Oyster juice source active peptide with osteogenesis promoting effect and application of oyster juice source active peptide

Abstract

The invention discloses an oyster juice source active peptide with an osteogenesis promoting effect and application, and relates to the technical field of biological medicine and functional food. A plurality of active small molecule peptides are separated and identified from the oyster juice for the first time through the following operations: opening a shell of a fresh oyster, sucking juice with an injector, centrifugally removing impurities, freeze-drying into powder, desalting, performing LC-MS / MS polypeptide identification and performing website predictive activity analysis, and functional verification finds that the peptides have the effects of remarkably promoting osteogenic differentiation and mineralization, and can be used for preparing the active small molecule peptides for the first time. The effects of improving bone mass and the like are shown in an osteoporosis animal model. Particularly, the amino acid sequence of the peptide SDK3 is SDKPDVKEVESFDKSKLK, and the peptide SDK3 has the best performance. A new research thought and application approach are provided for high-value utilization of oyster juice, prevention and treatment of bone metabolic diseases and the like.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and functional food technology, specifically involving active peptides obtained from oyster juice and their application in bone metabolism regulation, especially for the preparation of drugs, health products or foods for preventing and treating osteoporosis and promoting osteogenic differentiation and mineralization. Background Technology

[0002] Osteoporosis is a common metabolic bone disease characterized by decreased bone mass, deterioration of trabecular bone structure, and increased bone fragility, severely impacting patients' quality of life. Currently used clinical drug treatments, such as bisphosphonates, calcitonin, and hormone replacement therapy, while able to improve bone loss to some extent, suffer from significant side effects and poor patient compliance. Therefore, developing novel osteoproliferative substances that are naturally derived and highly safe is of great importance.

[0003] Oysters (Crassostrea gigas) are an important marine economic shellfish worldwide, rich in protein, polyunsaturated fatty acids, taurine, and other bioactive components. Previous studies have reported that various functional oyster peptides can be obtained through enzymatic hydrolysis of oyster meat tissue, exhibiting antioxidant, immunomodulatory, and anti-fatigue effects. However, these reports are all based on the edible muscle portion of the oyster, with limited research on components derived from oyster body fluids or juices.

[0004] Oyster fluid (also known as oyster shell fluid) is the liquid that flows out of the mantle cavity when an oyster opens its shell. It contains various proteins, small peptides, minerals, and immune-active substances. Existing literature reports that oyster internal fluid is mainly related to oyster immune defense and environmental stress response, but there are no reports on the application of peptides derived from oyster fluid in bone metabolism regulation or osteoporosis prevention. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides an oyster-derived bioactive peptide composition and its uses. The applicant has, for the first time, isolated and identified multiple bioactive small molecule peptides from oyster juice, and through functional verification, discovered that these peptides significantly promote osteogenic differentiation and mineralization, and exhibit effects such as improving bone mass in osteoporosis animal models. In particular, peptide SDK3 showed the best performance. This provides new research ideas and application pathways for the high-value utilization of oyster juice and the prevention and treatment of bone metabolic diseases.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] One objective of this invention is to provide an active peptide derived from oyster juice, wherein the amino acid sequence of the active peptide is selected from any one of SEQ ID NO.3-SEQ ID NO.5, namely any one of SDKPDVKEVESFDKSKLKK (SDK1), SDKPDVKEVESFDKSKL (SDK2), and SDKPDVKEVESFDKSKLK (SDK3).

[0008] Preferably, the amino acid sequence of the active peptide is SEQ ID NO.5, namely SDKPDVKEVESFDKSKLK (SDK3). SDKPDVKEVESFDKSKLK is an abbreviation, and the amino acids specifically represent Ser-Asp-Lys-Pro-Asp-Val-Lys-Glu-Val-Glu-Ser-Phe-Asp-Lys-Ser-Lys-Leu-Lys.

[0009] The second objective of this invention is to provide a method for preparing the above-mentioned active peptides, the method comprising the following steps: opening fresh oysters, drawing juice with a syringe, centrifuging to remove impurities, freeze-drying into powder, desalting, identifying the peptide by LC-MS / MS, and performing predictive activity analysis.

[0010] A third objective of this invention is to provide the application of the above-described active peptides in the preparation of products for the prevention and treatment of bone metabolic diseases and / or the improvement of bone health.

[0011] Preferably, the prevention and treatment of bone metabolic diseases and / or improvement of bone health are achieved through any one or more of the following pathways: promoting the expression of osteogenic transcription factors, promoting osteoblast differentiation, promoting osteoblast mineralization, improving bone metabolic indicators, and improving bone microstructure.

[0012] The bone metabolism indicators include osteocalcin (OPG) content, tartrate-resistant acid phosphatase 5b (TRAcp 5b) content, etc., and the bone microstructure includes bone density, trabecular structure (including the number and thickness of trabeculae), bone volume, etc.

[0013] Preferably, the bone metabolic diseases include osteoporosis, osteopenia, and fracture bone defect repair.

[0014] Preferably, the product form includes pharmaceuticals, functional foods, and / or health products.

[0015] Preferably, the product is administered via injection or oral administration.

[0016] The fourth objective of this invention is to provide a pharmaceutical composition comprising any of the above-described active peptides and a pharmaceutically acceptable carrier.

[0017] The fifth objective of this invention is to provide a functional food and / or health product, wherein the functional food and / or health product contains any of the above-mentioned active peptides and food-grade excipients.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. This invention is the first to report that multiple small molecule peptides (SDK1-SDK3) derived from oyster juice have osteogenic activity.

[0020] 2. Among them, SDK3 showed the best osteogenic effect, with a novel structural origin, and is a potential new natural osteogenic promoting factor.

[0021] 3. SDK3 significantly promotes osteoblast differentiation and mineralization, and enhances the expression of osteoblast-related transcription factors, with a stronger effect than other screening peptides.

[0022] 4. SDK3 has shown a significant effect in improving osteoporosis in animal models and has potential clinical application value.

[0023] 5. The active peptides in this invention are naturally derived, have high safety, and have application prospects in drug development and functional food development. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the process for collecting oyster juice and screening active peptides in Example 1 of the present invention.

[0025] Figure 2 This is a diagram showing the distribution of peptide chain length and quantity in oyster juice and the distribution of its active peptides (PeptideRanker score > 0.5) in Example 1 of the present invention.

[0026] Figure 3 This illustrates the effect of candidate peptides in Example 2 of the present invention on the expression of osteogenic-related transcription factors. A: Runx-2 expression; B: Osx expression; C: OPN expression.

[0027] Figure 4 This illustrates the promoting effects of SDK1, SDK2, and SDK3 on osteoblast differentiation and mineralization in Example 3 of the present invention. A: ALP staining results of osteoblasts after 4 days of treatment with SDK1, SDK2, and SDK3; B: Alizarin Red S staining results after 14 days of treatment with SDK1, SDK2, and SDK3.

[0028] Figure 5 This illustrates the protective effect of SDK3 on osteoporotic mice in Example 4 of the present invention. A: Micro-CT bone scan and bone morphometric analysis; B: Serum osteocalcin (OPG) content; C: Serum tartrate-resistant acid phosphatase 5b (TRAcp 5b) content. Detailed Implementation

[0029] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventional methods.

[0030] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0031] Example 1: Collection of oyster juice and identification of peptide composition

[0032] This embodiment provides a method for obtaining oyster juice from oysters and identifying its peptide composition. The specific steps are as follows:

[0033] Take fresh oysters, gently open the shells, and collect the liquid inside the shells using a sterile syringe. Place the collected liquid under low temperature conditions (4℃), and centrifuge at low speed (1000 rpm, 5 min) to remove cells and impurities. Obtain the supernatant as the oyster juice sample, and freeze-dry it into powder.

[0034] Desalting and Mass Spectrometry Identification of Peptide Sequences: Add an appropriate amount of 0.1% trifluoroacetic acid (TFA) to the sample, mix well, centrifuge at 20000g for 5 min, collect the supernatant, transfer to a 10 KD ultrafiltration centrifuge tube, and centrifuge at 12000g for 15 min. Add 200µL of 0.1% TFA, centrifuge at 12000g for 15 min, repeat twice, collect the filtrate, desalt using a C18 StageTip, and vacuum dry. After drying, reconstitute the peptides with 0.1% formic acid (FA), determine the peptide concentration, and then use high-resolution mass spectrometry (LC-MS / MS) to identify the peptide composition of the sample, obtaining peptide sequence and molecular weight information.

[0035] like Figure 2 As shown, oyster juice contains a large number of natural peptides. After desalting, the peptide lengths ranged from pentapeptides to 21 peptides, and the molecular weights ranged from 0.5 to 2.5 kDa. All peptides identified by mass spectrometry were input into the PeptideRanker website for activity scoring. It was found that some peptides showed potential biological activity in the PeptideRanker activity prediction tool, providing a basis for further screening of osteogenic active peptides.

[0036] Example 2: Screening and synthesis of osteogenic active peptides from oyster juice

[0037] The top 6 candidate peptides obtained from the PeptideRanker activity score in Example 1 are: LASGFSLEEPGTHASRIH (LAS, SEQ ID NO.1), MLYGPVAM (MLY, SEQ ID NO.2), SDKPDVKEVESFDKSKLKK (SDK1, SEQ ID NO.3), SDKPDVKEVESFDKSKL (SDK2, SEQ ID NO.4), SDKPDVKEVESFDKSKLK (SDK3, SEQ ID NO.5), and SDPAIGIAR (SDP, SEQ ID NO.6).

[0038] The above six peptides were prepared using a solid-phase peptide synthesis method, with a purity ≥95%, and were verified by HPLC and mass spectrometry.

[0039] The synthetic peptide was dissolved in a small amount of sterile water to prepare a peptide working solution, which was then added to the osteoblast culture system to achieve a concentration of 25 μmol / L. The osteoblast culture system consisted of 89% α-MEM basal medium, 10% fetal bovine serum, and 1% penicillin-dextrose antibody. Osteoblast MC3T3-E1 cells were treated with the 25 μmol / L peptide solution for 3 consecutive days. Cells were then collected for qPCR analysis to detect the effects of the candidate peptide on the expression levels of osteogenic transcription factors Runx2, Osterix (Osx), and OPN (reverse transcription kit: Genstar, A230; qPCR kit: Genstar, A304).

[0040] like Figure 3 As shown, compared with the control group, the SDK1, SDK2 and SDK3 intervention groups all significantly increased the expression of osteogenic transcription factors, with SDK3 showing the best effect and demonstrating excellent potential to promote the expression of osteogenic transcription factors.

[0041] Example 3: Effects of SDK1, SDK2, and SDK3 on osteoblast differentiation and mineralization

[0042] The mouse embryonic osteoblast precursor cell line (MC3T3-E1) was used. MC3T3-E1 cells were cultured at a rate of 5 × 10⁻⁶. 4Osteoblasts were seeded at a density of [number] cells / mL into 12-well plates and cultured to the logarithmic growth phase using osteogenic induction differentiation medium (ZQXZBIO, CSP080). Then, a 25 μmol / L peptide solution (SDK1, SDK2, SDK3) was added for intervention, while the control group received an equal volume of sterile water. The osteogenic induction differentiation medium was prepared from 85.9% α-MEM basal medium, 10% fetal bovine serum, 1% penicillin-antibiotic combination, 0.1% dexamethasone, 1% ascorbic acid, 1% sodium β-glycerophosphate, and 1% L-propionamide-glutamine solution.

[0043] like Figure 4 As shown in Figure A, osteoblasts were cultured and treated with SDK1, SDK2, and SDK3 consecutively. On day 4, ALP staining was performed using an alkaline phosphatase (ALP) staining kit (Beytime, C3206). The results showed that the ALP staining intensity in the SDK1, SDK2, and SDK3 intervention groups was significantly higher than that in the control group, indicating that SDK1, SDK2, and SDK3 have the ability to promote osteoblast differentiation, and SDK3 has the best osteogenic differentiation-promoting effect.

[0044] like Figure 4 As shown in Figure B, osteoblasts were cultured and treated with SDK1, SDK2, and SDK3 consecutively. On day 14, the cells were stained with Alizarin Red S mineralization nodules using an osteoblast mineralization nodule staining kit (Beytime, C0148S). The results showed that compared with the control group, the number and staining intensity of mineralization nodules formed in the SDK1, SDK2, and SDK3 intervention groups were significantly increased, indicating that SDK1, SDK2, and SDK3 have a significant ability to promote osteoblast mineralization, and SDK3 has the best bone mineralization-promoting effect.

[0045] The above results indicate that SDK1, SDK2, and SDK3 can significantly promote osteoblast differentiation and enhance the formation of mineralized nodules, with SDK3 exhibiting the best osteogenic activity.

[0046] Example 4: Effects of SDK3 on osteoporotic mice

[0047] Twelve-week-old female mice (C57BL / 6J) purchased from Vital Rivers (Beijing, China) were randomly divided into a sham-operated control group, an osteoporosis model group, and an SDK3 treatment group (n=6 per group). The osteoporosis model was established through bilateral ovariectomy (OVX), while the control group underwent only some adipose tissue removal around the ovaries. After model establishment, all mice recovered for one week, and then received daily intraperitoneal injections of SDK3 (10 mg / kg / body weight) for six weeks. The control and model groups received an equal volume of physiological saline.

[0048] After the intervention, mouse blood was collected to detect the levels of serum bone metabolism markers osteocalcin (OPG, Bioswamp, MU30199) and anti-tartrate acid phosphatase 5b (TRAcp 5b, Bioswamp, MU31006). At the same time, mouse femoral tissue was collected for Micro-CT bone scanning and bone morphometric analysis was performed to analyze changes in bone mineral density (BMD) and bone volume fraction (BV / TV) ratio.

[0049] like Figure 5 As shown, micro-CT bone scans revealed that, compared to the sham-operated group, the OVX-induced model group exhibited significantly decreased bone mineral density, trabecular bone number and thickness, and marked bone loss. However, the SDK3 treatment group improved OVX-induced bone loss in mice, with significantly higher bone mineral density, trabecular bone number and thickness, and bone volume compared to the model group. Furthermore, BMD and BV / TV levels were close to those of the normal control group. In addition, compared to the model group, the SDK3 intervention group showed a significant increase in serum OPG levels and a significant decrease in the osteoclast TRAcp5b content, indicating that SDK3 effectively improved OVX-induced bone metabolism disorders in mice.

[0050] The above results indicate that intraperitoneal injection of SDK3 peptide can significantly improve bone metabolism indicators and bone microstructure in osteoporotic mice, and has potential application value in the treatment of osteoporosis.

[0051] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An active peptide derived from oyster juice, characterized in that, Its amino acid sequence is selected from any one of SEQ ID NO.3-SEQ ID NO.

5.

2. The active peptide according to claim 1, characterized in that, Its amino acid sequence is SEQ ID NO.

5.

3. The method for preparing the active peptide according to any one of claims 1-2, characterized in that, Includes the following steps: Fresh oysters were opened, juice was extracted with a syringe, impurities were removed by centrifugation, freeze-dried into powder, desalted, and peptides were identified by LC-MS / MS and their activity was predicted by the website.

4. The use of the bioactive peptide according to any one of claims 1-2 in the preparation of products for the prevention and treatment of bone metabolic diseases and / or the improvement of bone health.

5. The application according to claim 4, characterized in that, The prevention and treatment of bone metabolic diseases and / or improvement of bone health are achieved through any one or more of the following pathways: promoting the expression of osteogenic transcription factors, promoting osteoblast differentiation, promoting osteoblast mineralization, improving bone metabolic indicators, and improving bone microstructure.

6. The application according to claim 5, characterized in that, The bone metabolic diseases include osteoporosis, osteopenia, and fracture bone defect repair.

7. The application according to claim 5, characterized in that, The product forms include pharmaceuticals, functional foods, and / or health supplements.

8. The application according to claim 7, characterized in that, The product can be taken by injection or orally.

9. A pharmaceutical composition, characterized in that, It includes the active peptide as described in any one of claims 1-2 and a pharmaceutically acceptable carrier.

10. A functional food and / or health product, characterized in that, It includes the active peptides as described in any one of claims 1-2 and food-acceptable excipients.

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