An eggshell membrane peptide and a preparation method thereof, and application of the eggshell membrane peptide in preparation of anti-inflammatory drugs
By using an enzymatic hydrolysis method combining pepsin and alkaline protease, and an ultrafiltration process to separate the hydrolysate, the problem of poor anti-inflammatory effects in eggshell membrane peptides was solved, and eggshell membrane peptides with good anti-inflammatory effects at low dosages without damaging cell activity were prepared.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- 四川牧舟科技有限公司
- Filing Date
- 2026-03-10
- Publication Date
- 2026-06-05
AI Technical Summary
In current eggshell membrane peptide preparation processes, the proportion of peptides with anti-inflammatory effects is relatively low, and excessive use of eggshell membrane peptides can lead to decreased cell activity.
An enzymatic hydrolysis method combining pepsin and alkaline protease was adopted. Enzymatic hydrolysis under different pH and temperature conditions was carried out, and ultrafiltration was used to separate hydrolysates of different molecular weights. Different hydrolysates were compounded to improve the anti-inflammatory effect. At the same time, sodium alginate was used to pretreat eggshell membrane powder to improve the enzymatic hydrolysis efficiency.
The content of anti-inflammatory peptides in eggshell membrane peptides was increased, ensuring good anti-inflammatory effects at lower dosages and reducing negative impacts on cell activity.
Smart Images
Figure CN122146826A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of eggshell membrane peptides, and more specifically, to an eggshell membrane peptide and its preparation method, and the application of eggshell membrane peptides in the preparation of anti-inflammatory drugs. Background Technology
[0002] The eggshell membrane is a thin film located between the eggshell and the egg white, mainly composed of proteins and containing small amounts of polysaccharides, lipids, and minerals. Studies have shown that nearly 500 proteins have been identified in the eggshell membrane, contributing anti-inflammatory, antibacterial, and antioxidant activities. Existing technologies have prepared various eggshell membrane peptides using different hydrolysis methods and verified their anti-inflammatory effects. However, the preparation process is usually a simple hydrolysis followed by product efficacy verification. Furthermore, many peptides in the product do not inherently possess anti-inflammatory properties. Therefore, how to enrich peptides with anti-inflammatory or adjunctive anti-inflammatory effects is a pressing technical problem that needs to be solved. Summary of the Invention
[0003] The purpose of this invention is to provide an eggshell membrane peptide and its preparation method, as well as the application of eggshell membrane peptide in the preparation of anti-inflammatory drugs, thereby solving the problem that the proportion of peptides with anti-inflammatory effects in the existing eggshell membrane peptide products obtained by eggshell membrane hydrolysis is relatively low.
[0004] The embodiments of the present invention are achieved through the following technical solutions:
[0005] A method for preparing eggshell membrane peptides includes the following steps:
[0006] S100. Add eggshell membrane powder to pure water at a ratio of 1:(20-40) and adjust the pH to 3.5-4.5. Then add pepsin and hydrolyze for 2-5 hours at a temperature of 35-38°C to obtain the first mixture.
[0007] S200, the first mixture is subjected to an enzyme inactivation process and an ultrafiltration process to obtain a first hydrolysis product and a first ultrafiltrate; the molecular weight of the first hydrolysis product is less than or equal to 1000 Da;
[0008] After adjusting the pH of the first ultrafiltrate to 8-9, add alkaline protease and hydrolyze for 6-10 hours at a temperature of 36-38°C to obtain the second mixture.
[0009] S400, the second mixture is subjected to an ultrafiltration process to obtain a second ultrafiltrate and a second hydrolysis product; the molecular weight of the second hydrolysis product is less than or equal to 1000 Da;
[0010] S500, the second hydrolysis product was dried to obtain the first polypeptide composition;
[0011] The eggshell membrane peptides include: a first polypeptide composition.
[0012] Existing research indicates that eggshell membrane peptides have certain effects in promoting wound healing, anti-oxidation, anti-inflammation, and lowering blood sugar. However, eggshell membranes contain various active substances such as proteins, chondroitin sulfate, and hyaluronic acid. Chondroitin sulfate is a common substance that improves osteoarthritis and inhibits joint inflammation, while hyaluronic acid can promote tissue repair and reduce external stimuli, thus aiding in inflammation reduction. Therefore, eggshell membranes possess certain anti-inflammatory effects. Furthermore, eggshell membranes contain a rich variety of proteins, each with different primary functions. For example, fibronectin primarily promotes wound healing; lysozyme is mainly used as an antibacterial or preservative agent; and histones are mainly used for anti-aging. The applicant hopes to improve the preparation method of eggshell membrane peptides to enrich substances with anti-inflammatory effects or those that aid in anti-inflammatory action, such as chitin-36, and to verify the effect of the enriched product on chondrocyte anti-inflammatory activity. In addition, the applicant discovered during the research process that when the dosage of eggshell membrane peptides exceeded a certain value, cell activity would decrease significantly. However, eggshell membrane peptides inhibit LPS-induced inflammatory response and apoptosis in chondrocytes in a dose-dependent manner. Therefore, how to increase the dosage of eggshell membrane peptides to enhance the anti-inflammatory effect while ensuring cell activity as much as possible is also one of the issues that the applicant needs to consider during the research and development process.
[0013] Common methods for hydrolyzing eggshell membranes include enzymatic hydrolysis, acid hydrolysis, and a combination of both. Enzymatic hydrolysis has the disadvantage of low efficiency but the advantage of obtaining characteristic peptides due to enzyme specificity. Acid hydrolysis has the advantage of high efficiency but the disadvantage of being difficult to control and prone to damaging some amino acids. This invention utilizes the stability and volume differences of different proteins under different hydrolysis conditions to separate hydrolysis products and explore the effects of different hydrolysis product components on the anti-inflammatory effects of chondrocytes. The purpose of using pepsin is that pepsin is not effective at hydrolyzing chitin-36 and β-defensins, but it can effectively hydrolyze collagen. First, pepsin is used to obtain the first hydrolysis product. After separation, alkaline protease is used to hydrolyze the remaining protein or peptide chains to obtain the second hydrolysis product. Since pepsin requires acidic conditions to maintain its activity, and the optimal pH is 1.5-2.5, but almost all proteins can be hydrolyzed by acid, it is necessary to appropriately increase the pH to ensure that the main components of the first hydrolysis product are different from those of the second hydrolysis product. Since different proteins have different hydrolysis efficiencies under different hydrolysis conditions, ultrafiltration can separate the hydrolysis products of different proteins to a certain extent. Small molecule peptides with a molecular weight of less than or equal to 1000 Da are more conducive to absorption and thus exert their effects.
[0014] The material-to-liquid ratio is expressed in g / mL. Alkaline protease can be trypsin.
[0015] Ultrafiltration is essentially a physical separation method. By controlling the pore size of the ultrafiltration membrane, the molecular weight of the retained substances can be adjusted. The solution can be driven through the ultrafiltration membrane by pressure, with a pressure of 0.2 MPa being a selectable value.
[0016] The hydrolysis products can be dried using conventional spray drying methods, with the temperature controlled at 40℃.
[0017] Preferably, the preparation process of the eggshell membrane powder includes:
[0018] A100. Dry the eggshell membrane and then pulverize it to obtain powder;
[0019] A200: Mix the powder, sodium alginate, calcium chloride and water, place at 10-15℃ for 3-5 hours, and filter to obtain the pre-made powder;
[0020] A300, pre-made powder is freeze-dried to obtain eggshell membrane powder;
[0021] The enzymatic hydrolysis time in S100 is 2-3 hours.
[0022] The eggshell membrane structure typically consists of an outer layer near the eggshell and an inner layer near the egg white. The outer layer is more porous than the inner layer. Therefore, without pretreatment, the proteins in the outer membrane are more readily accessible to enzymes, leading to a significant difference in hydrolysis efficiency between the two membrane layers. To ensure that the proteins in both membrane layers are in a similar hydrolysis environment, the applicant pretreated the eggshell membrane. Furthermore, the pretreated eggshell membrane powder exhibits higher enzymatic hydrolysis efficiency in S100, reducing the impact of the acidic environment on proteins that pepsin cannot hydrolyze.
[0023] Sodium alginate can form complex structures with some proteins, such as positively charged lysozyme, reducing disordered compaction and increasing the water content of the eggshell membrane before freeze-drying. This, in turn, increases the overall porosity of the eggshell membrane, especially the inner membrane structure. The formation of these complex structures can also reduce the impact of acidic environments on proteins that pepsin cannot digest.
[0024] Furthermore, experiments revealed that the anti-inflammatory effect of the product was improved after using the preparation method of eggshell membrane powder provided by this invention. The applicant hypothesizes that this is because the effective content of peptides with anti-inflammatory effects in the first polypeptide composition is increased.
[0025] Preferably, the freeze-drying process includes: pre-freezing the pre-made powder at -20℃ to -10℃ for 2-3 hours, then cooling it to -60℃ to -40℃ at a rate of 1-2℃ / min and holding it at that temperature for 1-2 hours; and then heating it to 5-10℃ at a rate of 4-6℃ / h under vacuum conditions and holding it at that temperature for 2-4 hours.
[0026] The size of ice crystals can be controlled by adjusting the cooling rate, while the pore size and uniformity can be controlled by adjusting the heating rate. Conventional freeze-drying processes typically use higher final temperatures to ensure low moisture content in the product. However, in this invention, eggshell membrane powder is not the final product, and the purpose of the freeze-drying process is not to obtain a dried product. Therefore, the final temperature in this invention is controlled at 0-10℃.
[0027] Preferably, the amount of pepsin in S100 is 3000-6000 U / g.
[0028] Preferably, the amount of alkaline protease in S300 is 5000-10000 U / g.
[0029] Preferably, the enzyme inactivation step in S200 includes: adjusting the pH of the first mixture to 7-8 and then letting it stand for 20-40 minutes.
[0030] High-temperature inactivation may damage the protein structure in the eggshell membrane. Adjusting the pH value can not only avoid the aforementioned problems, but also allow the system to quickly leave the acidic environment, reduce the types of peptide chains in the first hydrolysis product, and increase the enrichment effect.
[0031] Preferably, the first hydrolysis product is dried to obtain the second polypeptide composition;
[0032] By weight, the eggshell membrane peptide comprises: 80-100 parts of a first polypeptide composition and 10-30 parts of a second polypeptide composition.
[0033] During the experiment on the first hydrolysis product, the second hydrolysis product, and the product after drying the second ultrafiltrate, the applicant found that although the eggshell membrane peptide obtained after drying the second hydrolysis product had a better effect on inhibiting IL-1β, IL-18, and TNF-α, the activity of chondrocytes began to decrease significantly when the dosage exceeded 80 ug / mL. Furthermore, the eggshell membrane peptide inhibited the secretion of LPS-induced IL-1β, IL-18, and TNF-α in a dose-dependent manner. Therefore, the applicant hopes to increase the dosage of eggshell membrane peptide while ensuring the activity of chondrocytes, thereby improving the anti-inflammatory effect.
[0034] The dried peptides from the first hydrolysate exhibited significantly higher cell viability than those from the second hydrolysate and the second ultrafiltrate when used at concentrations exceeding 80 μg / mL. The peptides in both the first and second hydrolysates had molecular weights less than or equal to 1000 Da and similar chain lengths. The applicant hypothesizes that this is because the peptides in the first hydrolysate inherently possess relatively low cytotoxicity, or that the first hydrolysate contains protective peptides, such as reducing the effective concentration of more toxic peptides on cells through target competition, promoting the structure of more toxic peptides, or reducing oxidative damage through antioxidant effects. The applicant added a portion of the peptides obtained from the dried second hydrolysate while maintaining the same dosage, observing cell viability to determine if the cytotoxicity of the second hydrolysate could be reduced through the combination of the first and second hydrolysates. After confirming that the combination could reduce cytotoxicity, the applicant further experimented by changing the component ratios to obtain the optimal range for improving anti-inflammatory effects while ensuring cell viability to a certain extent.
[0035] An eggshell membrane peptide prepared according to the preparation method described above.
[0036] The application of the eggshell membrane peptide in the preparation of anti-inflammatory drugs.
[0037] When using eggshell membrane peptides in anti-inflammatory drugs, there are no restrictions on the dosage form, such as tablets, capsules, and injections. Furthermore, protein membrane peptides can be formulated into pharmaceutical compositions with other components, such as diluents, lubricants, swelling agents, disintegrants, or carriers. Pharmaceutical compositions facilitate the administration of compounds to a living organism to produce a major therapeutic effect. Various techniques for administering compounds exist in the art, including but not limited to oral, injection, inhalation, spray, parenteral, and topical administration. Pharmaceutical compositions can also be obtained by reacting the compound with inorganic or organic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and salicylic acid.
[0038] The term "carrier" defines a compound that facilitates its incorporation into cells or tissues. For example, dimethyl sulfoxide (DMSO) is a commonly used carrier because it helps many organic compounds take up into the cells or tissues of an organism.
[0039] The term "diluent" defines a compound diluted in water that dissolves a target compound and stabilizes the biologically active form of the compound. In this art, salts dissolved in buffer solutions are used as diluents. A commonly used buffer solution is a phosphate-buffered saline solution because it mimics the salt conditions of human blood. Because buffer salts can control the pH of the solution at low concentrations, buffer dilutions rarely alter the biological activity of the compound.
[0040] In some embodiments, the same substance can be used as a carrier, diluent, or excipient, or have any two or all three functions. Therefore, a single additive in a pharmaceutical composition can have multiple functions.
[0041] The pharmaceutical composition can be prepared using existing technologies, such as conventional methods of mixing, dissolving, granulation, making sugar-coated pills, grinding, emulsifying, encapsulating, embedding, or tableting.
[0042] Preferably, the concentration of the eggshell membrane peptide is 100-170 µg / mL.
[0043] The present invention has at least the following beneficial effects:
[0044] This invention utilizes the stability and volume differences of different proteins under different hydrolysis conditions to separate hydrolysis products and explores the effects of different hydrolysis product components on chondrocyte anti-inflammatory activity, aiming to screen for more effective eggshell membrane peptide compositions. The purpose of using pepsin is that while pepsin is not effective at hydrolyzing chitin-36 and β-defensins, it is effective at hydrolyzing collagen. First, pepsin is used to obtain the first hydrolysis product. After separation, alkaline protease is used to hydrolyze the remaining protein or peptide chains to obtain the second hydrolysis product. Since pepsin requires acidic conditions to maintain its activity, with an optimal pH of 1.5-2.5, but almost all proteins can be hydrolyzed by acid, it is necessary to appropriately increase the pH to ensure that the main components of the first hydrolysis product are different from those of the second hydrolysis product. Because the hydrolysis efficiency of different proteins varies under different hydrolysis conditions, ultrafiltration can separate the hydrolysis products corresponding to different proteins to a certain extent. Small molecule peptides with a molecular weight of less than or equal to 1000 Da are more easily absorbed, thus exerting their effects. Attached Figure Description
[0045] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.
[0046] Figure 1 Figure showing the results of LPS-induced release of inflammatory factors in primary chondrocytes;
[0047] Figure 2 This is a graph showing the effect of the first polypeptide composition on cell activity.
[0048] Figure 3The graph shows the effect of the first polypeptide composition on the IL-1β secretion level induced by LPS in primary chondrocytes.
[0049] Figure 4 The graph shows the effect of the first polypeptide composition on the IL-18 secretion level induced by LPS in primary chondrocytes.
[0050] Figure 5 The graph shows the effect of the first polypeptide composition on the TNF-α secretion level induced by LPS in primary chondrocytes.
[0051] Figure 6 This is a graph showing the effect of the second polypeptide composition on cell activity.
[0052] Figure 7 Figure showing the effect of the second polypeptide composition on LPS-induced IL-1β secretion levels in primary chondrocytes;
[0053] Figure 8 Figure showing the effect of the second polypeptide composition on LPS-induced IL-18 secretion levels in primary chondrocytes;
[0054] Figure 9 The graph shows the effect of the second polypeptide composition on the TNF-α secretion level induced by LPS in primary chondrocytes.
[0055] Figure 10 This is a graph showing the effect of the third polypeptide composition on cell viability.
[0056] Figure 11 Figure showing the effect of the third polypeptide composition on LPS-induced IL-1β secretion levels in primary chondrocytes;
[0057] Figure 12 Figure showing the effect of the third polypeptide composition on LPS-induced IL-18 secretion levels in primary chondrocytes;
[0058] Figure 13 The figure shows the effect of the third polypeptide composition on the TNF-α secretion level induced by LPS in primary chondrocytes.
[0059] Figure 14 The graph shows the effect of eggshell membrane peptides prepared in Examples 6-8 and Example 3 on cell viability.
[0060] Figure 15 This is a photograph of eggshell membrane peptides.
[0061] Figure 16 This is a graph showing the effect of eggshell membrane peptides prepared in Example 11 on cell viability.
[0062] Figure 17 The graph shows the effect of eggshell membrane peptides prepared in Example 11 on LPS-induced IL-1β secretion levels in primary chondrocytes.
[0063] Figure 18 The graph shows the effect of eggshell membrane peptides prepared in Example 11 on LPS-induced IL-18 secretion levels in primary chondrocytes.
[0064] Figure 19 The graph shows the effect of eggshell membrane peptides prepared in Example 11 on LPS-induced TNF-α secretion levels in primary chondrocytes.
[0065] Figure 20 Statistical analysis of Annexin V and PI fluorescence intensity in primary chondrocytes of the Control, LPS, and 140 µg / mL eggshell membrane peptide pretreatment groups;
[0066] Figure 21 Representative images of primary chondrocytes pretreated with Control, LPS, and 140 µg / mL eggshell membrane peptides, stained with Annexin V-PI to detect apoptosis. Detailed Implementation
[0067] To make the objectives, methods, and advantages of the embodiments of the present invention clearer, the methods in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments.
[0068] Example 1: A method for preparing eggshell membrane peptides, comprising the following steps:
[0069] S100. Eggshell membrane powder is added to pure water at a ratio of 1:20, the pH is adjusted to 3.5, then pepsin is added, the enzymatic hydrolysis time is 3 hours, the enzymatic hydrolysis temperature is 35℃, and the first mixture is obtained; the amount of pepsin is 3000U / g.
[0070] S200. After adjusting the pH of the first mixture to 7, let it stand for 20 minutes, and then obtain the first hydrolysis product and the first ultrafiltrate through an ultrafiltration process; the molecular weight of the first hydrolysis product is less than or equal to 1000 Da.
[0071] After adjusting the pH of the first ultrafiltrate to 8, S300 was added, and the enzymatic hydrolysis time was 6 hours and the enzymatic hydrolysis temperature was 36℃ to obtain the second mixture; the amount of alkaline protease used was 5000 U / g.
[0072] S400, the second mixture is subjected to an ultrafiltration process to obtain a second ultrafiltrate and a second hydrolysis product; the molecular weight of the second hydrolysis product is less than or equal to 1000 Da;
[0073] S500, the second hydrolysis product was dried to obtain the first polypeptide composition;
[0074] The eggshell membrane peptides include: a first polypeptide composition.
[0075] Example 2: A method for preparing eggshell membrane peptides, comprising the following steps:
[0076] S100. Eggshell membrane powder is added to pure water at a ratio of 1:40, the pH is adjusted to 4.5, then pepsin is added, the enzymatic hydrolysis time is 4 hours, the enzymatic hydrolysis temperature is 38℃, and the first mixture is obtained; the amount of pepsin is 6000U / g.
[0077] S200. After adjusting the pH of the first mixture to 8, let it stand for 40 minutes, and then obtain the first hydrolysis product and the first ultrafiltrate through an ultrafiltration process; the molecular weight of the first hydrolysis product is less than or equal to 1000 Da.
[0078] After adjusting the pH of the first ultrafiltrate to 9, S300 was added, and the enzymatic hydrolysis time was 10 hours at a temperature of 38°C to obtain the second mixture; the amount of alkaline protease used was 10000 U / g.
[0079] S400, the second mixture is subjected to an ultrafiltration process to obtain a second ultrafiltrate and a second hydrolysis product; the molecular weight of the second hydrolysis product is less than or equal to 1000 Da;
[0080] S500, the second hydrolysis product was dried to obtain the first polypeptide composition;
[0081] The eggshell membrane peptides include: a first polypeptide composition.
[0082] Example 3: A method for preparing eggshell membrane peptides, comprising the following steps:
[0083] S100. Eggshell membrane powder is added to pure water at a ratio of 1:30, the pH is adjusted to 4, then pepsin is added, the enzymatic hydrolysis time is 4 hours, the enzymatic hydrolysis temperature is 37℃, and the first mixture is obtained; the amount of pepsin is 5000U / g.
[0084] S200. After adjusting the pH value of the first mixture to 8, let it stand for 30 minutes, and then obtain the first hydrolysis product and the first ultrafiltrate through an ultrafiltration process; the molecular weight of the first hydrolysis product is less than or equal to 1000 Da.
[0085] After adjusting the pH of the first ultrafiltrate to 8, S300 was added, and the enzymatic hydrolysis time was 8 hours and the enzymatic hydrolysis temperature was 37℃ to obtain the second mixture; the amount of alkaline protease was 8000 U / g.
[0086] S400, the second mixture is subjected to an ultrafiltration process to obtain a second ultrafiltrate and a second hydrolysis product; the molecular weight of the second hydrolysis product is less than or equal to 1000 Da;
[0087] S500, the second hydrolysis product was dried to obtain the first polypeptide composition;
[0088] The eggshell membrane peptides include: a first polypeptide composition.
[0089] Test method:
[0090] Cell Extraction: Thirty healthy adult rats were purchased and euthanized. Hyaline cartilage was isolated from the knee joints of the rats, cut into small fragments, and pre-digested with 0.25% trypsin EDTA. Then, 2 g / L type II collagenase was added, and the cells were thoroughly digested in a water bath shaker for 6 hours. The cells were gently pipetted every 2 hours during digestion. After digestion, the single-cell suspension was filtered through a 70 µm cell sieve, centrifuged at 1000 r / min for 5 minutes to collect the cells, and washed twice with PBS. The obtained chondrocytes were resuspended in DMEM / F12 complete medium containing 10% FBS and 1% penicillin-streptomycin antibiotics, and incubated at 1×10⁶ cells / mL. 5 Cells were seeded at a density of 1 cell / cm² in six-well plates and cultured at 37°C in a 5% CO₂ incubator. When chondrocyte confluence reached approximately 80%, and first-generation chondrocytes in good condition were obtained, the cells were randomly divided into a control group and an LPS-treated group. An in vitro inflammation model was constructed using lipopolysaccharide (LPS) to simulate the pathological state of osteoarthritis. The LPS-treated group received LPS at a final concentration of 1 μg / mL, while the control group received an equal volume of PBS. The cells were cultured at 37°C in a 5% CO₂ incubator for 24 hours, followed by experimental analysis.
[0091] Toxicity testing: Primary chondrocytes in good growth condition were subjected to 5×10⁻⁶... 3 Cells / well were seeded at a density of [number] cells / well in 96-well plates and cultured for 24 hours until complete cell adhesion. Cells were then divided into a control group and treatment groups with different concentrations of eggshell membrane peptides (10, 20, 40, 80, 160, 320 µg / mL). The control group received an equal volume of complete culture medium containing the same concentration of DMSO. After 24 hours of treatment, 10 µL of CCK-8 reagent was added to each well, and the cells were incubated at 37°C for 2 hours. The absorbance of each well was measured at 450 nm using a microplate reader, and the chondrocyte viability of each group was calculated using the following formula:
[0092] Cell viability (%) = (Experimental group OD) 450 - Blank group OD 450 ) / (Control group OD 450 - Blank group OD 450 ) × 100%.
[0093] Immunofluorescence: Primary chondrocytes were inoculated at 2 × 10⁻⁶ cells / mL. 4 Cells were seeded at a density of 1 / well in 12-well plates pre-placed with sterile coverslips. After cell adhesion, appropriate treatments were performed, followed by gentle washing three times with pre-cooled PBS. Cells were then fixed with 4% paraformaldehyde at room temperature for 20 minutes and permeabilized with 0.1% Triton X-100 for 10 minutes. After blocking with PBS containing 3% BSA for 1 hour, the following assays were performed: For ASC body detection, rabbit anti-ASC primary antibody (1:200 dilution) was added and incubated overnight at 4°C. After washing with PBS, cells were incubated at room temperature in the dark for 1 hour before mounting and detection. For apoptosis detection, cells were incubated with Annexin V-FITC (1:150 dilution) in binding buffer at room temperature in the dark for 15 minutes, followed by staining with propidium iodide (PI, final concentration 1 µg / mL) for 5 minutes. After washing three times with PBS, cells were mounted with anti-fluorescence quenching mounting medium. Images were observed and acquired using a confocal laser scanning microscope. Six fields of view were randomly selected for image acquisition in each group, and Fiji software was used to quantitatively analyze fluorescence intensity and the proportion of positive cells. Each experiment was independently repeated three times.
[0094] Experimental results:
[0095] LPS significantly increased the release of inflammatory factors from primary chondrocytes.
[0096] Osteoarthritis (OA) is a chronic joint disease characterized primarily by degenerative changes in articular cartilage. Activation and persistence of intra-articular inflammation are crucial factors promoting disease progression in OA. Studies have shown that OA patients exhibit significantly elevated levels of pro-inflammatory cytokines within the joint cavity. These inflammatory mediators (IL-1β, IL-18, and TNF-α) not only reflect disease activity but also directly participate in the degradation of the cartilage matrix. Lipopolysaccharide (LPS), a classic inflammatory stimulant, can effectively mimic the inflammatory microenvironment in the pathological process of OA. To verify the effect of LPS treatment on the inflammatory response of primary chondrocytes, primary chondrocytes were treated with 1 µg / mL LPS for 24 hours. The secretion of inflammatory cytokines in the cell supernatant was then detected by ELISA. The results are shown below. Figure 1 As shown in the figure, compared with the control group, the expression levels of IL-1β increased by 7-fold (p < 0.001), IL-18 increased by 7.6-fold (p < 0.001), and TNF-α increased by 6.5-fold (p < 0.001) in the 1 µg / mL LPS group.
[0097] In this experiment, chondrocytes were pretreated with different concentrations of eggshell membrane peptides (10, 20, 40, 80, 160, 320 µg / mL), and cell viability was then measured using the CCK8 assay. The results are shown in [Figure number missing]. Figure 2 . Figure 2 The figure shows the cell viability assay of the eggshell membrane peptides prepared using the method provided in Example 3. As can be seen from the figure, when the concentration of eggshell membrane peptides exceeds 80 µg / mL, the cell viability is less than 90%, and the decline accelerates significantly.
[0098] In this experiment, LPS-induced chondrocytes were pretreated with eggshell membrane peptides at concentrations of 10, 20, 40, 80, 160, and 320 µg / mL, respectively. Cell supernatants were collected to detect the secretion levels of IL-1β, IL-18, and TNF-α. The results are shown in [Figure number missing]. Figure 3-5 As shown in the figure, eggshell membrane peptides inhibit LPS-induced secretion of IL-1β, IL-18, and TNF-α in a dose-dependent manner.
[0099] In summary, to ensure cell viability while maximizing the anti-inflammatory effect of eggshell membrane peptides, the dosage should ideally not exceed 80 µg / mL.
[0100] Example 4: The first hydrolysis product obtained in Example 3 was dried to obtain the second polypeptide composition.
[0101] Experiment: In this experiment, chondrocytes were pretreated with different concentrations of the second polypeptide composition (10, 20, 40, 80, 160, 320 µg / mL), and cell viability was then measured using the CCK8 assay. The results are shown in [Figure number missing]. Figure 6 The results showed that when the amount of the second peptide composition exceeded 80 µg / mL, the rate of decline in primary chondrocyte activity did not increase significantly and was less than that of the first peptide composition.
[0102] Subsequent cell experiments involved pretreating LPS-induced chondrocytes with the second polypeptide composition at concentrations of 10, 20, 40, 80, 160, and 320 µg / mL, respectively, and collecting cell supernatants to detect the secretion levels of IL-1β, IL-18, and TNF-α. The results are shown in [Figure number missing]. Figure 7-9 As can be seen from the figure, at the same concentration, the anti-inflammatory effect of the second polypeptide composition is weaker than that of the first polypeptide composition.
[0103] Example 5: The second ultrafiltrate obtained in Example 3 was concentrated and dried to obtain the third polypeptide composition.
[0104] In this experiment, chondrocytes were pretreated with different concentrations of the third polypeptide composition (10, 20, 40, 80, 160, 320 µg / mL), and cell viability was then measured using the CCK8 assay. The results are shown in [Figure number missing]. Figure 10 The results showed that when the amount of the third polypeptide composition exceeded 40 µg / mL, the rate of decline in primary chondrocyte activity began to increase.
[0105] Subsequent cell experiments involved pretreating LPS-induced chondrocytes with the third polypeptide composition at concentrations of 10, 20, 40, 80, 160, and 320 µg / mL, respectively. Cell supernatants were collected to detect the secretion levels of IL-1β, IL-18, and TNF-α. The results are shown below. Figure 11-13 As can be seen from the figure, at the same concentration, the anti-inflammatory effect of the third polypeptide composition is weaker than that of the first polypeptide composition.
[0106] In summary, the anti-inflammatory effect is better when the first polypeptide composition is selected as the anti-inflammatory drug component.
[0107] Example 6: Eggshell membrane peptides were obtained by compounding the first polypeptide composition obtained in Example 3 and the second polypeptide composition obtained in Example 4. By weight, the eggshell membrane peptides comprised 80 parts of the first polypeptide composition and 10 parts of the second polypeptide composition.
[0108] Example 7: Eggshell membrane peptides were obtained by compounding the first polypeptide composition obtained in Example 3 and the second polypeptide composition obtained in Example 4. By weight, the eggshell membrane peptides comprised 100 parts of the first polypeptide composition and 30 parts of the second polypeptide composition.
[0109] Example 8: Eggshell membrane peptides were obtained by compounding the first polypeptide composition obtained in Example 3 and the second polypeptide composition obtained in Example 4. By weight, the eggshell membrane peptides comprised 90 parts of the first polypeptide composition and 20 parts of the second polypeptide composition.
[0110] Experiment: In this experiment, chondrocytes were pretreated with eggshell membrane peptides prepared in Examples 6-8 and Example 3 (the concentrations of the first polypeptide composition used were 80, 160, and 320 µg / mL, respectively). Cell viability was then measured using the CCK8 assay. The results are shown in [Figure number missing]. Figure 14As shown in the figure, the rate of decrease in cell viability slowed down after the addition of the second polypeptide composition and the concentration of the first polypeptide composition exceeded 80 µg / mL. Subsequently, the inhibitory rates of different eggshell membrane peptides (with the first polypeptide composition used at a concentration of 320 µg / mL) on the secretion of inflammatory factors (pg / mL) were measured, and the results are shown in Table 1.
[0111] Inhibition rate = ( ) × 100%
[0112] Table 1
[0113]
[0114] As can be seen from the data in the table, the eggshell membrane peptides prepared using Examples 6-8 have good anti-inflammatory effects. Although the amount of the first polypeptide composition used in Example 6 is comparable to that in Example 3, the effect on cell activity is reduced and the anti-inflammatory effect is even slightly increased, especially the inhibitory effect on TNF-α secretion is improved.
[0115] Example 9: A method for preparing eggshell membrane peptides, comprising the following steps:
[0116] S100. Eggshell membrane powder is added to pure water at a ratio of 1:40, the pH is adjusted to 4, then pepsin is added, the enzymatic hydrolysis time is 2 hours, the enzymatic hydrolysis temperature is 37℃, and the first mixture is obtained; the amount of pepsin is 5000U / g.
[0117] S200. After adjusting the pH value of the first mixture to 8, let it stand for 30 minutes, and then obtain the first hydrolysis product and the first ultrafiltrate through an ultrafiltration process; the molecular weight of the first hydrolysis product is less than or equal to 1000 Da.
[0118] After adjusting the pH of the first ultrafiltrate to 8, S300 was added, and the enzymatic hydrolysis time was 8 hours and the enzymatic hydrolysis temperature was 37℃ to obtain the second mixture; the amount of alkaline protease was 8000 U / g.
[0119] S400, the second mixture is subjected to an ultrafiltration process to obtain a second ultrafiltrate and a second hydrolysis product; the molecular weight of the second hydrolysis product is less than or equal to 1000 Da;
[0120] S500, the second hydrolysis product is dried to obtain the first polypeptide composition; the first hydrolysis product is dried to obtain the second polypeptide composition;
[0121] By weight, the eggshell membrane peptides comprise: 90 parts of a first polypeptide composition and 20 parts of a second polypeptide composition;
[0122] The preparation process of the eggshell membrane powder includes:
[0123] A100. Dry the eggshell membrane and then pulverize it to obtain powder;
[0124] A200. Mix the powder, sodium alginate, calcium chloride and water, place at 10°C for 3 hours, and filter to obtain the pre-made powder.
[0125] A300, the pre-made powder is freeze-dried to obtain eggshell membrane powder; the freeze-drying process includes: pre-freezing the pre-made powder at -10℃ for 2 hours, then cooling it to -40℃ at a rate of 1℃ / min and holding it at that temperature for 1 hour; and then heating it to 5℃ at a rate of 4℃ / h under vacuum conditions and holding it at that temperature for 2 hours.
[0126] Example 10: A method for preparing eggshell membrane peptides, comprising the following steps:
[0127] S100. Eggshell membrane powder is added to pure water at a ratio of 1:40, the pH is adjusted to 4, then pepsin is added, the enzymatic hydrolysis time is 3 hours, the enzymatic hydrolysis temperature is 37℃, and the first mixture is obtained; the amount of pepsin is 5000U / g.
[0128] S200. After adjusting the pH value of the first mixture to 8, let it stand for 30 minutes, and then obtain the first hydrolysis product and the first ultrafiltrate through an ultrafiltration process; the molecular weight of the first hydrolysis product is less than or equal to 1000 Da.
[0129] After adjusting the pH of the first ultrafiltrate to 8, S300 was added, and the enzymatic hydrolysis time was 8 hours and the enzymatic hydrolysis temperature was 37℃ to obtain the second mixture; the amount of alkaline protease was 8000 U / g.
[0130] S400, the second mixture is subjected to an ultrafiltration process to obtain a second ultrafiltrate and a second hydrolysis product; the molecular weight of the second hydrolysis product is less than or equal to 1000 Da;
[0131] S500, the second hydrolysis product is dried to obtain the first polypeptide composition; the first hydrolysis product is dried to obtain the second polypeptide composition;
[0132] By weight, the eggshell membrane peptides comprise: 90 parts of a first polypeptide composition and 20 parts of a second polypeptide composition;
[0133] The preparation process of the eggshell membrane powder includes:
[0134] A100. Dry the eggshell membrane and then pulverize it to obtain powder;
[0135] A200. Mix the powder, sodium alginate, calcium chloride and water, place at 15°C for 5 hours, and filter to obtain the pre-made powder.
[0136] A300, the pre-made powder is freeze-dried to obtain eggshell membrane powder; the freeze-drying process includes: pre-freezing the pre-made powder at -20℃ for 3h, then cooling it to -60℃ at a rate of 2℃ / min and holding it for 2h; under vacuum conditions, heating it to 10℃ at a rate of 6℃ / h and holding it for 4h.
[0137] Example 11: A method for preparing eggshell membrane peptides, comprising the following steps:
[0138] S100. Eggshell membrane powder is added to pure water at a ratio of 1:40, the pH is adjusted to 4, then pepsin is added, the enzymatic hydrolysis time is 2 hours, the enzymatic hydrolysis temperature is 37℃, and the first mixture is obtained; the amount of pepsin is 5000U / g.
[0139] S200. After adjusting the pH value of the first mixture to 8, let it stand for 30 minutes, and then obtain the first hydrolysis product and the first ultrafiltrate through an ultrafiltration process; the molecular weight of the first hydrolysis product is less than or equal to 1000 Da.
[0140] After adjusting the pH of the first ultrafiltrate to 8, S300 was added, and the enzymatic hydrolysis time was 8 hours and the enzymatic hydrolysis temperature was 37℃ to obtain the second mixture; the amount of alkaline protease was 8000 U / g.
[0141] S400, the second mixture is subjected to an ultrafiltration process to obtain a second ultrafiltrate and a second hydrolysis product; the molecular weight of the second hydrolysis product is less than or equal to 1000 Da;
[0142] S500, the second hydrolysis product is dried to obtain the first polypeptide composition; the first hydrolysis product is dried to obtain the second polypeptide composition;
[0143] By weight, the eggshell membrane peptides comprise: 90 parts of a first polypeptide composition and 20 parts of a second polypeptide composition;
[0144] The preparation process of the eggshell membrane powder includes:
[0145] A100. Dry the eggshell membrane and then pulverize it to obtain powder;
[0146] A200. Mix the powder, sodium alginate, calcium chloride and water, place at 15°C for 4 hours, and filter to obtain the pre-made powder.
[0147] A300, the pre-made powder is freeze-dried to obtain eggshell membrane powder; the freeze-drying process includes: pre-freezing the pre-made powder at -15℃ for 2h, then cooling it to -50℃ at a rate of 2℃ / min and holding it at that temperature for 1.5h; and then heating it to 10℃ at a rate of 5℃ / h under vacuum conditions and holding it at that temperature for 3h.
[0148] Figure 15This is a photograph of the eggshell membrane powder prepared in Example 11.
[0149] Chondrocytes were pretreated with eggshell membrane peptides prepared in Example 11 (the first polypeptide composition was used at concentrations of 80, 100, 120, 140, and 160 µg / mL), and cell viability was then measured using the CCK8 assay. The test results are shown in [Figure 1]. Figure 16 As can be seen from the figure, when the concentration of the first polypeptide composition reaches 140 µg / mL, that is, when the concentration of eggshell membrane peptide reaches about 170 µg / mL, the cell activity is about 90%.
[0150] LPS-induced chondrocytes were pretreated with eggshell membrane peptides (the concentration of the first polypeptide composition was 140 µg / mL), and cell supernatants were collected to detect the secretion levels of IL-1β, IL-18, and TNF-α. Cell apoptosis was detected using Annexin V staining. The results are shown in the table below. Figure 17-21 .from Figure 17-19 As can be seen, the anti-inflammatory effect of the eggshell membrane peptides prepared using the method of Example 11 is improved compared to that of Example 8. Figure 20-21 It is evident that pretreatment with eggshell membrane peptides significantly inhibited LPS-induced chondrocyte apoptosis and reduced the proportion of Annexin V-positive cells.
[0151] Comparative Example 1: The difference from Example 11 is that the cooling rate is 0.5℃ / min.
[0152] Comparative Example 2: The difference from Example 11 is that the heating rate is 10°C / h.
[0153] When eggshell membrane peptides prepared in Example 11 and Comparative Examples 1-2 were used, the secretion levels (pg / mL) of LPS-induced IL-1β, IL-18 and TNF-α were measured. The experimental results are shown in Table 2.
[0154] Table 2
[0155]
[0156] As shown in the table, the anti-inflammatory effect of eggshell membrane peptides decreased when the cooling rate was too low or the heating rate was too high during the freeze-drying process. The applicant hypothesizes that the low porosity of the eggshell membrane structure means that when the enzymatic hydrolysis time in S100 is short, the proteins corresponding to the peptides in the first hydrolysis product are not completely hydrolyzed, resulting in a lower proportion of anti-inflammatory peptides in the eggshell membrane peptides. Alternatively, the increased difference in density between the inner and outer layers of the eggshell membrane may also lead to a decrease in the proportion of anti-inflammatory peptides in the eggshell membrane peptides.
[0157] The above are merely preferred embodiments of the present invention and are not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A method for preparing an eggshell membrane peptide, characterized by, Includes the following steps: S100. Add eggshell membrane powder to pure water at a ratio of 1:(20-40) and adjust the pH to 3.5-4.
5. Then add pepsin and hydrolyze for 2-5 hours at a temperature of 35-38°C to obtain the first mixture. S200, the first mixture is subjected to an enzyme inactivation process and an ultrafiltration process to obtain a first hydrolysis product and a first ultrafiltrate; the molecular weight of the first hydrolysis product is less than or equal to 1000 Da; After adjusting the pH of the first ultrafiltrate to 8-9, add alkaline protease and hydrolyze for 6-10 hours at a temperature of 36-38°C to obtain the second mixture. S400, the second mixture is subjected to an ultrafiltration process to obtain a second ultrafiltrate and a second hydrolysis product; the molecular weight of the second hydrolysis product is less than or equal to 1000 Da; S500, the second hydrolysis product was dried to obtain the first polypeptide composition; The eggshell membrane peptides include: a first polypeptide composition.
2. The production method according to claim 1, characterized by, The preparation process of the eggshell membrane powder includes: A100. Dry the eggshell membrane and then pulverize it to obtain powder; A200: Mix the powder, sodium alginate, calcium chloride and water, place at 10-15℃ for 3-5 hours, and filter to obtain the pre-made powder; A300, pre-made powder is freeze-dried to obtain eggshell membrane powder; The enzymatic hydrolysis time in S100 is 2-3 hours.
3. The preparation method according to claim 2, characterized in that, The freeze-drying process includes: pre-freezing the pre-made powder at -20℃ to -10℃ for 2-3 hours, then cooling it down to -60℃ to -40℃ at a rate of 1-2℃ / min and holding it at that temperature for 1-2 hours; and then heating it up to 5-10℃ at a rate of 4-6℃ / h under vacuum conditions and holding it at that temperature for 2-4 hours.
4. The preparation method according to claim 1, characterized in that, The amount of pepsin used in S100 is 3000-6000 U / g.
5. The preparation method according to claim 1, characterized in that, The amount of alkaline protease in the S300 is 5000-10000 U / g.
6. The preparation method according to claim 1, characterized in that, The enzyme inactivation process in S200 includes: adjusting the pH of the first mixture to 7-8 and then letting it stand for 20-40 minutes.
7. The preparation method according to any one of claims 1-6, characterized in that, The first hydrolysis product was dried to obtain the second polypeptide composition; By weight, the eggshell membrane peptide comprises: 80-100 parts of a first polypeptide composition and 10-30 parts of a second polypeptide composition.
8. An eggshell membrane peptide prepared by the method according to any one of claims 1-7.
9. The use of the eggshell membrane peptide of claim 8 in the preparation of an anti-inflammatory drug.
10. The application according to claim 9, characterized in that, The concentration of the eggshell membrane peptide used is 100-170 µg / mL.