Polypeptide with anti-osteoporosis activity and preparation method and application thereof

Abstract

The present application relates to the field of bioactive peptides, and specifically discloses a polypeptide with anti-osteoporosis activity and a preparation method thereof.The present application uses deer skin glue as a raw material to prepare the active polypeptide of deer skin glue with anti-osteoporosis effect, and the amino acid sequence is shown in Table SEQ.The preparation method comprises protease enzymolysis, high-temperature enzyme inactivation, and solid-phase extraction column separation of the enzymolysis product, etc.The present application uses prednisolone to induce zebrafish modeling, the polypeptide of the present application can significantly reverse the bone formation inhibition effect, increase the number of zebrafish vertebrae, has a good anti-osteoporosis effect, and can be used for preparing drugs, foods or health products for treating osteoporosis and / or carrying out bone repair.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically involving several anti-osteoporosis peptides, their preparation methods, and applications. Background Technology

[0002] Osteoporosis (OP) is a systemic disease characterized by decreased bone mass, deterioration of bone microstructure, increased bone fragility, decreased bone strength, and susceptibility to fractures. Its main clinical manifestations include pain, deformities, and fractures in the chest, back, and lower back. With the accelerating aging of the population, osteoporosis has become a significant public health problem in my country and worldwide. Currently used medications such as estrogen replacement therapy and teriparatide have shown certain side effects in preventing and treating osteoporosis. While the more widely accepted method of calcium supplementation combined with vitamin D may not be effective for everyone, excessive supplementation can increase the risk of kidney stones and cardiovascular disease.

[0003] Deer hide glue is known for its bone-strengthening properties. It contains a large amount of collagen, the most abundant protein in bones. Numerous studies have shown that collagen peptides can improve bone metabolism and have advantages such as wide availability, high bioavailability, and safety with no side effects, making it a good natural dietary source for preventing osteoporosis. Finding effective components in deer hide glue that can prevent and / or treat osteoporosis is an urgent problem to be solved. Summary of the Invention

[0004] Purpose of the invention: To overcome the problems existing in the prior art, the present invention selects and optimizes the anti-osteoporosis polypeptide and its preparation method through a large number of experimental screenings.

[0005] The technical solution of this invention is as follows: A polypeptide with anti-osteoporosis activity, wherein the amino acid sequences of the polypeptide as described in SEQ 1 are as follows: P2: GAP(+15.99)GDRGEP(+15.99)GPP(+15.99)GPAGF; P3: GSAGPP(+15.99)GATGFP(+15.99)GAAGR; P4: SGDRGETGPAGPAGPIGPVGAR; P6: GQAGVMGFP(+15.99)GPK.

[0006] The method for preparing the polypeptide with anti-osteoporosis activity according to the present invention includes the following steps: (1) Using deer hide glue as raw material, the raw material is heated and melted in a water bath, and the pH is adjusted to 7~8.0 to prepare a deer hide glue protein aqueous solution; (2) Add trypsin to the deer skin collagen solution, stir and keep at a constant temperature to obtain the enzymatic hydrolysis product; (3) After the enzymatic hydrolysis is completed, the enzymatic hydrolysis product obtained in step (2) is subjected to high temperature to inactivate the enzyme and obtain deer skin glue anti-osteoporosis enzymatic hydrolysate. (4) The enzymatic hydrolysate obtained in step (3) is separated by solid phase extraction column and eluted with pure water and acetonitrile of different concentrations in sequence; the eluent is collected and concentrated to obtain the peptide with anti-osteoporosis activity.

[0007] As a preferred embodiment, the method for preparing the above-mentioned polypeptide with anti-osteoporosis activity includes the following steps: (1) Using deer hide glue as raw material, the raw material is heated and melted in a water bath, and the pH is adjusted to 8.0 to prepare a deer hide glue protein aqueous solution; (2) Add 5% of 5000 U / g trypsin to the deer skin collagen solution, and stir and hydrolyze at a constant temperature of 37℃ and pH of 8.0 for 2 h to obtain the enzymatic hydrolysis product. (3) After the enzymatic hydrolysis is completed, the enzymatic hydrolysis product obtained in step (2) is subjected to high-temperature enzyme inactivation at a temperature of 95~105℃ for 10~20 min to obtain deer skin glue anti-osteoporosis enzymatic hydrolysate. (4) The enzymatic hydrolysate obtained in step (3) is separated by solid phase extraction column and eluted with pure water and acetonitrile of different concentrations of 5%, 10%, 15%, 20%, 40% and 80% respectively; the 10% and 15% acetonitrile eluents are collected and concentrated to obtain the peptide with anti-osteoporosis activity.

[0008] The use of the polypeptide described in this invention in the preparation of medicaments for treating osteoporosis and / or promoting bone repair. A polypeptide concentration of 20 μg / mL is particularly preferred. A drug with anti-osteoporosis activity comprising one or more polypeptides listed in SEQ1 above.

[0009] Beneficial effects

[0010] (1) The raw material used in the preparation method of the present invention is deer skin glue, which is relatively abundant.

[0011] (2) The preparation method described in this invention uses trypsin to enzymatically hydrolyze deer hide glue, which has a good enzymatic hydrolysis effect and can effectively hydrolyze deer hide glue into polypeptides with bone-forming effects.

[0012] (3) The deer skin glue bone-forming polypeptide described in this invention has high safety and strong bone-forming effect in vivo.

[0013] (4) The deer hide glue bone formation peptide described in this invention can be applied to the pharmaceutical industry, feed industry, etc., and has important economic value. Attached Figure Description

[0014] Figure 1 This is the secondary mass spectrum of the polypeptide P2 identified in Example 1 of this invention.

[0015] Figure 2 This is the secondary mass spectrum of the polypeptide P3 identified in Example 1 of the present invention.

[0016] Figure 3 This is the secondary mass spectrum of the polypeptide P4 identified in Example 1 of the present invention.

[0017] Figure 4 This is the secondary mass spectrum of the polypeptide P6 identified in Example 1 of this invention.

[0018] Figure 5 This is a statistical chart showing the effects of peptides P2, P3, P4, and P6 on bone formation in OP zebrafish in Example 2 of this invention. Note: Compared with Control, ### P < 0.001; compared with the Model, * P < 0.05, ** P < 0.01, *** P < 0.001. `x ± s, n≥ 12. Detailed Implementation

[0019] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0020] Example 1: Preparation of anti-osteoporosis peptides from deer hide glue (1) Pretreatment: Take 5 g of deer hide glue, heat it in a water bath to melt it, adjust the pH to 8.0, and prepare a 5% deer hide glue protein solution; (2) Trypsin hydrolysis: Add 5% of 5000 U / g trypsin to the deer skin collagen solution prepared in step (1), and stir and hydrolyze at a constant temperature of 37℃ and pH of 8.0 for 2 h to obtain the hydrolysis product. (3) High temperature enzyme inactivation: After the enzymatic hydrolysis is completed, the enzymatic hydrolysis product obtained in step (2) is subjected to enzyme inactivation at 100℃ for 10 min, centrifuged at 5000 r for 10 min, the supernatant is collected, concentrated and freeze-dried to obtain the inactivated powder. (4) Solid-phase extraction purification and separation: Take the lyophilized powder from step (3) and prepare a solution. Select Sep-Pak C 18Separation was performed using a Sep-Pak C18 solid-phase extraction column (6cc), with a loading concentration of 50 mg / mL and a loading volume of 2 mL. Elution was performed using acetonitrile (ACN) at different concentrations (0%, 5%, 10%, 15%, 20%, 40%, and 80%), and the fractions with the strongest anti-osteoporosis activity (10% and 15% ACN fractions) were collected. (4) Mass spectrometry sequencing. After separation and purification, nano-LC-MS / MS was used (chromatographic conditions: column: Reprosil C18 AQ (75 μm × 150 mm, 5 μm); injection volume 1 μL, flow rate 400 nL·min). -1 Mobile phase A (acetonitrile-formic acid-water, 2:0.2:98), mobile phase B (acetonitrile:formic acid:water, 80:0.2:20), 2%~30% B linear gradient elution for 90 min. Mass spectrometry conditions: electron energy 2.5 kV; ion transport capillary temperature 200 ℃; mass spectrometry first-stage full scan range m / z 300-2000; separation width 3. Total ion chromatograms (TIC) were obtained by tandem mass spectrometry analysis, and a series of MS / MS chromatograms were generated by collision-induced dissociation (CID) second-stage tandem mass spectrometry. Data were acquired using Xcalibur software. The structure and sequence of the more reactive components (10% and 15% ACN separation fractions) were identified; the second-stage mass spectra are shown in [reference needed]. Figures 1 to 4 After identification by secondary mass spectrometry, several anti-osteoporosis active peptides were obtained through analysis and screening. These are novel anti-osteoporosis peptides that had not been previously discovered. The specific amino acid sequences are as follows: P2: Gly-Ala-Hyp-Gly-Asp-Arg-Gly-Glu-Hyp-Gly-Pro-Hyp-Gly-Pro-Ala-Gly-Phe; P3: Gly-Ser-Ala-Gly-Pro-Hyp-Gly-Ala-Thr-Gly-Phe-Hyp-Gly-Ala-Ala-Gly-Arg; P4: Ser-Gly-Asp-Arg-Gly-Glu-Thr-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Pro-Ile-Gly-Pro-Val-Gly-Ala-Arg; P6: Gly-Gln-Ala-Gly-Val-Met-Gly-Phe-Hyp-Gly-Pro-Lys.

[0021] Example 2: Experiment on the anti-osteoporosis activity of deer hide gelatin active peptides 1. Experimental Materials 1.1 Reagents and Samples: The four bioactive peptides identified in Example 1 were synthesized by Nanjing Genscript Biotech Co., Ltd. The 10%-ACN separation buffer was obtained by separation and elution in Example 1.

[0022] 1.2 Drugs, Reagents and Instruments: Dimethyl sulfoxide (DMSO), Shanghai Maclean Biochemical Technology Co., Ltd., batch number: C12804515; Calcein, Shanghai Yuanye Biotechnology Co., Ltd., batch number: K22A9C68341; Prednisolone, Shanghai Yuanye Biotechnology Co., Ltd., batch number: T20J7H9291; Estradiol (17β-estradiol, E2), SIGMA-ALDRICH, batch number: 1812-0305; Stereofluorescence microscope, Leica, Germany, model: M205FA.

[0023] 2. Experimental methods: The anti-osteoporosis activity was tested using the following methods: Zebrafish larvae were grouped: Zebrafish embryos 72 h after fertilization were divided into three groups and placed in 12-well plates: Control group, Pred model group (10 μM), and E2 group (10 μM). -8 M), 10%-ACN separation solution group (20 μg·mL) -1 P2 group (20 μg·mL) -1 P3 group (20 μg·mL) -1 P4 group (20 μg·mL) -1 P6 group (20 μg·mL) -1 Each group had 3 wells, with 10 strips per well. All drugs were prepared using artificial seawater containing 0.5 mg / L methylene blue and DMSO as solvents. Starting from 3 days post-fertilization (3 dpf), the blank group was given control medium containing 0.2% DMSO, while other groups were given 10 μM Pred (containing 0.2% DMSO). At 5 dpf (day post-fertilization), each drug-treated group was given the corresponding drug-containing medium (containing 10 μM Pred), with half of the medium changed daily. At 7 dpf, 1 / 3 of the drug solution in the Pred group was discarded and replaced with the blank control solution, while 1 / 3 of the drug solution in each drug-treated group was discarded and replaced with the corresponding drug-containing medium without Pred. The 7 dpf procedure was repeated at 8 dpf. The experiment ended at 9 dpf, and calcein staining was performed. Bone formation was observed using a stereofluorescence microscope, and the number of joints was counted.

[0024] 3. Experimental Results Experimental results are as follows Figure 5 As shown, compared with the blank group, the proportion of zebrafish in the model group with the mean number of segments and the number of segments >3 (mean number of segments in the model group) was significantly reduced.P < 0.001); compared with the model group, the mean number of zebrafish segments in the estradiol E2, 10%-ACN and P2 groups was significantly increased ( P < 0.01, P < 0.05, P < 0.05), the proportion of zebrafish with more than 3 skeletal segments in groups E2, 10%-ACN, and P2, P3, P4, and P6 was significantly increased. P < 0.01, P < 0.001, P < 0.01, P <0.05, P < 0.05, P < 0.05). The above results show that Pred significantly inhibited zebrafish bone formation; estradiol E2, 10% ACN isolated peptides, and peptides P2, P3, P4, and P6 all promoted zebrafish bone formation, among which peptide P2 had a stronger bone-promoting effect.

[0025] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, any changes or variations to the above-described embodiments that are within the essential spirit of the present invention will fall within the scope of the claims of the present invention.

Claims

1. The use of the polypeptide in the preparation of a drug for treating osteoporosis and / or promoting bone repair; the amino acid sequence of the polypeptide is as follows: P2: GAP(+15.99)GDRGEP(+15.99)GPP(+15.99)GPAGF.

Images