A modified bovine bone collagen peptide, its preparation method, and a bone density-enhancing composition thereof.

Abstract

This invention provides a modified bovine bone collagen peptide, its preparation method, and a bone density-enhancing composition. The modified bovine bone collagen peptide forms a phosphoramide covalent bond between the phosphonic acid group (-PO3H2) in a bisphosphonic acid compound and the free amino group of the bovine bone collagen peptide. Furthermore, the phosphonic acid group, carboxyl group, and hydroxyl group form stable coordination chelates with calcium ions, making the modified bovine bone collagen peptide a stable calcium ion coordination chelate. The P-C-P bisphosphonic acid core in the modified bovine bone collagen peptide molecule of this invention is a bone-targeting core group, capable of specific and strong coordination binding with hydroxyapatite crystals in the mineralized matrix of bone tissue. This allows for precise targeting and enrichment in areas of active bone metabolism, overcoming the shortcomings of natural collagen peptides, such as lack of bone targeting and low systemic distribution efficiency, thus achieving highly efficient concentration of active ingredients in the local bone. The composition of this invention further promotes osteogenic differentiation of cells, increases bone density, and also promotes the maturation and cross-linking properties of type I bone collagen.

Description

Technical Field

[0001] This invention relates to the field of protein peptide technology, specifically to a modified bovine bone collagen peptide, its preparation method, and a bone density-increasing composition. Background Technology

[0002] Collagen is the most abundant protein in mammals, accounting for approximately 30% of total protein. To date, 28 different types of collagen have been discovered, with type I collagen being the most abundant, primarily found in animal bones, skin, and tendons. Bovine bone collagen peptides are peptides that break the ionic bonds and Schiff bases between collagen molecules, thereby opening aldehyde-amine cross-links. Yak bone collagen peptides GASGPMGPR and GLPGPM have effects on DPPH free radicals, ABTS cation free radicals, ·OH, and ·O. -2 Both peptides have good free radical scavenging effects. Studies have found that both peptides can significantly reduce the accumulation of ROS and malondialdehyde (MDA) in Caenorhabditis elegans and increase the activity of superoxide dismutase (SOD) and catalase (CAT), thereby achieving antioxidant and anti-aging effects. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a modified bovine bone collagen peptide, its preparation method, and a bone density-increasing composition.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows: the modified bovine bone collagen peptide is formed by the phosphonic acid group (-PO3H2) in the bisphosphonic acid compound forming a phosphoramide covalent bond with the free amino group of the bovine bone collagen peptide, and the phosphonic acid group, carboxyl group, and hydroxyl chelation site forming a stable coordination chelate with calcium ions, the modified bovine bone collagen peptide being a stable coordination chelate of calcium ions, and the bisphosphonic acid compound being alendronic acid, risedronic acid, or ibandronic acid.

[0005] The PCP bisphosphonate core in the modified bovine bone collagen peptide molecule is a core bone-targeting group that can specifically and strongly coordinate with hydroxyapatite crystals in the mineralized matrix of bone tissue, precisely targeting and enriching in areas of active bone metabolism (osteoporosis foci, bone defects). This overcomes the shortcomings of natural collagen peptides, such as lack of bone targeting and low systemic distribution efficiency, achieving highly efficient concentration of active ingredients in the local bone. On the one hand, the modified peptide retains the natural backbone sequence of bovine bone collagen peptides, which can mimic the type I collagen microenvironment of the extracellular matrix (ECM) in vivo, providing specific adhesion sites for BMSCs. Through integrin receptors, it mediates cell adhesion and spread, initiating the initial signal of osteogenic differentiation. On the other hand, the bisphosphonate group can promote the maturation of osteogenic phenotypes, such as alkaline phosphatase (ALP) activation and osteocalcin (OCN) secretion. At the same time, chelated calcium ions further amplify the osteogenic differentiation effect by regulating calcium-sensitive receptor (CaSR) signaling, synergistically inducing BMSCs to differentiate into osteoblasts and inhibiting adipogenic differentiation bias. The modified peptide uses phosphonic acid groups (-PO3H2), carboxyl groups, and hydroxyl groups as multiple chelating sites to form stable coordination chelates with calcium ions. It can slowly release free calcium under physiological conditions, continuously supplying calcium source for bone mineralization. At the same time, this chelation system can regulate the extracellular calcium microenvironment, induce the orderly nucleation and deposition of hydroxyapatite crystals, and improve the mineralization of bone matrix. In addition, the bisphosphonic acid groups can inhibit osteoclast proliferation and bone resorption activity, balance the homeostasis of bone formation and bone resorption metabolism, and synergistically improve bone density through three pathways: calcium supply, mineralization regulation, and bone metabolism balance. The collagen peptides in this modified peptide share high molecular homology with endogenous type I collagen, serving as biological templates for collagen molecule assembly and guiding the orderly arrangement of endogenous collagen molecules. Simultaneously, the hydroxyl and phosphonic acid groups in the modified peptide can form hydrogen bonds and electrostatic interactions with lysine and hydroxylysine residues in collagen molecules, stabilizing the triple helix structure of collagen and upregulating the activity of lysyl oxidase (LOX), promoting the formation of mature covalent cross-links such as pyridinoline and deoxypyridinoline between collagen molecules. Furthermore, calcium chelation sites can mediate the tight binding of collagen fibers to hydroxyapatite, constructing a complete "collagen-mineral" composite matrix, significantly enhancing the mature cross-linking properties of type I collagen and the stability of bone structure.

[0006] Preferably, the grafting rate of the PO3H2 group in the modified bovine bone collagen peptide is 350~550 μmol / g based on the bovine bone collagen peptide raw material; the molar ratio of PO3H2 to -NH2 in the modified bovine bone collagen peptide is 0.80~2.00; the molar ratio of PO3H2 to carboxyl groups in the modified bovine bone collagen peptide is 0.35~0.60; the phosphorus content in the modified bovine bone collagen peptide is 2.0%~3.2%; the chelated calcium content in the modified bovine bone collagen peptide is 6.0%~9.5%; the hydroxyl content in the modified bovine bone collagen peptide is 0.73~0.95 mmol / g; the weight-average molecular weight (Mw) of the modified bovine bone collagen peptide is 750~2000 Da; and the dispersion index (PDI) is 1.5~1.8.

[0007] The content of PO3H2 groups, hydroxyl groups, and carboxyl groups in modified bovine bone collagen peptides is the content of groups included in calcium ion chelation.

[0008] Preferably, the bisphosphonic acid compound is alendronic acid, riseronicacid, or ibandronic acid.

[0009] Preferably, the preparation method of the modified bovine bone collagen peptide includes the following steps:

[0010] (1) The phosphonic acid group (-PO3H2) in the bisphosphonic acid compound is activated by EDC / NHS and forms a phosphoramide covalent bond with the free amino group (-NH2) of bovine bone collagen peptide. The total hydroxyl content in the bovine bone collagen peptide raw material is 0.82~1.06 mmol / g, the total carboxyl content in the bovine bone collagen peptide raw material is 0.65~1.76 mmol / g, the total amino content in the bovine bone collagen peptide raw material is 0.55~1.32 mmol / g, the weight average molecular weight Mw of the bovine bone collagen peptide is 700~1700 Da, and the dispersion PDI is 1.5~1.8.

[0011] (2) 6%~8% (w / v) of the product aqueous solution of step (1) is heated to 40~45℃, and calcium lactate is slowly added. The amount of calcium lactate is 7.0%~10.5% of the weight of the product of step (1). The pH is controlled at 6.5~7.0, and the product is chelated at a constant temperature for 1.5~2 h to obtain modified bovine bone collagen peptide. Unreacted free small molecules are removed by dialysis bag.

[0012] Preferably, step (1) of the method for preparing modified bovine bone collagen peptides includes adjusting the pH of the bisphosphonic acid solution to 5.0~5.5, activating the bisphosphonic acid aqueous solution with EDC and NHS at a molar ratio of 1:(1.1~1.3):(1.1~1.3) at 25~30℃ in the dark to form a highly active bisphosphonic acid active ester, slowly adding the highly active bisphosphonic acid active ester solution dropwise to a 5%~10% bovine bone collagen peptide solution, controlling the temperature at 25~30℃ throughout the process, maintaining the pH at 5.5~6.0 with an acid-base adjuster, stirring the reaction for 3~4 hours, and then removing unreacted free small molecules using a dialysis bag.

[0013] This invention also provides a method for preparing any of the modified bovine bone collagen peptides described above, the method comprising the following steps:

[0014] (1) The phosphonic acid group (-PO3H2) in bisphosphonic acid compounds (containing PCP core) is activated by EDC / NHS and forms a phosphoramide covalent bond with the free amino group (-NH2) of bovine bone collagen peptide;

[0015] (2) 6%~8% (w / v) of the product aqueous solution of step (1) is heated to 40~45℃, and calcium lactate is slowly added. The amount of calcium lactate is 7.0%~10.5% of the weight of the product of step (1). The pH is controlled at 6.5~7.0, and the product is chelated at a constant temperature for 1.5~2 h to obtain modified bovine bone collagen peptide.

[0016] Preferably, step (1) of the method for preparing modified bovine bone collagen peptide includes adjusting the pH of the bisphosphonic acid solution to 5.0~5.5, activating the bisphosphonic acid aqueous solution with EDC and NHS at a molar ratio of 1:(1.1~1.3):(1.1~1.3) at 25~30℃ in the dark to form a highly active bisphosphonic acid active ester, slowly adding the highly active bisphosphonic acid active ester solution dropwise to a 5%~10% bovine bone collagen peptide solution, controlling the temperature at 25~30℃ throughout the process, maintaining the pH at 5.5~6.0 with an acid-base adjuster, stirring the reaction for 3~4 hours, and then removing free small molecules using a dialysis bag.

[0017] The present invention also provides a modified bovine bone collagen peptide composition comprising the following components in the following weight ratios: 5-15 parts by weight of any of the modified bovine bone collagen peptides described above, 0.1-1.0 parts by weight of salicumin, 20-30 parts by weight of maltodextrin, 25-35 parts by weight of starch, 0.2-1.2 parts by weight of citric acid, and 0.2-1.0 parts by weight of solid dosage form excipients, wherein the solid dosage form excipients are a combination of silica or magnesium stearate.

[0018] The combination of modified bovine bone collagen peptides, apigenin, and citric acid in the above composition can further enhance the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), increase bone density, and promote the maturation and cross-linking properties of type I bone collagen.

[0019] Preferably, the modified bovine bone collagen peptide composition comprises the following components in the following weight ratios: 8-12 parts by weight of the modified bovine bone collagen peptide as described above, 0.3-0.7 parts by weight of salicumin, 20-30 parts by weight of maltodextrin, 25-35 parts by weight of starch, 0.4-1.0 parts by weight of citric acid, and 0.2-1.0 parts by weight of solid dosage form excipients, wherein the solid dosage form excipients are a combination of silica or magnesium stearate.

[0020] Preferably, the weight ratio of any of the above-mentioned modified bovine bone collagen peptides, apigenin, and citric acid in the modified bovine bone collagen peptide composition is 1:(0.04~0.08):(0.05~0.1).

[0021] The beneficial effects of this invention are as follows: This invention provides a modified bovine bone collagen peptide, its preparation method, and a bone density-increasing composition. In the modified bovine bone collagen peptide molecule, the PCP bisphosphonate core is a bone-targeting core group that can specifically and strongly coordinate with hydroxyapatite crystals in the mineralized matrix of bone tissue, precisely targeting and enriching in areas of active bone metabolism (osteoporosis foci, bone defect areas). This overcomes the shortcomings of natural collagen peptides, such as lack of bone targeting and low systemic distribution efficiency, achieving highly efficient concentration of active ingredients in the local bone. The combination of modified bovine bone collagen peptide, salicumin, and citric acid in the composition of this invention can further enhance the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), increase bone density, and simultaneously promote the maturation and cross-linking properties of type I bone collagen. Attached Figure Description

[0022] Figure 1 This is a GPC chromatogram of the modified bovine bone collagen peptide of Example 1 of the present invention.

[0023] Figure 2 The image shows the infrared spectrum of the modified bovine bone collagen peptide from Example 1 of this invention.

[0024] Figure 3 The image shows the infrared spectrum of the modified bovine bone collagen peptide from Example 2 of this invention.

[0025] Figure 4 This is a chromatogram of the modified bovine bone collagen peptides from Example 1 of the present invention, obtained by detecting the mature cross-linking performance of type I bone collagen (HPLC method). Detailed Implementation

[0026] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0027] Example 1

[0028] As an embodiment of the present invention, a modified bovine bone collagen peptide is formed by the phosphonic acid group (-PO3H2) in a bisphosphonic acid compound (containing a PCP core) forming a phosphoramide covalent bond with the free amino group of the bovine bone collagen peptide, and the phosphonic acid group, carboxyl group, and hydroxyl chelation site forming a stable coordination chelate with calcium ions, thus the modified bovine bone collagen peptide is a stable coordination chelate of calcium ions.

[0029] The grafting rate of the PO3H2 group in the modified bovine bone collagen peptide, based on the bovine bone collagen peptide raw material, is 350~550 μmol / g. The molar ratio of PO3H2 to -NH2 in the modified bovine bone collagen peptide is 0.80~2.00, the molar ratio of PO3H2 to carboxyl groups in the modified bovine bone collagen peptide is 0.35~0.60, the phosphorus content in the modified bovine bone collagen peptide is 2.0%~3.2%, the chelated calcium content in the modified bovine bone collagen peptide is 6.0%~9.5%, the hydroxyl content in the modified bovine bone collagen peptide is 0.73~0.95 mmol / g, the weight-average molecular weight (Mw) of the modified bovine bone collagen peptide is 750~2000 Da, and the dispersion PDI is 1.5~1.8.

[0030] Bisphosphonic acid compounds include riseronic acid.

[0031] The preparation method of the modified bovine bone collagen peptide in this embodiment includes the following steps:

[0032] 1) Prepare risedronic acid aqueous solution and adjust the pH to 5.0 with 0.1 mol / L hydrochloric acid solution; mix risedronic acid aqueous solution, EDC, and NHS in a molar ratio of 1:1.2:1.2 and activate at 25±2℃ in the dark for 30 min to form highly active risedronic acid active ester; slowly add the active ester solution dropwise to an 8% (w / w) bovine bone collagen peptide aqueous solution, maintain the temperature at 30±1℃ throughout, and maintain the pH of the system at 5.5 using 0.1 mol / L hydrochloric acid solution and 0.1 mol / L sodium hydroxide solution, and stir the reaction for 3.5 h; put the reaction solution into a 500 Da dialysis bag and dialyze with deionized water for 24 h to remove unreacted free small molecules, and obtain risedronic acid grafted modified bovine bone collagen peptide;

[0033] (2) Prepare the product obtained in step (1) into a 7% (w / v) aqueous solution, heat it to 42°C, slowly add calcium lactate, the amount of calcium lactate added is 8.5% of the dry weight of the product in step (1) (based on calcium ion content), adjust the pH to 6.7 with an acid-base regulator, stir and chelate at a constant temperature for 1.8 h, put the reaction solution into a 500 Da dialysis bag, dialyze with deionized water for 24 h to remove unreacted free small molecules, and obtain the modified bovine bone collagen peptide (calcium chelate) of this embodiment. The amount of risedronic acid is 2.8% of the mass of the bovine bone collagen peptide raw material.

[0034] I. Characterization and Detection

[0035] 1. Weight-average molecular weight (Mw) and dispersion (PDI)

[0036] Detection method: Gel permeation chromatography (GPC)

[0037] Chromatographic conditions: A TSKgel G2000 SWXL column (7.8 mm × 300 mm, aqueous gel permeation column) was used. The mobile phase was 0.1 mol / L sodium nitrate aqueous solution, the flow rate was 1.0 mL / min, the column temperature was 30℃, and the detection was performed using a differential refractive index detector. A standard curve was plotted using polyethylene glycol (PEG) as the molecular weight standard, and the weight-average molecular weight and dispersity of the samples were calculated.

[0038] 2. Total amino, total carboxyl, and total hydroxyl content

[0039] Detection method: Potentiometric titration

[0040] (1) Amino content: The ninhydrin colorimetric method combined with potentiometric titration was used, with glycine as the standard. The absorbance was measured at 570 nm, and the free amino content was quantitatively calculated.

[0041] (2) Carboxyl content: Titrate with 0.1 mol / L NaOH standard solution using potentiometric titration until pH=8.2, and calculate the molar content of carboxyl groups;

[0042] (3) Hydroxyl content: The molar content of hydroxyl groups was quantitatively calculated by combining periodic acid oxidation method with potentiometric titration and the oxidation products.

[0043] 3. Phosphorus content

[0044] Detection method: ICP-OES samples were pretreated by microwave digestion, a phosphorus standard curve was plotted, and the mass fraction of phosphorus in the samples was quantitatively determined by external standard method.

[0045] 4. Grafting rate

[0046] Detection method: Phosphorus content was determined by inductively coupled plasma optical emission spectrometry (ICP-OES). The amount of phosphonic acid grafting was calculated based on the molecular structure of risedronic acid. The grafting rate (μmol / g) was calculated based on the mass of raw bovine bone collagen peptide.

[0047] 5. Chelated calcium content

[0048] Detection method: ICP-OES

[0049] Detection method: ICP-OES samples were pretreated by microwave digestion, a calcium standard curve was plotted, and the mass fraction of phosphorus in the samples was quantitatively determined by external standard method.

[0050] The test results showed that the total hydroxyl content of the raw bovine bone collagen peptide was 0.94 mmol / g, the total carboxyl content was 1.20 mmol / g, the total amino content was 0.93 mmol / g, the weight-average molecular weight (Mw) of the raw bovine bone collagen peptide was 1218 Da, and the dispersion index (PDI) was 1.63.

[0051] The grafting rate of the PO3H2 group in the modified bovine bone collagen peptide, based on the bovine bone collagen peptide raw material, is 422 μmol / g. The molar ratio of PO3H2 to -NH2 in the modified bovine bone collagen peptide is 1.38, the molar ratio of PO3H2 to carboxyl groups is 0.48, the phosphorus content is 2.83%, the chelated calcium content is 8.23%, the hydroxyl content is 0.83 mmol / g, the weight-average molecular weight (Mw) is 1384 Da, and the dispersion index (PDI) is 1.68.

[0052] Example 2

[0053] As a modified bovine bone collagen peptide in this embodiment of the invention, the only difference between this embodiment and Example 1 is that the bisphosphonate compound is alendronic acid.

[0054] The grafting rate of the PO3H2 group in the modified bovine bone collagen peptide, based on the bovine bone collagen peptide raw material, is 415 μmol / g. The molar ratio of PO3H2 to -NH2 in the modified bovine bone collagen peptide is 1.35, the molar ratio of PO3H2 to carboxyl groups is 0.47, the phosphorus content is 2.78%, the chelated calcium content is 8.15%, the hydroxyl content is 0.82 mmol / g, the weight-average molecular weight (Mw) is 1362 Da, and the dispersion index (PDI) is 1.66.

[0055] Example 3

[0056] As a modified bovine bone collagen peptide in this embodiment of the invention, the only difference between this embodiment and Example 1 is the amount of riseronic acid used.

[0057] Risedronic acid dosage: 2.3% of the mass of bovine bone collagen peptide raw material. The grafting rate of PO3H2 groups of modified bovine bone collagen peptide based on bovine bone collagen peptide raw material is 358 μmol / g.

[0058] Example 4

[0059] As a modified bovine bone collagen peptide in this embodiment of the invention, the only difference between this embodiment and Example 1 is the amount of riseronic acid used.

[0060] Risedronic acid dosage: 3.0% of the mass of bovine bone collagen peptide raw material. The grafting rate of PO3H2 groups of modified bovine bone collagen peptide based on bovine bone collagen peptide raw material is 487 μmol / g.

[0061] Example 5

[0062] As a modified bovine bone collagen peptide in this embodiment of the invention, the only difference between this embodiment and Example 1 is the amount of riseronic acid used.

[0063] Risedronic acid dosage: 3.2% of the mass of bovine bone collagen peptide raw material. The grafting rate of PO3H2 groups of the modified bovine bone collagen peptide is 537 μmol / g based on the bovine bone collagen peptide raw material.

[0064] Example 6

[0065] As a modified bovine bone collagen peptide in this embodiment of the invention, the only difference between this embodiment and Example 1 is the amount of calcium lactate added. The amount of calcium lactate added is 6.3% of the dry weight of the product in step (1) (based on calcium ion content).

[0066] The modified bovine bone collagen peptide contains 6.08% chelated calcium.

[0067] Example 7

[0068] As a modified bovine bone collagen peptide in this embodiment of the invention, the only difference between this embodiment and Example 1 is the amount of calcium lactate added. The amount of calcium lactate added is 7.4% of the dry weight of the product in step (1) (based on calcium ion content).

[0069] The modified bovine bone collagen peptide contains 7.19% chelated calcium.

[0070] Example 8

[0071] As a modified bovine bone collagen peptide in this embodiment of the invention, the only difference between this embodiment and Example 1 is the amount of calcium lactate added. The amount of calcium lactate added is 9.1% of the dry weight of the product in step (1) (based on calcium ion content).

[0072] The modified bovine bone collagen peptide contains 8.82% chelated calcium.

[0073] Example 9

[0074] As a modified bovine bone collagen peptide in this embodiment of the invention, the only difference between this embodiment and Example 1 is the amount of calcium lactate added. The amount of calcium lactate added is 9.8% of the dry weight of the product in step (1) (based on calcium ion content).

[0075] The modified bovine bone collagen peptide contains 9.46% chelated calcium.

[0076] Example 10

[0077] As an embodiment of the present invention, a modified bovine bone collagen peptide composition comprises the following components in the following weight ratio: 10 parts by weight of the modified bovine bone collagen peptide described in Example 1, 0.5 parts by weight of apigenin, 25 parts by weight of maltodextrin, 30 parts by weight of starch, 0.6 parts by weight of citric acid, and 0.5 parts by weight of magnesium stearate.

[0078] Comparative Example 1

[0079] As a comparative example of the present invention, this modified bovine collagen peptide differs from Example 1 only in that calcium ion chelation is not performed. That is, the dialysis purification product of step (1) of Example 1.

[0080] Comparative Example 2

[0081] As a comparative example of the present invention, this modified bovine bone collagen peptide differs from Example 1 only in that it does not undergo a bisphosphonate compound reaction, but directly chelates the bovine bone collagen peptide raw material with calcium ions.

[0082] Experimental methods

[0083] I. Material Characterization

[0084] (a) GPC characterization of molecular distribution

[0085] Chromatographic conditions: A TSKgel G2000 SWXL column (7.8 mm × 300 mm, aqueous gel permeation column) was used. The mobile phase was 0.1 mol / L sodium nitrate aqueous solution, the flow rate was 1.0 mL / min, the column temperature was 30℃, and the detection was performed using a differential refractive index detector. A standard curve was plotted using polyethylene glycol (PEG) as the molecular weight standard, and the weight-average molecular weight and dispersity of the samples were calculated.

[0086] The GPC chromatogram of the product of Example 1 is shown below. Figure 1As shown. Results: The product sample from the example exhibited a continuous distribution of multiple characteristic peaks within the range of 15–22 min, with no macromolecular components >3000 Da. The main peak was located around 17.2 min, corresponding to a weight-average molecular weight Mw = 1384 Da and a dispersion PDI = 1.68. The molecular weight distribution was as follows: <500 Da accounted for 21.9%, 500–1000 Da accounted for 41.6%, 1000–2000 Da accounted for 25.2%, and 2000–3000 Da accounted for 11.3%, demonstrating that the modification reaction was uniform and the purification was thorough.

[0087] (ii) Infrared characterization

[0088] Fourier transform infrared spectroscopy (FTIR) was used to measure the sample in the mid-infrared band (4000~400 cm⁻¹). -1 The infrared absorption spectrum of the sample was obtained. The sample was prepared by potassium bromide tableting. 1~2 mg of sample was mixed with 100~150 mg of dry KBr powder and ground evenly in an agate mortar (particle size ≤2μm); pressure was applied on a tablet press (8~10 MPa) for 1~2 min to form a transparent thin film, which was then placed in the sample cell for testing.

[0089] The infrared results of the sample in Example 1 are shown below. Figure 2 As shown, at 1650 cm -1 (Amide I band), 1540 cm -1 (Amide II band), 1240 cm -1 The characteristic absorption peak of collagen peptides appeared at the (amide III band), proving that the modification process did not destroy the peptide bonds and secondary structure of bovine bone collagen peptides, thus preserving the bioactive framework of collagen. At 1240 cm⁻¹ -1 (P=O stretching), 1080 cm -1 (POC / POH telescopic), 960 cm -1 The presence of characteristic phosphonic acid group peaks at the (PO stretching) position indicates that risedronic acid is covalently grafted onto the amino group of the peptide chain via a phosphoramide bond. At 870 cm⁻¹ -1 The presence of a Ca-O coordination vibration characteristic peak at the point indicates that calcium ions form stable coordination chelates with the carboxyl, hydroxyl, and phosphonic acid groups of the peptide chain, thus successfully constructing the modified structure described in this invention.

[0090] The infrared results of the sample in Example 2 are shown below. Figure 3 As shown, at 1650 cm -1 (Amide I band), 1540 cm -1 (Amide II band), 1240 cm -1The characteristic absorption peak of collagen peptides appeared at the (amide III band), proving that the modification process did not destroy the peptide bonds and secondary structure of bovine bone collagen peptides, thus preserving the bioactive framework of collagen. At 1240 cm⁻¹ -1 (P=O stretching), 1080 cm -1 (POC / POH telescopic), 960 cm -1 The presence of characteristic phosphonic acid group peaks at the (PO stretching) position indicates that risedronic acid is covalently grafted onto the amino group of the peptide chain via a phosphoramide bond. At 870 cm⁻¹ -1 The presence of a Ca-O coordination vibration characteristic peak at the point indicates that calcium ions form stable coordination chelates with the carboxyl, hydroxyl, and phosphonic acid groups of the peptide chain, thus successfully constructing the modified structure described in this invention.

[0091] II. Performance Testing

[0092] (a) Osteoblast proliferation assay (quantitative detection using CCK-8 assay)

[0093] 1. Experimental Methods

[0094] Mouse osteoblasts MC3T3-E1 were taken and subjected to a concentration of 1×10⁻⁶ cells. 4 One cell per well was inoculated into a 96-well plate and cultured for 24 hours for adhesion.

[0095] Groups: Control group (complete culture medium), sample group (culture medium + 100 μg / mL modified collagen peptide). After culturing for 24, 48, and 72 h, 10 μL CCK-8 reagent was added to each well, and the mixture was incubated at 37℃ for 1.5 h. The absorbance (OD) value of each well was measured at 450 nm using a microplate reader.

[0096] The average value of the three parallel experiments was taken.

[0097] 2. Calculation of Quantitative Results

[0098] Cell proliferation rate = (OD of sample group - OD of control group) / OD of control group × 100%

[0099] The experimental results are shown in Figure 1:

[0100] Table 1. Osteoblast proliferation properties of modified bovine bone collagen peptides

[0101] The proliferation rates of the two examples at 24h, 48h, and 72h were significantly higher than those of the comparative examples 1 and 2 (p<0.05), indicating that the synergistic structure of bisphosphonate grafting and calcium ion chelation can significantly promote the growth and proliferation of osteoblasts MC3T3-E1.

[0102] (II) Quantitative Detection of Alizarin Red S Mineralization

[0103] 1. Reagents and Instruments

[0104] Alizarin Red S staining solution, 10% formaldehyde fixative, cetylpyridine chloride (CPC) solution, and microplate reader.

[0105] 2. Experimental Methods

[0106] Cell fixation: After 21 days of induction, the culture medium was discarded, the cells were washed twice with PBS, and fixed with 10% formaldehyde at room temperature for 30 min.

[0107] Staining: Discard the fixative, wash with PBS, add 0.1% Alizarin Red S staining solution, and stain at room temperature in the dark for 30 min.

[0108] Washing: Discard the staining solution, wash thoroughly with PBS until no free dye remains, and record the red calcium nodules under a microscope.

[0109] Quantitative determination: Add 10% CPC solution and incubate at room temperature for 15 min to fully dissolve the calcium nodules; take the solution and measure the absorbance (OD value) at 562 nm to characterize the mineralization deposition level.

[0110] Mineralization deposition rate (%) = OD value of sample group / OD value of complete culture medium in control group × 100%

[0111] The average value of the three parallel experiments was taken.

[0112] (III) Determination of the maturation and cross-linking properties of type I bone collagen (HPLC method)

[0113] 1. Sample pretreatment

[0114] The cell matrix after induced differentiation was taken and purified by pepsin enzymatic hydrolysis, hydrochloric acid hydrolysis, and solid-phase extraction to obtain type I collagen hydrolysate.

[0115] 2. Chromatographic conditions

[0116] High performance liquid chromatography (HPLC) instrument with a C18 column; the mobile phase is acetonitrile-water system, 0-5 min 5% acetonitrile, 5-20 min linearly boosted to 20% acetonitrile, 20-25 min 25% acetonitrile; detection wavelength: 295 nm; column temperature: 30℃, flow rate: 1.0 mL / min.

[0117] 3. Detection indicators

[0118] Quantitative determination of mature collagen cross-linked compounds: pyridinium (PYD) and deoxypyridinium (DPD), calculation of cross-linking concentration and PYD / DPD ratio, characterizing the degree of mature cross-linking of type I collagen.

[0119] The chromatogram of the sample in Example 1 is shown below. Figure 4As shown in the figure. Based on pyridinoline (PYD) and deoxypyridinoline (DPD) standards, the peak at 12.2 min is deoxypyridinoline and the peak at 13.4 min is pyridinoline (PYD). The retention times of these two peaks are consistent with those of the pyridinoline (PYD) and deoxypyridinoline (DPD) standards, and they are the target characteristic peaks. The remaining peaks are matrix impurities and hydrolysis byproducts, which may include free amino acids, hydroxyproline, glycosaminoglycan fragments, non-crosslinked pyridine derivatives, collagen peptide fragments, hydrophobic pigments, lipid byproducts, etc.

[0120] The average value of the three parallel experiments was taken.

[0121] Table 2. Bone mineralization and deposition properties of modified bovine bone collagen peptides and maturation and cross-linking properties of type I bone collagen.

[0122] The PYD / DPD ratios in the example groups remained at the normal bone tissue level of 2.64–2.87, while those in the comparative group were significantly lower than this range. These results confirm that the present invention, by constructing a synergistic structure of bisphosphonate-grafted and calcium ion-chelated polymeric modified peptides, not only significantly enhances bone mineralization deposition capacity but also effectively optimizes the mature cross-linking structure of type I collagen, endowing the bone matrix with excellent toughness and structural stability.

[0123] (iv) Testing of the properties of the composition

[0124] Sample 1: 10 parts by weight of the modified bovine bone collagen peptide described in Example 1, 0.5 parts by weight of apigenin, and 0.6 parts by weight of citric acid.

[0125] Sample 2: 10 parts by weight of the modified bovine bone collagen peptide described in Example 1 and 1.1 parts by weight of succinate.

[0126] Sample 3: 10 parts by weight of the modified bovine bone collagen peptide described in Example 1 and 1.1 parts by weight of citric acid.

[0127] The detection method is the same as above, sample group (culture medium + 100 μg / mL composition).

[0128] Table 3. Bone mineralization deposition properties and maturation cross-linking properties of modified bovine bone collagen peptide compositions.

[0129] The combination of modified bovine bone collagen peptides, apigenin, and citric acid can further enhance the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), increase bone density, and promote the maturation and cross-linking properties of type I bone collagen.

[0130] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A modified bovine bone collagen peptide, characterized in that, The modified bovine bone collagen peptide is formed by the phosphonic acid group (-PO3H2) in the bisphosphonic acid compound forming a phosphoramide covalent bond with the free amino group of the bovine bone collagen peptide. Furthermore, the phosphonic acid group, carboxyl group, and hydroxyl chelation sites form a stable coordination chelate with calcium ions. The modified bovine bone collagen peptide is a stable coordination chelate with calcium ions. The bisphosphonic acid compound is alendronic acid, risedronic acid, or ibandronic acid.

2. The modified bovine collagen peptide according to claim 1, characterized in that, The grafting rate of the PO3H2 group in the modified bovine bone collagen peptide is 350~550 μmol / g based on the bovine bone collagen peptide raw material. The molar ratio of PO3H2 to -NH2 in the modified bovine bone collagen peptide is 0.80~2.00, and the molar ratio of PO3H2 to carboxyl group in the modified bovine bone collagen peptide is 0.35~0.

60.

3. The modified bovine collagen peptide according to claim 1, characterized in that, The modified bovine bone collagen peptides contain 2.0% to 3.2% phosphorus, 6.0% to 9.5% chelated calcium, 0.73 to 0.95 mmol / g hydroxyl groups, and have a weight-average molecular weight (Mw) of 750 to 2000 Da and a dispersion index (PDI) of 1.5 to 1.

8.

4. The modified bovine collagen peptide according to claim 1, characterized in that, The preparation method of the modified bovine bone collagen peptide includes the following steps: (1) The phosphonic acid group (-PO3H2) in the bisphosphonic acid compound is activated by EDC / NHS and forms a phosphoramide covalent bond with the free amino group (-NH2) of bovine bone collagen peptide. The total hydroxyl content in the bovine bone collagen peptide raw material is 0.82~1.06 mmol / g, the total carboxyl content in the bovine bone collagen peptide raw material is 0.65~1.76 mmol / g, the total amino content in the bovine bone collagen peptide raw material is 0.55~1.32 mmol / g, the weight average molecular weight (Mw) of the bovine bone collagen peptide is 700~1700 Da, and the dispersion (PDI) is 1.5~1.

8. (2) 6%~8% (w / v) of the product aqueous solution of step (1) is heated to 40~45℃, and calcium lactate is slowly added. The amount of calcium lactate is 7.0%~10.5% of the weight of the product of step (1). The pH is controlled at 6.5~7.0, and the product is chelated at a constant temperature for 1.5~2 h to obtain modified bovine bone collagen peptide. Unreacted free small molecules are removed by dialysis bag.

5. The modified bovine collagen peptide according to claim 3, characterized in that, The preparation method of modified bovine bone collagen peptides includes the following steps (1): adjusting the pH of the bisphosphonic acid solution to 5.0~5.5, activating the bisphosphonic acid aqueous solution with EDC and NHS at a molar ratio of 1:(1.1~1.3):(1.1~1.3) at 25~30℃ in the dark to form a highly active bisphosphonic acid active ester, slowly adding the highly active bisphosphonic acid active ester solution to a 5%~10% bovine bone collagen peptide solution, controlling the temperature at 25~30℃ throughout the process, maintaining the pH at 5.5~6.0 with an acid-base adjuster, stirring the reaction for 3~4 hours, and then removing unreacted free small molecules using a dialysis bag.

6. The method for preparing the modified bovine bone collagen peptide according to any one of claims 1 to 4, characterized in that, The method includes the following steps: (1) The phosphonic acid group (-PO3H2) in bisphosphonic acid compounds is activated by EDC / NHS and forms a phosphoramide covalent bond with the free amino group (-NH2) of bovine bone collagen peptide; (2) 6%~8% (w / v) of the product aqueous solution of step (1) is heated to 40~45℃, and calcium lactate is slowly added. The amount of calcium lactate is 7.0%~10.5% of the weight of the product of step (1). The pH is controlled at 6.5~7.0, and the product is chelated at a constant temperature for 1.5~2 h to obtain modified bovine bone collagen peptide.

7. The method for preparing modified bovine bone collagen peptides according to claim 5, characterized in that, The preparation method of modified bovine bone collagen peptides includes the following steps (1): adjusting the pH of the bisphosphonic acid solution to 5.0~5.5, activating the bisphosphonic acid aqueous solution with EDC and NHS at a molar ratio of 1:(1.1~1.3):(1.1~1.3) at 25~30℃ in the dark to form a highly active bisphosphonic acid active ester, slowly adding the highly active bisphosphonic acid active ester solution to a 5%~10% bovine bone collagen peptide solution, controlling the temperature at 25~30℃ throughout the process, maintaining the pH at 5.5~6.0 with an acid-base adjuster, stirring the reaction for 3~4 hours, and then removing free small molecules using a dialysis bag.

8. A modified bovine bone collagen peptide composition, characterized in that, The modified bovine bone collagen peptide composition comprises the following components in the following weight ratios: 5-15 parts by weight of the modified bovine bone collagen peptide as described in any one of claims 1-5, 0.1-1.0 parts by weight of apigenin, 20-30 parts by weight of maltodextrin, 25-35 parts by weight of starch, 0.2-1.2 parts by weight of citric acid, and 0.2-1.0 parts by weight of solid dosage form excipients, wherein the solid dosage form excipients are a combination of silica or magnesium stearate.

9. The modified bovine bone collagen peptide composition according to claim 8, characterized in that, The modified bovine bone collagen peptide composition comprises the following components in the following weight ratios: 8-12 parts by weight of the modified bovine bone collagen peptide as described in any one of claims 1-5, 0.3-0.7 parts by weight of apigenin, 20-30 parts by weight of maltodextrin, 25-35 parts by weight of starch, 0.4-1.0 parts by weight of citric acid, and 0.2-1.0 parts by weight of solid dosage form excipients, wherein the solid dosage form excipients are a combination of silica or magnesium stearate.

10. The modified bovine bone collagen peptide composition according to claim 8, characterized in that, The modified bovine bone collagen peptide composition contains, in any one of claims 1 to 5, modified bovine bone collagen peptide, apigenin, and citric acid in a weight ratio of 1:(0.04 to 0.08):(0.05 to 0.1).

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