Preparation method and application of anti-cell-aging and anti-oxidation bovine bone peptide
Bovine bone peptides were prepared by combining enzymatic hydrolysis and acid treatment, and their antioxidant and anti-aging effects were verified through various experiments. This solved the problem of low release efficiency of bovine bone nutrients and opened up the application of bovine bone peptides in the field of anti-aging.
Patent Information
- Application Number
- CN202510941346.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-09
- Publication Date
- 2025-11-14
AI Technical Summary
Existing technologies cannot effectively utilize the bioactive components in bovine bones, resulting in low nutrient release efficiency, and there are no reports on the application of bovine bone peptides in anti-cellular aging.
Bovine bone peptides were prepared using a combination of enzymatic hydrolysis and acid treatment. Their antioxidant and anti-aging effects were verified through a series of experiments, including MTT assay for cell viability, DCFDA fluorescence assay for ROS levels, and β-galactosidase staining for senescent cells.
The study significantly improved the release efficiency of bovine bone peptides, discovered its effects in anti-cellular aging, and provided new evaluation methods and application prospects.
Smart Images
Figure CN120943882A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of bovine bone, and more particularly to a method for preparing an anti-cellular aging and antioxidant bovine bone peptide and its application. Background Technology
[0002] Beef bones are a natural resource rich in various nutrients, primarily consisting of two categories: inorganic and organic matter. In terms of inorganic matter, beef bones are rich in calcium, mainly in the form of calcium phosphate, accounting for approximately 86% of the total weight of the bones. In addition, beef bones also contain important minerals such as phosphorus, magnesium, and fluorine. In terms of organic matter, beef bones contain protein, especially collagen, as well as fats, carbohydrates, and other nutrients. Furthermore, beef bones are rich in various vitamins, such as B vitamins, and trace elements such as iron and zinc.
[0003] However, while traditional physical grinding processes can break down the physical structure of bovine bones, they cannot fully release their bioactive components. The dense bone matrix in bovine bones binds minerals such as calcium and phosphorus, which are encapsulated within hydroxyapatite crystals, while collagen forms a stable cross-linked network with these minerals. Therefore, conventional physical grinding processes cannot fully dissolve these nutrients, resulting in insufficient dissolution rates. Furthermore, the absorption rate of calcium from directly consumed bovine bone meal is far lower than that of soluble calcium sources such as calcium lactate, and protein, due to its large molecular structure, is difficult for the intestines to absorb directly, leading to low bioavailability.
[0004] To overcome these limitations, modern processes employ various methods to enhance the release efficiency of nutrients from bovine bones. Enzymatic hydrolysis is an effective method; by using type II collagenase and subtilis neutral protease for synergistic hydrolysis, collagen can be degraded into small peptide molecules, significantly increasing the degree of hydrolysis to over 50% and enhancing its absorption efficiency. Furthermore, acid treatment optimization is also an effective method; treating bovine bones with citric acid solution promotes the dissolution of calcium ions. Experiments show that the calcium dissolution rate of acid-treated bovine bones can reach 75%, far exceeding the dissolution rate of simple pulverization processes.
[0005] Beyond their efficient release of nutrients, bovine bone hydrolysates also possess other functional applications. For instance, collagen peptides have been shown to adsorb heavy metals such as Pb²⁺ and can be used to remediate contaminated soils, such as those contaminated with cadmium. These functional applications offer new possibilities for the use of bovine bone in the environmental and medical fields.
[0006] Scientific research evidence shows that biotransformed bovine bone marrow peptide powder contains small molecule peptides that can enhance immunity, promote bone development, and have been experimentally proven to accelerate metabolism and wound healing. Furthermore, its industrial development potential should not be overlooked. Existing studies have used bovine bone powder in functional foods, such as bone meal noodles; when added at a level of 5%, it can significantly increase protein and calcium content while controlling cooking losses within a reasonable range.
[0007] In conclusion, although the physical grinding process of bovine bone meal presents a bottleneck in release efficiency, its nutritional and functional value can be fully activated through bio-enzymatic hydrolysis and chemical extraction technologies. The application of these technologies not only provides novel raw materials for the food industry but also opens up new application prospects in the environmental protection and pharmaceutical fields.
[0008] However, there are no reports of bovine bone peptides being used as an anti-cellular aging agent, nor are there any similar evaluation methods. Summary of the Invention
[0009] Purpose of the invention: To provide a more effective method for preparing bovine bone peptides with anti-cellular aging and antioxidant properties and their applications. Specific objectives are detailed in the specific implementation section, which outlines several substantive technical effects.
[0010] To achieve the above objectives, the present invention adopts the following technical solution:
[0011] A method for preparing anti-cellular aging and antioxidant bovine bone peptides, characterized in that...
[0012] The process includes the following steps: the bovine bone peptide is dissolved in double-distilled water to achieve a final concentration of 200 mg / ml;
[0013] Thiazol blue (MTT), as a hydrogen ion-accepting dye, acts on the respiratory chain in the mitochondria of living cells. Under the action of succinate dehydrogenase and cytochrome C, thiazol blue (MTT) is reduced to water-insoluble blue-purple formazan, which is deposited in the cells. Dead cells do not have this function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells. The absorbance value of MTT at 490 nm using an enzyme-linked immunosorbent assay (ELISA) can indirectly reflect the number of live cells. MRC5 cells were digested and seeded into 96-well plates at 5000 cells / well. After overnight cell attachment, the cells were treated according to different experimental designs and cultured for 72 hours. Then, MTT was added and incubated at 37°C for 4 hours. After dissolving in 150 u DMSO, the OD value was measured at 490 nm. Cell viability (%) = (OD value of experimental group - OD value of negative control) / (OD value of blank control - OD value of negative control) x 100%.
[0014] tert-butyl hydroperoxide is an oxidant that also induces cellular senescence by inducing oxidative stress. Dichlorofluorescein diacetate (HDCFDA) is a chemically reduced fluorescein. After intracellular esterases cleave the acetate group and oxidize it, the non-fluorescent HDCFDA is converted into 2',7-difluorofluorescein DCF, which emits high-intensity fluorescence, thus reflecting the intracellular ROS content. Cells were cultured in a medium containing bovine bone peptides or yak bone peptides and seeded in 6-well plates. After overnight cell adhesion, tert-butyl hydroperoxide was added to the cells for 1 hour, followed by the addition of 1 μM HDCFDA. The cells were stained in the dark for 30 minutes, digested, centrifuged, and washed three times with PBS. The intracellular fluorescence intensity was then detected by flow cytometry.
[0015] After MRC5 cells were passaged and adhered, they were treated with 100 mM tert-butyl peroxide for 2 h, then the tert-butyl peroxide was removed and the culture medium was replaced with fresh medium for continued culture. This treatment was repeated 3 times. For the bovine bone peptide treatment group, bovine bone peptide was added during the tert-butyl peroxide-induced cell senescence process to observe whether bovine bone peptide had a protective effect against hydrogen peroxide-induced cell senescence.
[0016] Using X-Gal as a substrate, a deep blue substance is generated under the catalysis of the aging-specific protein β-galactosidase. Cells that show positive staining under an optical microscope are senescent cells. Cells treated with the above-mentioned senescence-inducing method were processed according to the kit instructions: the cell culture medium was removed, the cells were washed once with PBS, 0.5 ml of senescence fixative was added, and the cells were fixed at room temperature for 15 min. After washing twice with PBS, 0.5 ml of staining working solution was added. The 0.5 ml staining working solution contained 5 ml of staining solution A, 5 ml of staining solution B, 465 μg of staining solution at ℃, and 25 ml of X-gal. The solution was prepared fresh before use. Staining was performed overnight at 37℃. The next day, the cells were observed and photographed under an optical microscope. Three fields of view were randomly selected from each group of cells, and the number of cells and positive cells in each field of view were counted using Image-Pro Plus. The positive cell rate (%) was calculated as: (Number of positive cells / Number of cells) x 100%.
[0017] Cells treated with the above-mentioned senescence-induced method were collected, washed three times with PBS, and the supernatant was discarded. An appropriate amount of RIPA lysis buffer (RIPA formulation: 50 mM pH 8.0 Tris-HCl, 2% NP-40, 150 mM NaCl, 0.2% SDS, 0.5% sodium deoxycholate) containing 1% protease cocktail was added to resuspend the cell pellet. The pellet was then lysed on ice for 30 min, centrifuged at 12000 mpm at 49°C for 15 min, and the supernatant was transferred to a new 1.5 ml EP tube. 2 L of protein extraction buffer was taken and protein concentration was determined according to the instructions of the BCA protein concentration assay kit. Based on the protein concentration, quantification was performed at 1 μg / mL, and 5x loading buffer was added and mixed well. The mixture was then heated in a 100°C metal bath for denaturation for 10 min before use for SDS-PAGE electrophoresis. Remaining samples were stored at -80°C.
[0018] Based on the molecular weight of the target protein, select an appropriate concentration of separating gel for electrophoresis. Add the boiled sample to the loading wells, add 1x electrophoresis buffer to the electrophoresis tank, and perform electrophoresis at a constant voltage of 70V for 30 min. After all the samples in the wells are level, switch to a constant voltage of 120V for electrophoresis. Select the stop time for electrophoresis based on the position of the protein marker. After the SDS-PAGE electrophoresis is completed, place the gel and PVDF membrane into the transfer device, and then transfer the membrane at a constant current of 400 mA for 40 min.
[0019] After transfer, the target protein band was cut according to the protein marker position and blocked with rapid protein blocking buffer for 5 min. The primary antibody was diluted with primary antibody dilution buffer, and the protein band was immersed in the primary antibody and incubated overnight on a shaker at 4°C. The next day, the PVDF membrane was washed 3 times with TBS-T for 5 min each time, and then incubated at room temperature for 1 h with horseradish peroxidase-labeled secondary antibody dilution buffer. It was washed 3 times with TBS-T for 5 min each time. Finally, it was developed and photographed in a gel imaging system using ECL chemiluminescence solution. For semi-quantitative analysis of the protein bands, the protein bands were scanned in grayscale using Image-J software. The grayscale value of the control group protein was 100% to quantify the target protein.
[0020] A further technological development of this invention is that the bovine bone peptide is either red bovine bone peptide or yak bone peptide.
[0021] A further technological development of this invention is that 0.88 g of red bovine bone peptide is weighed and dissolved in 4.4 ml of double-distilled water to achieve a final concentration of 200 mg / ml.
[0022] Weigh 0.85 g of yak bone peptides and dissolve them in 4.25 ml of double-distilled water to obtain a final concentration of 200 mg / ml.
[0023] A further technological development of this invention lies in the fact that the method for preparing bovine bone peptides involves crushing, washing, steaming, draining and settling, hydrolyzing and filtering the broth, concentrating and drying it into powder.
[0024] A further technological development of this invention lies in the effect of bovine bone peptide on the proliferation of MRC5 cells: MRC5 cells were seeded in 96-well plates and cultured in either basal medium or medium containing a certain concentration of bovine bone peptide. After 96 hours, the cell proliferation rate was detected using the MTT assay. The cell proliferation rate of cells treated with bovine bone peptide or yak bone peptide was significantly higher than that of the control cells. The cell proliferation was monitored using a HoloMonitor M4 live-cell imaging system. Every 12 hours, images were taken in the same field of view, and the number of cells in each field of view was counted. Five fields of view were used in each group, and the data were collected for five consecutive days before a cell proliferation curve was plotted. The results showed that the cell proliferation rate of cells cultured using bovine bone peptide or yak bone peptide was slightly higher than that of cells cultured in basal medium.
[0025] A further technological development of this invention lies in,
[0026] Bovine bone peptides mitigate the effects of hydrogen peroxide on MRC5 cell proliferation.
[0027] MRC5 cells were seeded in 96-well plates and cultured in basal medium or medium containing a certain concentration of red bovine bone peptide or yak bone peptide, respectively. After cell attachment, cells were treated with a gradient of tert-butyl peroxide. After 2 hours, the tert-butyl peroxide was removed, and cells were cultured in the corresponding medium for another 72 hours. Cell viability was assessed using the MTT assay. Compared with cells treated with tert-butyl peroxide alone, cells treated with bovine bone peptide showed increased viability, with a significant difference observed in the 200 μg / ml HNG group. 100 μg / ml and 200 μg / ml red bovine bone peptide significantly increased the viability of cells treated with 200 μM tert-butyl peroxide from 7.3% to 28.3% and 31.3%, respectively. 200 μg / ml HNG significantly increased the viability of cells treated with 100 μM tert-butyl peroxide from 21.67% to 48.3%. The survival rate of cells treated with tert-butyl hydroperoxide increased significantly from 4.3% to 29.0%.
[0028] A further technological development of this invention lies in,
[0029] Bovine bone peptides significantly inhibited hydrogen peroxide-induced intracellular ROS levels:
[0030] Cells were seeded in 6-well plates and treated with medium containing bovine bone peptides or yak bone peptides. After cell adhesion, the cells were treated with 100 μM tert-butyl peroxide for 1 h and incubated with a DCF probe for half an hour. Cell fluorescence intensity was detected by flow cytometry. The intracellular ROS level in the tert-butyl peroxide-treated group was significantly increased, reaching about 2.5 times that of the control group. However, after using medium containing bovine bone peptides, the intracellular ROS level decreased significantly to 1-1.5 times, showing a significant difference.
[0031] Uses of red bovine bone peptides or yak bone peptides in the preparation of foods, health products, or medicines that prevent cell aging.
[0032] The present invention, which adopts the above technical solution, has the following beneficial effects compared with the prior art: it provides a novel evaluation method and innovatively discovers experimental methods and related products for the anti-aging effects of bovine bone peptides and yak bone peptides. Attached Figure Description
[0033] To further illustrate the present invention, the following description is provided in conjunction with the accompanying drawings:
[0034] Figure 1 The effects of red yak bone peptide (HNG) and yak bone peptide (MNG) on the proliferation of MRC5 cells were investigated. a, c: The proliferation rate of HNG- or MNG-treated cells and control cells was detected by MTT assay. b, d: The changes in the proliferation rate of HNG- or MNG-treated cells were monitored by live-cell imaging system. *P<0.05.
[0035] Figure 2 To mitigate the effects of bovine bone peptides and yak bone peptides on the proliferation of MRC5 cells; a, c: MTT assay to detect the effect of HNG or MNG combined with hydrogen peroxide on the proliferation of MRC5 cells; b, d: Statistical analysis of the inhibitory effect of HNG or MNG treatment on hydrogen peroxide cell killing. *P<0.05, **P<0.01, ***P<0.001.
[0036] Figure 3 To reduce ROS levels induced by hydrogen peroxide (TBHP) from bovine bone peptides; a, c: Flow cytometry analysis of the effect of HNG or MNG on the increase in cellular ROS levels induced by tert-butyl peroxide (TBHP) at a concentration of 100 μM. HNG or MNG concentrations were 100 μg / ml (+) and 200 μg / ml (++); b, d: Statistical results of ROS levels, **P<0.01, ***P<0.001.
[0037] Figure 4Bovine bone peptide significantly reduced the number of hydrogen peroxide-induced senescent cells; a: β-galactosidase staining to detect the effect of HNG or MNG on tert-butyl peroxide-induced cell senescence, bar 100 μM, tert-butyl peroxide concentration was 100 μM. HNG or MNG concentration was 100 μg / ml (+), 200 μg / ml (++); b, c: Statistical results of the number of positive cells in five fields of view in figure a. ***P<0.001.
[0038] Figure 5 To reduce the expression of aging-related proteins by bovine bone peptide; a: Detection of the effect of HNG / MNG combined with tert-butyl hydrogen peroxide on the expression of aging-related proteins by protein immunoblotting; b: Statistical results of grayscale scanning of three parallel experiments, *P<0.05, **P<0.01.
[0039] Figure 6 and Figure 7 For the source of reagents and equipment. Detailed Implementation
[0040] The present invention will be further illustrated below with reference to the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.
[0041] This experiment used a peroxidase-induced senescence model of MRC-5 human lung fibroblasts to evaluate whether the bovine bone peptides provided by the client have anti-cellular senescence effects. Evaluation experiment begins.
[0042] Sample to be tested
[0043] The dried powders of red bovine bone peptide (HNG) and yak bone peptide (MNG) used in the experiment were provided by Gansu Zhongtai Biotechnology Co., Ltd. 0.88 g of red bovine bone peptide was weighed and dissolved in 4.4 ml of double-distilled water to a final concentration of 200 mg / ml. 0.85 g of yak bone peptide was weighed and dissolved in 4.25 ml of double-distilled water to a final concentration of 200 mg / ml.
[0044] MTT assay for cell proliferation rate
[0045] Thiazol blue (MTT) is a hydrogen ion-accepting dye that acts on the respiratory chain in the mitochondria of living cells. Under the action of succinate dehydrogenase and cytochrome C, exogenous MTT is reduced to water-insoluble blue-purple formazan, which is deposited in the cells. Dead cells lack this function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells. The absorbance value is measured at 490 nm using an enzyme-linked immunosorbent assay (ELISA) scanner, indirectly reflecting the number of viable cells. MRC5 cells were digested and seeded into 96-well plates at 5000 cells / well. After overnight cell attachment, the cells were treated according to the experimental design and cultured for another 72 hours. MTT was then added and incubated at 37°C for 4 hours. After dissolving in 150 u DMSO, the OD value was measured at 490 nm. Cell viability (%) = (OD value of experimental group - OD value of negative control) / (OD value of blank control - OD value of negative control) x 100%.
[0046] Detection of intracellular reactive oxygen species (ROs)
[0047] Reactive oxygen species (ROS) refer to the collective term for oxygen-containing free radicals and peroxides that readily form free radicals, all of which are involved in oxygen metabolism in organisms. During oxidative stress, ROS levels increase dramatically, damaging cell structure. Tert-butyl hydroperoxide is an oxidant that also induces cellular senescence by triggering oxidative stress. Dichlorofluorescein diacetate (HDCFDA) is a chemically reduced fluorescein. After intracellular esterases cleave the acetate group and oxidize it, the non-fluorescent HDCFDA is converted into the highly fluorescent 2,7'-dichlorofluorescein (DCF), thus reflecting the intracellular ROS content. Cells were cultured in medium containing HNG or MNG and seeded in 6-well plates. After overnight cell attachment, tert-butyl hydroperoxide was added to the cells for 1 hour, followed by the addition of 1 μM HDCFDA. The cells were stained in the dark for 30 minutes, digested, centrifuged, and washed three times with PBS. Intracellular fluorescence intensity was then detected by flow cytometry.
[0048] Hydrogen peroxide induces MRC5 cell senescence:
[0049] This evaluation used tert-butyl hydroperoxide (TMH), a compound that induces cell senescence through peroxidation. After MRC5 cells were passaged and adhered, they were treated with 100 mM TMH for 2 h, then the TMH was removed and replaced with fresh medium. This treatment was repeated three times. For the HNG or MNG treatment groups, the TMH-induced cell senescence process was observed...
[0050] In this study, a certain amount of HNG or MNG was added to observe whether bovine bone peptides had a protective effect against hydrogen peroxide-induced cell senescence.
[0051] 1.7 β-Galactosidase staining detection:
[0052] Using X-Gal as a substrate, a deep blue substance is generated under the catalysis of the aging-specific protein β-galactosidase. Cells that show positive staining under an optical microscope are senescent cells. Cells treated with the above-mentioned senescence-inducing method were processed according to the kit instructions: the cell culture medium was removed, the cells were washed once with PBS, 0.5 mL of senescence fixative was added, and the cells were fixed at room temperature for 15 min. The cells were then washed twice with PBS, and 0.5 mL of staining working solution (containing 5 mL of staining solution A, 5 mL of staining solution B, 465 mL of staining solution C, and 25 mL of X-gal, freshly prepared) was added. The cells were stained overnight at 37°C. The next day, the cells were observed and photographed under an optical microscope. Three fields of view were randomly selected from each cell group, and the number of cells and positive cells in each field of view was counted using Image-Pro Plus. The positive cell rate (%) was calculated as: (Number of positive cells / Number of cells) x 100%.
[0053] Western blot assay:
[0054] Cells treated with the above-mentioned senescence-induced method were collected, washed three times with PBS, and the supernatant was discarded. An appropriate amount of RIPA lysis buffer (RIPA formulation: 50 mM pH 8.0 Tris-HCl, 2% NP-40, 150 mM NaCl, 0.2% SDS, 0.5% sodium deoxycholate) containing 1% protease cocktail was added to resuspend the cell pellet. The pellet was then lysed on ice for 30 min, centrifuged at 12000 rpm at 4°C for 15 min, and the supernatant was transferred to a new 1.5 ml EP tube. 2 μL of protein extraction buffer was taken and protein concentration was determined according to the BCA protein concentration assay kit instructions. Based on the protein concentration, quantification was performed at 1 μg / mL, and 5x loading buffer was added and mixed well. The mixture was then heated in a 100°C metal bath for denaturation for 10 min before use for SDS-PAGE electrophoresis. The remaining sample was stored at -80°C.
[0055] Based on the molecular weight of the target protein, select an appropriate concentration of separating gel for electrophoresis. Add the boiled sample to the wells, and add 1x electrophoresis buffer to the electrophoresis tank. First, perform electrophoresis at a constant voltage of 70V for 30 min. After all wells are level with the sample, switch to a constant voltage of 120V. Select the stop time based on the protein marker position. After SDS-PAGE electrophoresis is complete, place the gel and PVDF membrane into the transfer apparatus, and then transfer the membrane at a constant current of 400mA for 40 min.
[0056] After transfer, the target protein band was cut according to the protein marker positions and blocked with rapid protein blocking buffer for 5 min. The primary antibody was diluted with primary antibody dilution buffer, and the protein bands were immersed in the primary antibody and incubated overnight on a shaker at 4°C. The next day, the PVDF membrane was washed three times with TBS-T for 5 min each time, then incubated at room temperature for 1 h with horseradish peroxidase-labeled secondary antibody dilution buffer, washed three times with TBS-T for 5 min each time, and finally developed and photographed using ECL chemiluminescence buffer in a gel imaging system. For semi-quantitative analysis of the protein bands, Image-J software was used to perform grayscale scanning of the protein bands, with the grayscale value of the control group protein set at 100%, to quantify the target protein.
[0057] Statistical analysis:
[0058] Statistical analysis was performed using GraphPAD Prism8 software. Data are presented as mean ± standard deviation. Normality was tested using the Shapiro-Wilk method. For normally distributed data (P>0.05), independent t-tests (for comparisons between two groups) or one-way ANOVA (for comparisons of three or more groups) were used. For non-normally distributed data (P<0.05), nonparametric tests were used. * P<0.05 indicates a significant difference, ** P<0.01 indicates a highly significant difference, and *** P<0.001 indicates an extremely significant difference.
[0059] Evaluation results:
[0060] Effects of bovine bone peptides on MRC5 cell proliferation:
[0061] MRC5 cells were seeded in 96-well plates and cultured in either basal medium or medium containing a certain concentration of bovine bone peptide. Cell proliferation was assessed using the MTT assay after 96 hours. Cells treated with HNG or MNG showed significantly higher proliferation rates than control cells (Figure 1a, c). Similarly, cell proliferation was monitored using a HoloMonitor M4 live-cell imaging system. Images were taken every 12 hours in the same field of view, and the number of cells in each field was counted. Five fields of view were used per group, and cell proliferation curves were plotted after five consecutive days (Figure 1b, d). The results showed that cells cultured with HNG or MNG had a slightly higher proliferation rate than those cultured in basal medium, but the difference was not statistically significant.
[0062] MRC5 cells were seeded in 96-well plates and cultured in basal medium or medium containing a certain concentration of HNG or MNG, respectively. After cell attachment, the cells were treated with a gradient of tert-butyl peroxide. After 2 hours, the tert-butyl peroxide was removed, and the cells were cultured in the corresponding medium for another 72 hours. Cell viability was assessed by MTT assay. The results showed that the cell viability was increased compared with cells treated with tert-butyl peroxide alone, with a significant difference observed in the 200 μg mHNG group (Figure 2a, c). 100 μgm and 200 μgm HNG significantly increased the cell survival rate of cells treated with 200 μM tert-butyl peroxide from 7.3% to 28.3% and 31.3%, respectively. Similarly, 200 μgm IMNG significantly increased the cell survival rate of cells treated with 100 μM tert-butyl peroxide from 21.67% to 48.3% and with 200 μM tert-butyl peroxide from 4.3% to 29.0%. Figure 2 (b, d). The above results indicate that appropriate concentrations of bovine bone peptides can protect cells from damage caused by hydrogen peroxide.
[0063] Cells were seeded in 6-well plates and treated with medium containing HNG or MNG. After cell attachment, the cells were treated with 100 μM tert-butyl hydroperoxide for 1 hour, and then incubated with a DCF probe for half an hour. Cell fluorescence intensity was detected by flow cytometry. Figure 3 In the group treated with tert-butyl hydrogen peroxide alone, the intracellular ROS level was significantly increased, reaching approximately 25 times that of the control group. However, after using a culture medium containing bovine bone peptides, the intracellular ROS level decreased significantly to 1-1.5 times, showing a significant difference.
[0064] Bovine bone peptides inhibit hydrogen peroxide-induced cellular senescence:
[0065] Cells were processed according to the procedure in Method 1.7, and β-galactosidase staining was performed according to the kit instructions (Figure 4). In the cell group treated with tert-butyl hydrogen peroxide alone, cell volume increased, cell bodies spread out, and became flattened; over 90% of the cells were stained blue-green. In contrast, cells cultured with different concentrations of HNG or MNG showed a significant reduction in senescent cells, ranging from 20% to 40%. These results indicate that bovine bone peptides can effectively protect cells and prevent hydrogen peroxide-induced cell senescence.
[0066] Bovine bone peptides reduce the expression of proteins related to cellular senescence.
[0067] After processing and collecting cells according to the procedure in Method 1.7, protein immunoblotting experiments were performed to detect the expression of aging-related proteins p16, p21, p27, and p53. Figure 5 When cells were treated with tert-butyl hydrogen peroxide alone, the expression of aging-related proteins increased significantly. However, when cells were cultured with different concentrations of HNG and MNG, the expression of these marker proteins decreased significantly in a dose-dependent manner.
[0068] This patent may also be titled: A method for preparing anti-cellular aging and antioxidant bovine bone peptides and its application.
[0069] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claims.
Claims
1. A method for preparing anti-cellular aging and antioxidant bovine bone peptides, characterized in that, The process includes the following steps: the bovine bone peptide is dissolved in double-distilled water to achieve a final concentration of 200 mg / ml; Thiazol blue (MTT), as a hydrogen ion-accepting dye, acts on the respiratory chain in the mitochondria of living cells. Under the action of succinate dehydrogenase and cytochrome C, thiazol blue (MTT) is reduced to water-insoluble blue-purple formazan, which is deposited in the cells. Dead cells do not have this function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells. The absorbance value of MTT at 490 nm using an enzyme-linked immunosorbent assay (ELISA) can indirectly reflect the number of live cells. MRC5 cells were digested and seeded into 96-well plates at 5000 cells / well. After overnight cell attachment, the cells were treated according to different experimental designs and cultured for 72 hours. Then, MTT was added and incubated at 37°C for 4 hours. After dissolving in 150 u DMSO, the OD value was measured at 490 nm. Cell viability (%) = (OD value of experimental group - OD value of negative control) / (OD value of blank control - OD value of negative control) x 100%. tert-butyl hydroperoxide is an oxidant that also induces cellular senescence by inducing oxidative stress. Dichlorofluorescein diacetate (HDCFDA) is a chemically reduced fluorescein. After intracellular esterases cleave the acetate group and oxidize it, the non-fluorescent HDCFDA is converted into 2',7-difluorofluorescein DCF, which emits high-intensity fluorescence, thus reflecting the intracellular ROS content. Cells were cultured in a medium containing bovine bone peptides or yak bone peptides and seeded in 6-well plates. After overnight cell adhesion, tert-butyl hydroperoxide was added to the cells for 1 hour, followed by the addition of 1 μM HDCFDA. The cells were stained in the dark for 30 minutes, digested, centrifuged, and washed three times with PBS. The intracellular fluorescence intensity was then detected by flow cytometry. After MRC5 cells were passaged and adhered, they were treated with 100 mM tert-butyl peroxide for 2 h, then the tert-butyl peroxide was removed and the culture medium was replaced with fresh medium for continued culture. This treatment was repeated 3 times. For the bovine bone peptide treatment group, bovine bone peptide was added during the tert-butyl peroxide-induced cell senescence process to observe whether bovine bone peptide had a protective effect against hydrogen peroxide-induced cell senescence. Using X-Gal as a substrate, a deep blue substance is generated under the catalysis of the aging-specific protein β-galactosidase. Cells that show positive staining under an optical microscope are senescent cells. Cells treated with the above-mentioned senescence-inducing method were processed according to the kit instructions: the cell culture medium was removed, the cells were washed once with PBS, 0.5 ml of senescence fixation solution was added, and the cells were fixed at room temperature for 15 min. After washing twice with PBS, 0.5 ml of staining working solution was added. The 0.5 ml staining working solution contained 5 ml of staining solution A, 5 ml of staining solution B, 465 μL of staining solution at ℃, and 25 ml of X-gal. The solution was prepared fresh before use. Staining was performed overnight at 37℃. The next day, the cells were observed and photographed under an optical microscope. Three fields of view were randomly selected from each group of cells, and the number of cells and positive cells in each field of view were counted using Image-Pro Plus. The positive cell rate (%) was calculated as: (Number of positive cells / Number of cells) x 100%. Cells treated with the above-mentioned senescence-induced method were collected, washed three times with PBS, and the supernatant was discarded. An appropriate amount of RIPA lysis buffer (RIPA formulation: 50 mM pH 8.0 Tris-HCl, 2% NP-40, 150 mM NaCl, 0.2% SDS, 0.5% sodium deoxycholate) containing 1% protease cocktail was added to resuspend the cell pellet. The pellet was then lysed on ice for 30 min, centrifuged at 12000 mpm at 49°C for 15 min, and the supernatant was transferred to a new 1.5 ml EP tube. 2 L of protein extraction buffer was taken and protein concentration was determined according to the instructions of the BCA protein concentration assay kit. Based on the protein concentration, quantification was performed at 1 μg / mL, and 5x loading buffer was added and mixed well. The mixture was then heated in a metal bath at 100°C for 10 min to denature the protein before SDS-PAGE electrophoresis. The remaining sample was stored at -80 degrees Celsius. Based on the molecular weight of the target protein, select an appropriate concentration of separating gel for electrophoresis. Add the boiled sample to the sample well, add Ix electrophoresis buffer to the electrophoresis tank, and first electrophore at a constant voltage of 70V for 30 min. After all the samples in the wells are level, switch to a constant voltage of 120V for electrophoresis. Select the time to stop electrophoresis according to the position of the protein marker. After SDS-PAGE electrophoresis is completed, place the gel and PVDF membrane into the transfer apparatus and transfer the membrane at a constant current of 400 mA for 40 min. After transfer, the target protein band was cut according to the protein marker position and blocked with rapid protein blocking buffer for 5 min. The primary antibody was diluted with primary antibody dilution buffer, and the protein band was immersed in the primary antibody and incubated overnight on a shaker at 4°C. The next day, the PVDF membrane was washed 3 times with TBS-T for 5 min each time, and then incubated at room temperature for 1 h with horseradish peroxidase-labeled secondary antibody dilution buffer. It was washed 3 times with TBS-T for 5 min each time. Finally, it was developed and photographed in a gel imaging system using ECL chemiluminescence solution. For semi-quantitative analysis of the protein bands, the protein bands were scanned in grayscale using Image-J software. The grayscale value of the control group protein was 100% to quantify the target protein.
2. The preparation method of the anti-cellular aging and antioxidant bovine bone peptide as described in claim 1, characterized in that, Bovine bone peptides are either red bovine bone peptides or yak bone peptides.
3. The method for preparing an anti-cellular aging and antioxidant bovine bone peptide as described in claim 2, characterized in that, Weigh 0.88 g of red bovine bone peptide and dissolve it in 4.4 ml of double-distilled water to a final concentration of 200 mg / ml. Weigh 0.85 g of yak bone peptides and dissolve them in 4.25 ml of double-distilled water to obtain a final concentration of 200 mg / ml.
4. The method for preparing an anti-cellular aging and antioxidant bovine bone peptide as described in claim 1, characterized in that, The preparation method of bovine bone peptides involves crushing, washing, steaming, draining and settling, hydrolyzing and filtering the broth, concentrating and drying to produce powder.
5. The method for preparing an anti-cellular aging and antioxidant bovine bone peptide as described in claim 1, characterized in that, Effects of bovine bone peptide on MRC5 cell proliferation: MRC5 cells were seeded in 96-well plates and cultured in either basal medium or medium containing a certain concentration of bovine bone peptide. After 96 hours, cell proliferation rate was detected using the MTT assay. Cells treated with bovine bone peptide or yak bone peptide showed significantly higher proliferation rates than control cells. Cell proliferation was monitored using a HoloMonitor M4 live-cell imaging system. Images were taken in the same field of view every 12 hours, and the number of cells in each field was counted. Five fields of view were used for each group, and cell proliferation curves were plotted after five consecutive days of data collection. The results showed that cells cultured with bovine bone peptide or yak bone peptide had a slightly higher proliferation rate than cells cultured in basal medium.
6. The method for preparing an anti-cellular aging and antioxidant bovine bone peptide as described in claim 1, characterized in that, Bovine bone peptides mitigate the effects of hydrogen peroxide on MRC5 cell proliferation. MRC5 cells were seeded in 96-well plates and cultured in basal medium or medium containing a certain concentration of red bovine bone peptide or yak bone peptide, respectively. After cell attachment, cells were treated with a gradient of tert-butyl peroxide. After 2 hours, the tert-butyl peroxide was removed, and cells were cultured in the corresponding medium for another 72 hours. Cell viability was assessed using the MTT assay. Compared with cells treated with tert-butyl peroxide alone, cells treated with bovine bone peptide showed increased viability, with a significant difference observed in the 200 μg / ml HNG group. 100 μg / ml and 200 μg / ml red bovine bone peptide significantly increased the viability of cells treated with 200 μM tert-butyl peroxide from 7.3% to 28.3% and 31.3%, respectively. 200 μg / ml HNG significantly increased the viability of cells treated with 100 μM tert-butyl peroxide from 21.67% to 48.3%. The survival rate of cells treated with tert-butyl hydroperoxide increased significantly from 4.3% to 29.0%.
7. The method for preparing an anti-cellular aging and antioxidant bovine bone peptide as described in claim 1, characterized in that, Bovine bone peptides significantly inhibited hydrogen peroxide-induced intracellular ROS levels: Cells were seeded in 6-well plates and treated with medium containing bovine bone peptides or yak bone peptides. After cell adhesion, the cells were treated with 100 μM tert-butyl peroxide for 1 h and incubated with a DCF probe for half an hour. Cell fluorescence intensity was detected by flow cytometry. The intracellular ROS level in the tert-butyl peroxide-treated group was significantly increased, reaching about 2.5 times that of the control group. However, after using medium containing bovine bone peptides, the intracellular ROS level decreased significantly to 1-1.5 times, showing a significant difference.
8. Uses of red bovine bone peptides or yak bone peptides in the preparation of foods, health products, or medicines that prevent cell aging.