidate peptide solution, dissolved in PBS, concentration 10 μM) per well was seeded into 96-well plates. A blank control group was added with 100 μL of culture medium per well. The plates were incubated at 37 ℃ in a 5% CO2 incubator for 24 h. 10 μL of CCK-8 solution was added to each well, and the plates were incubated at 37 ℃ in a 5% CO2 incubator for 1 h. The absorbance was measured at 450 nm using a microplate reader, and the cell proliferation rate was calculated. The cell proliferation rate results of the 25 candidate peptides listed in Table 1 for osteoblasts are shown in the figure below. Figure 1 As shown:

[0060] Depend on Figure 1 It was found that different candidate peptides had significantly different effects on osteoblast activity. Compared with the control group, some candidate peptides had little effect on osteoblast activity or a slight promoting effect (G29, G30, G31, G32, G33, G34), while most candidate peptides showed a significant promoting effect on osteoblast proliferation (G26, G27, G28, G35, G36, G37, G38, G39, G40, G41, G42, G43, G44, G45, G46, G47, G48, G49, G50). Among them, the osteoblast proliferation rate after intervention with antler peptide G35 exceeded 150%. The four candidate peptides with the most significant promotion of osteoblast activity (antler peptides: G35, G40, G44, G45) were selected for subsequent experiments.

[0061] Example 5

[0062] Effects of different concentrations of antler osteopetrokinase on osteoblasts

[0063] (1) The culture and passage of osteoblasts (MC3T3-E1) were the same as in Example 4;

[0064] (2) Take cells in the logarithmic growth phase, digest them, and then use 2×10⁻⁶ cells in the experimental group. 3 Cells were seeded at 100 μL / well in 96-well plates, with a blank control group added at 100 μL / well. Incubation was performed at 37 ℃ in a 5% CO2 incubator for 24 h. After adding 10 μL of CCK-8 solution to each well and incubating at 37 ℃ in a 5% CO2 incubator for 1 h, absorbance was measured at 450 nm using a microplate reader to calculate cell proliferation rate. The cell proliferation rate of osteoblasts at four different concentrations of antler-derived osteopeptide is shown in the figure below. Figure 2 As shown;

[0065] Depend on Figure 2It was found that different concentrations of antler-derived osteoblast peptides had significantly different effects on osteoblast activity. The promoting effects of the four antler-derived osteoblast peptides on osteoblast activity all showed a concentration-dependent trend. Among them, antler-derived osteoblast peptides G35 and G45 showed the strongest promoting effect on osteoblast activity at a peptide concentration of 8 μM, which was much higher than the other two antler-derived peptides. Therefore, a concentration of 8 μM for G35 and G45 was selected for subsequent experiments.

[0066] Example 6

[0067] Effects of antler-derived osteogenic peptides on osteoblast growth curves

[0068] (1) The culture and passage of osteoblasts (MC3T3-E1) were the same as in Example 4;

[0069] (2) Take cells in the logarithmic growth phase, digest them, and then use 2×10⁻⁶ cells in the experimental group. 3 Cells were seeded at 100 μL / well in 96-well plates, with a blank control group added at 100 μL / well. Incubation was performed at 37 ℃ in a 5% CO2 incubator for 24 h. Then, 10 μL of CCK-8 solution was added to each well, and incubation was continued at 37 ℃ in a 5% CO2 incubator for 1 h. Absorbance was measured at 450 nm using a microplate reader. The same measurements were performed at the same time on days 1, 2, and 3. Cell proliferation curves were plotted based on the absorbance values. The osteoblast growth curve after intervention with antler-derived bone-strengthening peptide G35 / G45 is shown below. Figure 3 As shown; by Figure 3 It can be seen that antler-derived osteopeptide G35 has a significant effect on osteoblast proliferation.

Claims

1. A deer antler bone-strengthening peptide, characterized in that, The aforementioned deer antler bone-strengthening peptide is G35; The amino acid sequence of G35 is shown in SEQ ID NO.

10.

2. The use of the antler-strengthening peptide according to claim 1 in the preparation of bone repair drugs.

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