Eggshell membrane nonapeptide and application thereof in prevention and treatment of osteoporosis

Abstract

The invention discloses an eggshell membrane nonapeptide and application thereof in prevention and treatment of osteoporosis, and belongs to the technical field of biology. The eggshell membrane nonapeptide is characterized in that the amino acid sequence of the eggshell membrane nonapeptide is shown as SEQ ID NO.1, and the amino acid sequence of the eggshell membrane nonapeptide is specifically Val-Pro-Pro-Gly-Asp-Tyr-Leu-Phe-Gln. The eggshell membrane nonapeptide provided by the invention can repair and improve the oxidation resistance, differentiation activity and bone formation ability of osteoblasts influenced by oxidative stress, and provides a new treatment means for preventing and treating osteoporosis caused by oxidative stress.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to an eggshell membrane nonapeptide and application thereof in preventing and treating osteoporosis. Background Art

[0002] Increased bone fragility caused by osteoporosis has become the main cause of fractures in the elderly, with high disability and mortality rates, and has imposed a heavy burden on society. It is generally believed that hormone imbalance, calcium and vitamin D deficiency, and normal aging are the main causes of osteoporosis. However, in recent years, oxidative stress, as another important factor, has also begun to be confirmed in the literature. Recent studies have shown that oxidative stress may be the main factor leading to the functional separation of osteoblasts and osteoclasts in osteoporosis. Therefore, regulating oxidative stress has become a key approach to treat osteoporosis. Although many drugs that prevent osteoclastic bone resorption or promote bone formation have been developed, their therapeutic effects are relatively poor and have certain limitations and side effects. For example, long-term use of anti-bone resorption drugs can lead to interrupted coupling of bone resorption and formation, thereby increasing bone fragility and osteonecrosis. Therefore, it is of great significance to develop a new treatment method that can reverse the imbalance of bone homeostasis while promoting osteogenesis and reducing osteoclasts.

[0003] As a byproduct of egg processing, eggshell membrane contains a variety of proteins such as collagen and keratin, as well as bioactive substances such as hyaluronic acid and chondroitin sulfate, and is a potential source of active peptides. However, eggshell membrane is usually used as feed or landfill, which is a serious waste of resources. Eggshell membrane has been proven to have a certain antioxidant effect, so the development of active peptides in eggshell membrane for the prevention and treatment of osteoporosis is of great significance. Summary of the invention

[0004] The purpose of the present invention is to provide an eggshell membrane nonapeptide and its application in preventing and treating osteoporosis to solve the problems existing in the above-mentioned prior art. The eggshell membrane nonapeptide of the present invention can repair and improve the antioxidant capacity, differentiation activity and bone formation ability of osteoblasts affected by oxidative stress, and provides a new treatment method for preventing and treating osteoporosis caused by oxidative stress.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides an eggshell membrane nonapeptide, wherein the amino acid sequence of the eggshell membrane nonapeptide is shown in SEQ ID NO.1, specifically: Val-Pro-Pro-Gly-Asp-Tyr-Leu-Phe-Gln.

[0007] The eggshell membrane nonapeptide of the present invention can be synthesized by solid phase synthesis, liquid phase synthesis, biosynthesis or enzymatic synthesis; in some embodiments of the present invention, the eggshell membrane nonapeptide is synthesized by solid phase synthesis.

[0008] The present invention also provides the use of the eggshell membrane nonapeptide in preparing a medicine for preventing and treating osteoporosis.

[0009] Preferably, the osteoporosis is osteoporosis caused by oxidative stress.

[0010] The present invention also provides the use of the eggshell membrane nonapeptide in preparing a medicine for improving the antioxidant capacity of osteoblasts.

[0011] The present invention also provides the use of the eggshell membrane nonapeptide in preparing a drug for promoting osteoblast differentiation.

[0012] The present invention also provides the use of the eggshell membrane nonapeptide in preparing a drug for promoting osteoblast mineralization.

[0013] The present invention also provides the use of the eggshell membrane nonapeptide in preparing a medicine for promoting new bone formation.

[0014] The present invention also provides a medicine for preventing and treating osteoporosis, wherein the active ingredient of the medicine is the eggshell membrane nonapeptide according to claim 1.

[0015] Preferably, the drug also includes a pharmaceutically acceptable carrier.

[0016] The present invention discloses the following technical effects:

[0017] The eggshell membrane nonapeptide Val-Pro-Pro-Gly-Asp-Tyr-Leu-Phe-Gln of the present invention is derived from eggshell membrane, and the raw material source is wide, highly safe, and easy to obtain. The present invention verifies through in vitro antioxidant experiments and in vitro oxidative stress osteogenesis model experiments that the eggshell membrane nonapeptide has extremely high antioxidant effect, has a repairing effect on osteoblasts affected by oxidative stress, can improve the antioxidant capacity, differentiation activity and bone formation ability of osteoblasts, and shows significant bone repair ability, providing a new technical means for treating osteoporosis caused by oxidative stress, and showing excellent application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1It is the secondary mass spectrum of the peptide segment Val-Pro-Pro-Gly-Asp-Tyr-Leu-Phe-Gln;

[0020] Figure 2 It is the ABTS and DPPH free radical scavenging rate of the peptide segment Val-Pro-Pro-Gly-Asp-Tyr-Leu-Phe-Gln;

[0021] Figure 3 is the activity of superoxide dismutase (T-SOD) of osteoblasts under different treatments;

[0022] Figure 4 is the activity of alkaline phosphatase (AKP) of osteoblasts under different treatments;

[0023] Figure 5 Fluorescence microscopy images of osteoblast mineralization staining under different treatments;

[0024] Figure 6 SEM images of osteoblasts after mineralization under different treatments. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] The technical solution of the present invention is as follows:

[0031] Eggshell membrane was used as raw material, and eggshell membrane hydrolysate was prepared by enzymatic hydrolysis technology. The polypeptide components with molecular weight less than 3K Da in the eggshell membrane hydrolysate were separated by ultrafiltration and other technologies. Then, a highly active nonapeptide Val-Pro-Pro-Gly-Asp-Tyr-Leu-Phe-Gln was identified by HPLC-MS / MS identification, bioinformatics analysis and AutoDock Vina molecular simulation docking. The nonapeptide was subjected to in vitro antioxidant experiments and in vitro oxidative stress osteogenesis model experiments to detect its efficacy on osteoblasts affected by oxidative stress.

[0032] The preparation process of the eggshell membrane hydrolysate is as follows:

[0033] 1. Take dried and pure eggshell membranes, add purified water according to the material-liquid ratio of 100 g: (600-1000) mL, add 40 mmol / L of one or more of arginine, proline, serine, and cysteine ​​and mix with the eggshell membranes to obtain an eggshell membrane suspension.

[0034] 2. Transfer the eggshell membrane suspension to an enzymolysis tank, and add 3000U / g keratinase and 4000U / g collagenase to the eggshell membrane substrate, and perform a segmented or synchronous composite enzymolysis reaction. The reaction temperature of keratinase is 30-60℃, pH 6-10, and the reaction time is 1-5h; the reaction temperature of collagenase is 30-60℃, pH 6-10, and the reaction time is 1-4h. After the reaction is completed, the temperature is raised to 80-90℃ for 10-30min to inactivate the enzymes and obtain the eggshell membrane hydrolyzate.

[0035] 3. Centrifuge the eggshell membrane hydrolyzate and dry it to obtain a powdered eggshell membrane hydrolyzate.

[0036] Example 1 Preparation of eggshell membrane nonapeptide

[0037] 1. Take dried and pure eggshell membranes, add purified water according to the solid-liquid ratio of 100 g: 800 mL, add 40 mmol / L cysteine ​​and mix with the eggshell membranes to obtain an eggshell membrane suspension.

[0038] 2. Transfer the eggshell membrane suspension to an enzymolysis tank, and add 3000U / g keratinase and 4000U / g collagenase of eggshell membrane substrate to it in turn, and perform a segmented composite enzymolysis reaction. The reaction temperature of the keratinase enzymolysis process is 50°C, pH 7.0, and the reaction time is 4h; the reaction temperature of the collagenase enzymolysis process is 50°C, pH 7.0, and the reaction time is 3h. After the reaction is completed, the temperature is raised to 90°C for 30min to inactivate the enzymes and obtain the eggshell membrane hydrolyzate.

[0039] 3. Centrifuge the eggshell membrane hydrolysate, dry it, and extract the powdered eggshell membrane polypeptide.

[0040] 4. Screening and identification of target peptides: After the shell membrane polypeptide was dissolved in sterile water, it was ultrafiltered and dried to separate the polypeptide components with a molecular weight of less than 3K Da. After preparing the polypeptide solution, HPLC-MS / MS identification and bioinformatics analysis were performed, i.e., the peptide property calculator online tool was used to predict the function of the polypeptide, and then AutoDockVina was used for molecular simulation docking to identify a highly active nonapeptide Val-Pro-Pro-Gly-Asp-Tyr-Leu-Phe-Gln (SEQ ID NO.1). The secondary mass spectrum of the nonapeptide is shown in the following figure. Figure 1 shown.

[0041] Example 2 Functional verification of eggshell membrane nonapeptide

[0042] The nonapeptide Val-Pro-Pro-Gly-Asp-Tyr-Leu-Phe-Gln selected in Example 1 was synthesized by solid phase synthesis, and the function of the nonapeptide was verified. The process is as follows:

[0043] 1. In vitro antioxidant experiment: The nonapeptide was made into a peptide solution with a concentration of 1 mg / mL, and the free radical scavenging rate was measured in ABTS and DPPH reagents. Figure 2 As shown, the ABTS free radical scavenging rate of the peptide segment can reach more than 80%, and the DPPH free radical scavenging rate can reach more than 85%.

[0044] 2. Intracellular activity evaluation experiment: First, 400mmol / L H 2 O 2Mouse embryonic osteoblasts MC3T3-E1 (purchased from ATCC) were treated for 24 hours to obtain an oxidative stress osteoblast model. Subsequently, the oxidative stress osteoblasts were treated with complete medium containing 100 μg / mL of the above peptide solution as the nonapeptide group (VPPGDYLFQ group), and the complete medium without the above peptide solution was used as the model group (Model group). Normal MC3T3-E1 cells were used as controls (Control group), and the superoxide dismutase T-SOD activity and alkaline phosphatase AKP activity of each group of cells were measured.

[0045] The results of T-SOD activity test are as follows Figure 3 As shown, H 2 O 2 The treated osteoblasts showed a significant decrease in T-SOD activity compared to normal cells, indicating that the oxidative stress osteoblast model was successfully constructed. Compared with the model group, the nonapeptide group significantly improved the decrease in T-SOD activity caused by oxidative stress, indicating that the nonapeptide has a certain antioxidant repair ability in cells.

[0046] AKP activity test results Figure 4 As shown, H 2 O 2 The treated osteoblasts showed a significant decrease in AKP activity compared with normal cells. Compared with the model group and the control group, the nonapeptide group significantly increased AKP activity, indicating that it can alleviate the inhibitory effect of oxidative stress on the differentiation function of osteoblasts.

[0047] 3. Mineralized nodule staining image: Osteoblasts are the main functional cells of bone formation and are responsible for new bone formation. The extracellular matrix secreted by osteoblasts can produce mineralization. Mineralized nodules are a sign of osteoblast differentiation and maturation, and are also the main morphological characteristics of osteoblasts in performing their osteogenic function. Observing the mineralized nodules of osteoblasts is one of the common methods for studying osteoblast differentiation. Calcium salts produced by osteoblasts cultured in vitro combine with alizarin red to form a stable heme complex, which appears red.

[0048] The cells in the model group and the nonapeptide group cultured for 21 days were washed three times with PBS, and fixed with 4% paraformaldehyde for 1 hour; the fixative was discarded, and 1 mL of 0.1% Alizarin Red staining working solution was added to each well of the 6-well plate after washing with PBS and stained for 15 minutes; the staining solution was discarded, and the stained cells were rinsed with deionized water on a shaker for 5 times, each time for 3 minutes; dried in a 37°C incubator overnight; and the stained cells were scanned and imaged.

[0049] The results are as follows Figure 5As shown, the osteoblasts in the nonapeptide group produced obvious mineralized nodules, and the osteoblasts had begun to play a role in new bone synthesis, while the model group had almost no formation of mineralized nodules, which showed that the new bone synthesis of osteoblasts was inhibited by oxidative stress, and the nonapeptide of the present invention can improve the differentiation ability of osteoblasts by regulating oxidative stress, indicating that to a certain extent, the nonapeptide can alleviate the differentiation function damage of oxidative stress-type osteoblasts and promote the formation of their mineralized nodules.

[0050] 4. Scanning electron microscopy: The osteoblasts that produced mineralized nodules in the model group and the nonapeptide group were washed three times with ultrapure water, freeze-dried, spray-coated with gold, and placed in a scanning electron microscope chamber for vacuum pumping. The acceleration voltage was set to 15 kV, and the structure of the osteoblast mineralized nodules under different treatments was observed under a JSM.6390 / LV scanning electron microscope. The results are as follows Figure 6 As shown, the osteoblast mineralization nodules in the model group were smaller, while after culture with the nonapeptide, the cell mineralization nodules enlarged and showed an accumulation state, indicating that it can effectively promote the biomineralization of osteoblasts.

[0051] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An eggshell membrane nonapeptide, characterized in that The amino acid sequence of the eggshell membrane nonapeptide is shown in SEQ ID NO.

1.

2. Use of the eggshell membrane nonapeptide according to claim 1 in the preparation of a medicament for preventing and treating osteoporosis.

3. The use according to claim 2, characterized in that: The osteoporosis is osteoporosis caused by oxidative stress.

4. Use of the eggshell membrane nonapeptide according to claim 1 in the preparation of a medicament for improving the antioxidant capacity of osteoblasts.

5. Use of the eggshell membrane nonapeptide according to claim 1 in the preparation of a drug for promoting osteoblast differentiation.

6. Use of the eggshell membrane nonapeptide according to claim 1 in the preparation of a drug for promoting osteoblast mineralization.

7. Use of the eggshell membrane nonapeptide according to claim 1 in the preparation of a drug for promoting new bone formation.

8. A drug for preventing and treating osteoporosis, characterized in that: The active ingredient of the medicine is the eggshell membrane nonapeptide according to claim 1.

9. The drug according to claim 8, characterized in that The medicine also includes a pharmaceutically acceptable carrier.

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