Bovine lactoferrin compound and preparation method thereof

Abstract

The invention belongs to the field of biological materials, and particularly relates to a bovine lactoferrin complex and a preparation method thereof.The preparation method comprises the steps that bovine lactoferrin is subjected to deglycosylation, and deglycosylated bovine lactoferrin is obtained; carrying out dehydration condensation on the de-glycosylated bovine lactoferrin and chitosan to obtain a composite precursor; and compounding the compound precursor with chlorogenic acid to obtain the bovine lactoferrin compound. Compared with single bovine lactoferrin and chlorogenic acid, the bovine lactoferrin compound prepared by the preparation method disclosed by the invention can show a remarkable effect of promoting osteoblast proliferation under a very small concentration, and has an obvious synergistic interaction capability.

Description

Technical Field

[0001] The invention belongs to the field of biomaterials, and particularly relates to a bovine lactoferrin complex and a preparation method thereof. Background Art

[0002] Bone health is crucial for maintaining human health and relies on a dynamic balance of bone metabolism—the delicate equilibrium between bone formation and bone resorption. Disruption of this balance can lead to various bone-related diseases, such as osteoporosis, osteoarthritis, rheumatoid arthritis, and bone tumors. While traditional drug therapies (such as bisphosphonates and parathyroid hormone analogs) can slow bone loss to some extent, long-term use can lead to side effects such as mandibular osteonecrosis and cardiovascular complications. Therefore, the development of safer and more effective bone protection strategies is urgently needed.

[0003] In recent years, natural active ingredients have attracted considerable attention for their potential to regulate bone metabolism through multiple pathways. Chlorogenic acid (CGA) and bovine lactoferrin (BLF) have been particularly well-recognized for their roles in promoting osteoblastic activity. BLF is an iron-binding glycoprotein with a molecular weight of approximately 80 kDa. Studies have shown that BLF not only possesses antimicrobial and immunomodulatory properties but also promotes osteoblast proliferation by activating the MAPK / ERK and PI3K / Akt pathways and inhibits osteoclast differentiation by regulating the OPG / RANKL ratio. However, the functional domains of BLF are highly dependent on their intact protein structure. Due to hydrolysis by digestive enzymes and pH-induced degradation in the human digestive environment, oral BLF is destroyed during gastric digestion, severely impairing its functional properties. This, combined with the high cost of obtaining BLF, has limited its application in functional foods. Chlorogenic acid is a polyphenolic compound composed of caffeic acid and quinic acid linked by an ester bond and is widely found in plants such as coffee and honeysuckle. CGA has been shown to promote osteoblast activity, inhibit osteoclast activity, increase bone mineralization, improve bone strength, and improve bone density loss induced by ovariectomy. It also upregulates osteocalcin and deoxypyridinium and stimulates alkaline phosphatase activity in primary osteoblasts. Furthermore, CGA inhibits the expression of osteoclast-specific genes (such as TRAP and CTSK) and reduces bone resorption. However, CGA's ester bond is susceptible to hydrolysis in the gastrointestinal tract, and its oral bioavailability is less than one-third, severely limiting its in vivo efficacy. Summary of the Invention

[0004] To address the problems of poor stability and low bioavailability of chlorogenic acid and bovine lactoferrin in the prior art, the present invention provides a method for preparing a bovine lactoferrin complex. The technical solution is as follows: A method for preparing a bovine lactoferrin complex comprises the following steps: deglycosylation of bovine lactoferrin to obtain deglycosylated bovine lactoferrin; dehydration condensation of the deglycosylated bovine lactoferrin with chitosan to obtain a complex precursor; and then compounding the complex precursor with chlorogenic acid to obtain the bovine lactoferrin complex.

[0005] Furthermore, the molecular weight of the chitosan is 50-100 kDa.

[0006] Furthermore, the mass ratio of the deglycosylated bovine lactoferrin to chitosan is 1:1-3; the mass ratio of the composite precursor to chlorogenic acid is 5-8:1.

[0007] Further, the following steps are included: a. Place deglycosylated bovine lactoferrin in a buffer solution at pH 6-7, add chitosan, mix thoroughly, add carbodiimide, and react at 25-35°C for 16-30 hours to obtain a reaction solution. b. Dissolve N-hydroxysuccinimide in dimethyl sulfoxide to prepare an N-hydroxysuccinimide solution, place chlorogenic acid in the N-hydroxysuccinimide solution to obtain a mixed solution; add the mixed solution to the reaction solution, stir thoroughly, and mix thoroughly; c. Control the pH of the system to 5-6, stir thoroughly, and then react in the dark for 10-20 hours. Purify the reaction solution by dialyzing and freeze-drying to obtain the bovine lactoferrin complex.

[0008] Furthermore, the mass ratio of the deglycosylated bovine lactoferrin to carbodiimide in step a is 1:0.025-0.04.

[0009] Furthermore, the mass ratio of chlorogenic acid to N-hydroxysuccinimide in step b is 0.7-1.5:1; and the sufficient stirring in step b is stirring at room temperature for 15-30 minutes, and then standing at 0-5°C for 0.5-1 hour.

[0010] Furthermore, the dialysis purification in step c is performed at 3-10° C. for 48-72 hours, with water changed every 6-12 hours; the molecular weight cut-off of the dialysis is 8000-14000 Da.

[0011] Furthermore, the deglycosylation of bovine lactoferrin comprises the following steps: placing bovine lactoferrin in a buffer solution and dispersing it uniformly to obtain a protein solution; adding N The enzyme solution of -glycoamidase was placed in a 37°C incubator and incubated for 12 to 20 hours; the deglycosylated bovine lactoferrin in the reaction solution was separated and collected by ultrafiltration.

[0012] Furthermore, the concentration of bovine lactoferrin in the protein solution is 1-10 mg / mL; and the pH of the buffer solution is 7-8.5.

[0013] A bovine lactoferrin complex prepared by the above preparation method has a cell proliferation rate greater than that of bovine lactoferrin at a concentration of 5-100 μg / mL.

[0014] A bovine lactoferrin complex prepared by the above preparation method.

[0015] By adopting the above scheme, the method of the present invention has the following advantages: 1. Compared to bovine lactoferrin and chlorogenic acid alone, the bovine lactoferrin complex produced by the present invention exhibits a significant osteoblast proliferation-promoting effect at minimal concentrations, demonstrating significant synergistic enhancement. This improves both accessibility and stability. Furthermore, the combination of bovine lactoferrin, chitosan, and chlorogenic acid modulates the hydrophilicity of the complex, enhancing its bioavailability.

[0016] 2. The present invention removes the surface of bovine lactoferrin N -chain oligosaccharide structure, weakens the steric hindrance between trypsin and the enzyme action site on the ligand, thereby enhancing the effective contact between bovine lactoferrin protein and protease, and promoting the absorption and conversion of bovine lactoferrin. And chitosan with uniform amino distribution is more convenient than chitosan with complex structure and large volume. N -chain oligosaccharides, after entering the body, are more likely to come into contact with enzymes under the alkaline conditions of the intestines and separate from bovine lactoferrin.

[0017] 3. The preparation method of the present invention first deglycosylates bovine lactoferrin to improve the bioaccessibility of bovine lactoferrin. At the same time, by compounding with chitosan, the problem of poor stability of deglycosylated bovine lactoferrin is solved, and its environmental resistance is improved.

[0018] 4. After the amino groups on the chitosan of the present invention are condensed with the carboxyl groups on bovine lactoferrin, the amino groups on the chitosan can be prevented from affecting the binding of chlorogenic acid during the subsequent esterification of chlorogenic acid with the hydroxyl groups on the composite precursor. After entering the intestine, chlorogenic acid is more easily released, and the entire composite can be rapidly decomposed. By controlling the composite strength of the composite, the problem of multiple composites causing the chlorogenic acid to be excreted from the body before it has time to be absorbed can be avoided.

[0019] 5. The chain chitosan of the present invention can form a protective enclosure for bovine lactoferrin after combining with bovine lactoferrin, thereby preventing excessive reaction of bovine lactoferrin caused by chlorogenic acid, increasing the stability of bovine lactoferrin while ensuring its biological activity. The bovine lactoferrin and chlorogenic acid are controlled in an appropriate spatial connection relationship without affecting their respective activities, and can also connect with each other and act synchronously on osteoblasts, forming a synergistic effect.

[0020] 6. The present invention utilizes the excess carbodiimide in the previous step to react in the subsequent esterification step, omitting the filtration and drying steps, reducing the loss of raw materials and operational complexity. The preparation method and principle are simple and easy to control. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 A comparison chart of osteoblast proliferation rates between various embodiments and comparative examples; Figure 2 The figure is a comparison of the average particle size of each embodiment and the comparative example; Figure 3 Surface hydrophobicity comparison diagram of each embodiment and comparative example. DETAILED DESCRIPTION

[0022] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0023] Example 1: (1) Bovine lactoferrin was placed in a 0.5 mol / L PBS buffer solution with a pH of 7.5 and dispersed evenly to obtain a 5 mg / mL protein solution; N -glycoamidase enzyme solution was placed in a 37°C incubator and incubated for 16 hours; deglycosylated bovine lactoferrin in the reaction solution was separated and collected by ultrafiltration; (2) Deglycosylated bovine lactoferrin was placed in a MES buffer solution with a pH of 6.5, chitosan was added in an amount twice that of the deglycosylated bovine lactoferrin, the mixture was mixed evenly, carbodiimide was added in an amount of 0.03 times that of the deglycosylated bovine lactoferrin, and the mixture was reacted at 30°C for 20 h to obtain a reaction solution; (3) Dissolve N-hydroxysuccinimide in dimethyl sulfoxide to prepare an N-hydroxysuccinimide solution; take chlorogenic acid 0.3 times the mass of deglycosylated bovine lactoferrin, and place the chlorogenic acid in the N-hydroxysuccinimide solution at a mass ratio of 1:1 to obtain a mixed solution; add the mixed solution to the reaction solution of step (2), stir at room temperature for 30 minutes, and then stand in an ice bath for 0.5 hours to mix evenly; (4) The pH of the system was adjusted to 5.5 with hydrochloric acid, and the mixture was stirred thoroughly. The mixture was then allowed to react in the dark for 15 h. The reaction solution was dialyzed continuously at 4 °C for 48 h using a dialysis bag with a molecular weight cutoff of 8000-14000 Da, and the water was replaced every 6 h. The obtained sample was freeze-dried to obtain a bovine lactoferrin complex.

[0024] Example 2: The difference from Example 1 is that: (1) The deglycosylated bovine lactoferrin is placed in a MES buffer solution with pH of 6.5, 1 times of the mass of the deglycosylated bovine lactoferrin of chitosan is added, mixed uniformly, 0.03 times of the mass of the deglycosylated bovine lactoferrin of carbodiimide is added, and the reaction is carried out at 30°C for 20h to obtain a reaction liquid.

[0025] Example 3: The difference from Example 1 is that: (1) The deglycosylated bovine lactoferrin is placed in a MES buffer solution with pH of 6.5, 3 times of the mass of the deglycosylated bovine lactoferrin of chitosan is added, mixed uniformly, 0.03 times of the mass of the deglycosylated bovine lactoferrin of carbodiimide is added, and the reaction is carried out at 30°C for 20h to obtain a reaction liquid.

[0026] Example 4: The difference from Example 1 is that: (3) N-hydroxysuccinimide is dissolved in dimethyl sulfoxide to prepare an N-hydroxysuccinimide solution; 0.4 times of the mass of the deglycosylated bovine lactoferrin of chlorogenic acid is placed in the N-hydroxysuccinimide solution at a mass ratio of 1:1 to obtain a mixed solution; the mixed solution is added to the reaction liquid of step (2), stirred at room temperature for 30min, and then placed in an ice bath for 0.5h, and mixed uniformly.

[0027] Example 5: The difference from Example 1 is that: (3) N-hydroxysuccinimide is dissolved in dimethyl sulfoxide to prepare an N-hydroxysuccinimide solution; 0.25 times of the mass of the deglycosylated bovine lactoferrin of chlorogenic acid is placed in the N-hydroxysuccinimide solution at a mass ratio of 1:1 to obtain a mixed solution; the mixed solution is added to the reaction liquid of step (2), stirred at room temperature for 30min, and then placed in an ice bath for 0.5h, and mixed uniformly.

[0028] Comparative Example 1: (1) The bovine lactoferrin is placed in a PBS buffer solution with pH of 7.5 and a concentration of 0.5 mol / L, and is uniformly dispersed to obtain a protein solution with a concentration of 5mg / mL; the enzyme solution of glycosamidase is added to the protein solution, and the mixture is incubated in a 37°C incubator for 16h; the deglycosylated bovine lactoferrin in the reaction liquid is separated by ultrafiltration and collected; N (2) The deglycosylated bovine lactoferrin is placed in a MES buffer solution with pH of 6.5, 2 times of the mass of the deglycosylated bovine lactoferrin of chitosan is added, mixed uniformly, 0.03 times of the mass of the deglycosylated bovine lactoferrin of carbodiimide is added, and the reaction is carried out at 30°C for 20h to obtain a reaction liquid; the product is collected after dialysis in a dialysis bag, and is freeze-dried to obtain the product.

[0029] ​Comparative Example 2: (1) N-hydroxysuccinimide was dissolved in dimethyl sulfoxide to prepare an N-hydroxysuccinimide solution; chlorogenic acid was placed in the N-hydroxysuccinimide solution at a mass ratio of 1:1 to obtain a mixed solution; 0.01 times the mass of chlorogenic acid was added to the reaction solution of step (2), and the mixture was stirred at room temperature for 30 minutes, and then allowed to stand in an ice bath for 0.5 hours to mix evenly; (2) Adjust the pH of the system to 5.5 with hydrochloric acid, stir and mix thoroughly, and then react in the dark for 15 hours; use a dialysis bag with a molecular weight cutoff of 8000~14000 Da to continuously dialyze the reaction solution at 4°C for 48 hours, and replace the water every 6 hours; the obtained sample is freeze-dried.

[0030] Comparative Example 3: (2) Deglycosylated bovine lactoferrin was placed in a MES buffer solution with a pH of 6.5, 0.3 times the amount of chlorogenic acid as that of deglycosylated bovine lactoferrin was added, the mixture was mixed evenly, 0.02 times the amount of carbodiimide as that of deglycosylated bovine lactoferrin was added, and the mixture was reacted at 30°C for 20 hours to obtain a reaction solution; the product was collected after dialyzing with a dialysis bag and freeze-dried.

[0031] The cells were suspended at 5×10 3 Cells were seeded at a density of 100 cells / well in a 96-well plate. After 12 hours of adherent culture, the original culture medium was replaced with fresh culture medium containing 20 μg / mL of the complex of each example and comparative example. After 24 hours of culture, 5 mg / mL of MTT solution was added to each well and incubated at 37°C in the dark for 4 hours. Dimethyl sulfoxide was added to dissolve the formazan crystals, and the absorbance was measured at 490 nm. The results are shown in Figure 2. Figure 1 shown.

[0032] Depend on Figure 1 As can be seen, Example 1 has the best osteoblast proliferation ability. In Examples 2 and 3, where chitosan is reduced or increased, excessive chitosan content may increase the burden of complex decomposition. However, too little chitosan content can also affect the protective effect of bovine lactoferrin, failing to effectively control the relationship between bovine lactoferrin and chlorogenic acid, which is both separated and connected. Meanwhile, in Examples 4 and 5, where chlorogenic acid is increased or decreased, the osteoblast proliferation ability is reduced. This decrease in osteoblastic effect caused by changes in chlorogenic acid content also demonstrates the synergistic relationship between chlorogenic acid and bovine lactoferrin. Combined with the lower cell proliferation rate of Comparative Example 3, which does not contain chitosan, this indicates that the bovine lactoferrin, chlorogenic acid, and chitosan of the present invention need to be controlled in an appropriate ratio to achieve the best osteoblastic effect.

[0033] The particle size of the sample solution (2 mg / mL) was measured using a Zetasizer Nano ZS. Figure 2As shown in the figure, the presence of chitosan promotes the combination of chitosan and chlorogenic acid, but the particle size of each example does not increase exponentially, which indicates that the method of the present application for preparing bovine lactoferricin complex does not affect the dispersibility of the key active ingredient, which is beneficial to the dispersion of the complex in the cell culture environment and the contact with osteoblasts, thereby enhancing its osteogenic activity.

[0034] Surface hydrophobicity was determined using 8-anilino-1-naphthalene sulfonic acid (ANS) as a probe. Protein samples were diluted with PBS (0.01 M) to a concentration of 0.01-0.05 mg / mL, and 3 mL of the protein sample was mixed with 20 μL of 8 mM ANS solution under light-avoiding conditions for 1 min. The fluorescence intensity was measured using a fluorescence spectrophotometer, with λex set to 380 nm, λem set to 510 nm, and the slit set to 5 nm. The surface hydrophobicity was determined from the slope of the linear regression graph.

[0035] The results, as shown in Figure 3 Chlorogenic acid with phenolic hydroxyl structure binds to bovine lactoferricin, and the introduction of additional hydroxyl groups increases the hydrophilicity of the protein. Compared to Comparative Example 1 without the participation of chlorogenic acid, the hydrophobicity of the remaining examples and comparative examples is significantly smaller, and at the same time, Figure 1 In the cell culture environment, the proliferation rate of osteoblasts in Comparative Example 1 is also the smallest, indicating that the regulation of the surface hydrophobicity of the complex can also significantly affect the stability and bioavailability of the complex in the cell environment, and affect its osteogenic activity.

[0036] For those skilled in the art, based on the above described technical solutions and concepts, various corresponding changes and modifications can be made, and all of these changes and modifications should be within the scope of protection of the claims of the present application.

Claims

1. A method for preparing a bovine lactoferrin complex, characterized in that: The following steps are involved: The bovine lactoferrin is deglycosylated to obtain deglycosylated bovine lactoferrin; the deglycosylated bovine lactoferrin is dehydrated and condensed with chitosan to obtain a composite precursor; and the composite precursor is then compounded with chlorogenic acid to obtain a bovine lactoferrin complex.

2. The method for preparing the bovine lactoferrin complex according to claim 1, wherein The molecular weight of the chitosan is 50-100 kDa.

3. The method for preparing the bovine lactoferrin complex according to claim 1, wherein The mass ratio of the deglycosylated bovine lactoferrin to chitosan is 1:1-3; the mass ratio of the composite precursor to chlorogenic acid is 5-8:

1.

4. The method for preparing the bovine lactoferrin complex according to any one of claims 1 to 3, characterized in that: The following steps are involved: a. Place deglycosylated bovine lactoferrin in a buffer solution at pH 6-7, add chitosan, mix thoroughly, add carbodiimide, and react at 25-35°C for 16-30 hours to obtain a reaction solution. b. Dissolve N-hydroxysuccinimide in dimethyl sulfoxide to prepare an N-hydroxysuccinimide solution, place chlorogenic acid in the N-hydroxysuccinimide solution to obtain a mixed solution; add the mixed solution to the reaction solution, stir thoroughly, and mix thoroughly; c. Control the pH of the system to 5-6, stir thoroughly, and then react in the dark for 10-20 hours. Purify the reaction solution by dialyzing and freeze-drying to obtain the bovine lactoferrin complex.

5. The method for preparing the bovine lactoferrin complex according to claim 4, wherein The mass ratio of the deglycosylated bovine lactoferrin to carbodiimide in step a is 1:0.025-0.

04.

6. The method for preparing the bovine lactoferrin complex according to claim 4, characterized in that: The mass ratio of chlorogenic acid to N-hydroxysuccinimide in step b is 0.7-1.5:1; the sufficient stirring in step b is stirring at room temperature for 15-30 minutes, and then standing at 0-5°C for 0.5-1 hour.

7. The method for preparing the bovine lactoferrin complex according to claim 4, characterized in that: The dialysis purification in step c is performed at 3-10° C. for 48-72 hours, with water changed every 6-12 hours; the molecular weight cut-off for dialysis is 8000-14000 Da.

8. The method for preparing the bovine lactoferrin complex according to claim 1, wherein The deglycosylation of bovine lactoferrin comprises the following steps: placing bovine lactoferrin in a buffer solution and dispersing it uniformly to obtain a protein solution; adding N The enzyme solution of -glycoamidase was placed in a 37°C incubator and incubated for 12 to 20 hours; the deglycosylated bovine lactoferrin in the reaction solution was separated and collected by ultrafiltration.

9. The method for preparing the bovine lactoferrin complex according to claim 8, characterized in that: The concentration of bovine lactoferrin in the protein solution is 1-10 mg / mL; and the pH of the buffer solution is 7-8.

5.

10. A bovine lactoferrin complex prepared by the preparation method according to any one of claims 1 to 3, characterized in that: At concentrations of 5-100µg / mL, the cell proliferation rate was greater than that of bovine lactoferrin.

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