Camel placenta immunologically active peptide, its preparation method and application

Abstract

The application discloses a camel placenta immunologically active peptide and a preparation method and application thereof, and belongs to the technical field of bioactive peptides. The technical problem to be solved by the application is that there is a lack of camel placenta-derived immunologically active peptides with a definite amino acid sequence, high immunological activity, low toxicity and good water solubility in the prior art. To solve the problem, the application provides a camel placenta immunologically active peptide with an amino acid sequence as shown in SEQ ID NO: 1, 2, 3, 4 or 5. The peptide has high binding energy with an immune receptor, and is non-toxic and has good water solubility after activity score, toxicity prediction, water solubility prediction and molecular docking screening. The camel placenta immunologically active peptide overcomes the shortcomings of an unknown composition and large batch difference of an existing active peptide mixture, and can be used for preparing an immunopotency-enhancing drug, a functional food or a health product.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide technology. More specifically, this invention relates to a camel placental immunomodulatory peptide, its preparation method, and its applications. Background Technology

[0002] Camel placenta, as a mammalian placenta, is rich in proteins, amino acids, and bioactive substances. In recent years, research on extracting immunomodulatory peptides from animal placentas to enhance the body's immune function has attracted attention. However, current technologies still have the following problems and shortcomings.

[0003] First, most known immunologically active peptides are derived from the placenta of common livestock such as cattle, sheep, and pigs, or from raw materials such as whey protein and soy protein. Research on immunologically active peptides derived from camel placenta is relatively limited. The composition and sequence of placental proteins differ between different sources, making it difficult to efficiently obtain peptides with specific immune-enhancing functions unique to camel placenta by directly applying the active peptide sequences or preparation methods from other species.

[0004] Secondly, existing methods for preparing placental bioactive peptides typically involve enzymatic hydrolysis, separation, and purification. However, these methods generally suffer from unstable yields and unclear target peptides. For example, in conventional enzymatic hydrolysis processes, the type and combination of enzymes, as well as the hydrolysis conditions, significantly affect the released polypeptide sequences. Insufficient or excessive hydrolysis can lead to low yields of specific immunologically active peptides. Furthermore, subsequent separation and purification often rely solely on a rough cutoff based on molecular weight ranges (e.g., less than 3 kDa or less than 5 kDa), resulting in a mixture of polypeptides where the content of truly immunologically active peptides is unknown, leading to significant batch-to-batch variations. This makes product quality difficult to standardize, thus limiting its stable application in functional foods, health products, or pharmaceuticals.

[0005] Furthermore, screening for highly active, low-toxicity immunomodulatory peptides from complex enzymatic hydrolysis products presents technical challenges. Traditional methods for screening bioactive peptides largely rely on in vitro chemical activity assays or cell experiments, which are time-consuming and have limited throughput. Verifying each of a large number of potentially active peptides individually is costly and inefficient. Simultaneously, some peptides, while exhibiting immunomodulatory activity, may suffer from poor water solubility, easy degradation by the gastrointestinal tract, or potential cytotoxicity, leading to unsatisfactory in vivo results. Current technologies lack a rapid and comprehensive screening strategy that combines bioinformatics prediction with molecular interaction analysis, making it difficult to accurately identify candidate peptides from a large pool that can effectively bind to immune-related receptors while possessing good safety and water solubility.

[0006] Furthermore, existing studies rarely report the specific amino acid sequences of immunomodulatory peptides from camel placenta. Since the immunomodulatory function of peptides is highly dependent on their primary structure and spatial conformation, the lack of clear sequence information makes it impossible to accurately characterize and quality control the active ingredients of related products. How to systematically resolve bioactive peptides with well-defined sequences, high immunomodulatory activity, and low toxicity from camel placenta remains a pressing technical problem to be solved in this field. Summary of the Invention

[0007] One object of the present invention is to provide a camel placental immunoactive peptide, its preparation method and application, so as to at least solve the above-mentioned problems.

[0008] To achieve the objectives and other advantages of this invention, a camel placenta immunoactive peptide is provided, the amino acid sequence of which is shown in SEQ ID NO: 1, 2, 3, 4 or 5.

[0009] The present invention also provides the application of camel placental immune-active peptides in the preparation of drugs, functional foods or health products that help enhance immunity.

[0010] The present invention also provides a camel placental immunoactive peptide composition comprising one or more of the above-mentioned camel placental immunoactive peptides.

[0011] The present invention also provides the application of a camel placental immunomodulatory peptide composition in the preparation of drugs, functional foods or health products that help enhance immunity.

[0012] This invention also provides a method for preparing camel placental immunoactive peptides, comprising the following steps: S1. After crushing the camel placenta, it is degreased and freeze-dried to obtain camel placenta powder; S2. Suspend camel placenta powder in phosphate buffer, add protease for enzymatic hydrolysis, and obtain hydrolysate. S3. After filtering or centrifuging the enzymatic hydrolysate, take the supernatant. Separate the supernatant through an ultrafiltration membrane to retain solutions with a molecular weight of less than 3kDa. Then freeze-dry the supernatant to obtain active peptide powder. S4. Prepare the active peptide powder into a sample peptide solution and identify the peptide sequence of the sample peptide solution using liquid chromatography-mass spectrometry. S5. The bioactivity of the polypeptide sequences obtained in S4 is scored, and peptides with scores >0.8 are screened out. Then, the toxicity, water solubility and pharmacokinetic properties of the peptides are predicted, and non-toxic and water-soluble active peptides are screened out. S6. The binding energy between the active peptide and the receptor was evaluated using molecular docking. Based on the binding energy and the predicted pharmacokinetic properties, five active peptides were screened out, which were then obtained as camel placental immunoactive peptides.

[0013] Preferably, the protease is papain, and one or more of neutral protease, alkaline protease, flavor protease or trypsin, and the mass ratio of papain to other proteases is 1:1 to 3:1.

[0014] Preferably, the enzymatic hydrolysis conditions are: enzyme addition of 3000 U / g to 7000 U / g, hydrolysis time of 2 h to 6 h, and material-to-liquid ratio of 1:1 to 1:3.

[0015] The present invention has at least the following beneficial effects: First, this invention discloses the amino acid sequences of camel placental immunoactive peptides as shown in SEQ ID NO: 1, 2, 3, 4 or 5. These sequences are obtained for the first time from camel placenta, clarifying the primary structure of the active peptides and providing a sequence basis for the characterization of active ingredients, quality control and large-scale preparation of related products.

[0016] Secondly, the immune-active peptides screened by this invention through bioinformatics activity scoring, toxicity prediction, water solubility prediction and pharmacokinetic properties have high immunomodulatory activity, low cytotoxicity and good water solubility. Compared with mixed peptides obtained without such comprehensive screening, the target peptides are more likely to exert stable and safe immune-enhancing effects in in vivo and in vitro applications.

[0017] Third, the preparation method of this invention, based on enzymatic hydrolysis and ultrafiltration, further employs liquid chromatography-mass spectrometry to identify peptide sequences, and combines activity scoring, safety and water solubility prediction. Finally, molecular docking is used to evaluate the binding energy between peptides and receptors, and camel placental immunoactive peptides such as SEQ ID NO: 1, 2, 3, 4 or 5 are screened out. This method combines experimental identification with computational simulation, avoiding the low efficiency and high cost of traditional methods that require one-by-one experimental verification, and realizing the rapid and accurate identification of high-affinity immunoactive peptides from a large number of peptides.

[0018] Fourth, the immunomodulatory peptides and their compositions of the present invention can be used to prepare drugs, functional foods or health products that enhance immunity. Due to their well-defined peptide sequences, good safety and good water solubility, they provide new functional components for the development of immunomodulatory products derived from camel placenta.

[0019] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0020] Figure 1 This is a graph showing the results of the immunological activity assessment of ultrafiltration components of camel placental enzymatic hydrolysate; Figure 2 This is a graph showing the effect of temperature on the stability of the F3 component; Figure 3 This is a graph showing the effect of pH on the stability of component F3; Figure 4 This is a graph showing the effect of simulated gastrointestinal digestion on the stability of the F3 component; Figure 5 This is a diagram showing the results of polypeptide sequence identification for component F3; Figure 6 This is the mass spectrum of the peptide WFR; Figure 7 This is the mass spectrum of the peptide RFGPA; Figure 8 This is the mass spectrum of the peptide FSGDPNWFPK; Figure 9 This is the mass spectrum of the peptide FHGDPRLPYG; Figure 10 This is the mass spectrum of peptide FGYDGDFYR; Figure 11 This is a graph showing the effect of camel placental immunomodulatory peptides on the proliferation of RAW264.7 cells; Figure 12 The figure shows the effect of camel placental immunomodulatory peptides on the phagocytic rate of RAW264.7 cells. Figure 13 This is a graph showing the effect of camel placental immunomodulatory peptides on NO secretion in RAW264.7 cells. Detailed Implementation

[0021] The present invention will now be described in further detail with reference to the embodiments and accompanying drawings, so that those skilled in the art can implement it based on the description.

[0022] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0023] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0024] Example 1: This invention provides a camel placenta immunomodulatory peptide, the preparation method of which is as follows: (1) Preparation of camel placenta enzymatic hydrolysate: Camel placenta was crushed, defatted and freeze-dried to obtain camel placenta powder; the camel placenta powder was suspended in phosphate buffer at a material-to-liquid ratio of 1:1, and papain and complex protease (purchased from Beijing Solarbio Technology Co., Ltd., product number C8800) were added. The mass ratio of papain to complex protease was 1:1, the amount of enzyme added was 4967 U / g, and the enzymatic hydrolysis time was 4.31 h to prepare camel placenta enzymatic hydrolysate.

[0025] (2) Ultrafiltration separation of camel placental enzymatic hydrolysate: After filtering the enzymatic hydrolysate through a 0.45 μm filter membrane, the hydrolysate was centrifuged sequentially at 4 ℃ and 3000 × g for 10 min using ultrafiltration centrifuge tubes with molecular weight cutoffs of 10 kDa and 3 kDa, respectively, to separate the hydrolysate into three fractions: >10 kDa, 3-10 kDa, and <3 kDa, named F1, F2, and F3, respectively. Each fraction was freeze-dried and stored at -20 ℃. Sample solutions of 50, 250, 500, 750, and 1000 μg / mL were prepared from the above three fractions, respectively.

[0026] (3) Immunological activity assessment of ultrafiltration fractions of camel placental enzymatic hydrolysate ① The effect of ultrafiltration fractions of camel placental enzymatic hydrolysate with different molecular weights on the proliferation of RAW264.7 cells was determined using the MTT assay. RAW264.7 cells in the logarithmic growth phase were divided into groups of 1.5 × 10⁻⁶ cells. 4 Seeds were inoculated at a density of 100 μL / well in 96-well plates and incubated at 37 °C with 5% CO2 for 24 h. After discarding the culture medium, 100 μL / well of different concentrations of test sample solutions (final concentrations of 50, 250, 500, 750, and 1000 μg / mL), as well as blank control and positive control LPS (1.5 μg / mL), were added to each well and incubated for 24 h. Then, 10 μL of MTT (5 mg / mL) was added to each well and incubated in the dark for 4 h. The supernatant was discarded, and 150 μL of LDMSO was added. The plates were shaken in the dark for 10 min to dissolve the formazan, and the absorbance was measured at 490 nm. All experiments were independently repeated three times. Data were analyzed by one-way ANOVA using GraphPad Prism 10.1.2 software. Duncan's multiple test was used for comparisons between groups. p < 0.05 was considered statistically significant.

[0027] Cell proliferation rate (%) = (OD of experimental group - OD of blank group) / OD of blank group × 100%.

[0028] ② The effect of ultrafiltration fractions of camel placental enzymatic hydrolysate with different molecular weights on the phagocytic function of RAW264.7 cells was evaluated using the neutral red uptake method. RAW264.7 cells in the logarithmic growth phase were divided into groups of 1.5 × 10⁻⁶ cells. 4Cells were seeded at a density of 100 μL / well in 96-well plates and cultured at 37 °C with 5% CO2 for 24 h. After discarding the culture medium, 100 μL of different concentrations of test sample solutions (final concentrations of 50, 250, 500, 750, and 1000 μg / mL), as well as blank control and positive control LPS (1.5 μg / mL) were added to each well and incubated for 24 h. The supernatant was discarded, and 100 μL of neutral red staining solution was added to each well. The plates were incubated at 37 °C for 30 min. The staining solution was discarded, and the plates were washed 1-2 times with PBS. 150 μL of neutral red lysis buffer was added to each well, and the plates were lysed at room temperature with shaking for 10 min. The absorbance was measured at 540 nm to evaluate phagocytic activity.

[0029] Neutral red phagocytosis rate (%) = (Experimental group OD - Blank group OD) / Blank group OD × 100%.

[0030] Experimental results are as follows Figure 1 As shown. By Figure 1 (A) It can be seen that within the concentration range of 50-500 μg / mL, each component can promote the proliferation of RAW264.7 cells in a concentration-dependent manner. Among them, the F3 component has the best effect. Compared with the blank control group, its proliferation rate at 500 μg / mL reached 53.90%, which is significantly higher than that of other components (p<0.05). Figure 1 As shown in (B), further neutral red phagocytosis experiments revealed that the F3 component also significantly enhanced the phagocytic function of RAW264.7 cells, achieving a phagocytosis rate of 50.21% at 500 μg / mL, significantly higher than other components (p < 0.05). This may be because the low molecular weight component has better membrane permeability, receptor affinity, and bioavailability, further confirming that the immunologically active substances in camel placental enzymatic hydrolysates are mainly enriched in the low molecular weight range. These results indicate that when the drug concentration is in the range of 50-1000 μg / mL, the cell proliferation rate and phagocytosis rate of the three ultrafiltration components with different molecular weights all show a trend of first increasing and then decreasing, reaching the highest at 500 μg / mL, with the F3 component showing the best effect. Therefore, in the subsequent experiments, the 500 μg / mL F3 component was selected for the stability investigation.

[0031] (4) Stability analysis of F3 component ① Temperature 10 mg of the lyophilized powder of component F3 was weighed and dissolved in distilled water to prepare a 10 mg / mL solution. The solutions were then incubated at constant temperatures of -20, 4, 25, 65, and 100 °C for 2 h at pH 7.0. After cooling to room temperature, each component was collected and lyophilized into powder. The untreated sample served as the control group. The stability of component F3 under different temperature conditions was evaluated using the proliferation rate and neutrophil phagocytosis rate of RAW264.7 cells as indicators. The experimental results are as follows: Figure 2 As shown.

[0032] ②pH 10 mg of the lyophilized powder of component F3 was weighed and dissolved in distilled water to prepare a 10 mg / mL solution. The pH of each solution was adjusted to 3, 5, 7, 9, and 11 using 0.5 mol / L HCl and NaOH, respectively. After standing at room temperature for 2 h, the pH was adjusted to 7.0. Each component was collected and lyophilized into powder. The untreated sample served as the control group. The stability of component F3 under different pH conditions was evaluated using the proliferation rate of RAW264.7 cells and the phagocytic rate of neutral red cells as indicators. The experimental results are as follows: Figure 3 As shown.

[0033] ③ Simulate human gastrointestinal digestion Simulated gastric digestion: 10 mg of the lyophilized powder of component F3 was weighed and reconstituted in artificial gastric fluid to prepare a 10 mg / mL solution. The pH was adjusted to 2.0 with 0.5 mol / L HCl. Pepsin was added at a 2% (w / w) enzyme-to-protein ratio, vortexed, and hydrolyzed in a 37 °C water bath for 2 h to simulate small intestinal digestion. The enzyme was inactivated by boiling in a water bath for 10 min. After cooling to room temperature, a portion of the digested solution was centrifuged at 8000 rpm for 10 min, and the supernatant was the gastric digestion sample. Simulated intestinal digestion: After 2 h of gastric digestion of component F3, the pH was adjusted to 7.5 with 0.5 mol / L NaOH. An equal volume of artificial intestinal fluid and 2% (w / w) trypsin were added, and the mixture was hydrolyzed in a 37 °C water bath for 2 h to simulate small intestinal digestion. The enzyme was then inactivated by boiling in a water bath for 10 min. After cooling, the mixture was centrifuged at 8000 rpm for 10 min, and the supernatant was the gastrointestinal digestion sample. Undigested, gastric digested, and gastrointestinal digested components were collected and lyophilized into powder. Using the undigested group as a control, the stability of the F3 fraction under gastrointestinal digestion conditions was assessed by RAW264.7 cell proliferation rate and neutral red phagocytosis rate. The experimental results are as follows: Figure 4 As shown.

[0034] Depend on Figure 2It was found that after treating the F3 component within a temperature range of -20 to 100 ℃, its effect on the proliferation and phagocytic rate of RAW264.7 cells did not change significantly compared with the untreated group. Although the promoting effect of the F3 component on cell proliferation and phagocytosis decreased slightly under 100 ℃ treatment conditions (p < 0.05), it still maintained 34.99% and 39.29% respectively, indicating that the immune activity was not completely lost and showing good heat tolerance potential. Figure 3 It was found that within the experimentally defined pH range, the effect of component F3 on cell proliferation and phagocytosis showed a decreasing trend with increasing pH, peaking at neutral condition (pH 7.0) at 53.51% and 67.48%, respectively, which was significantly different from other pH treatment groups (p < 0.05). Compared with the untreated group, at acidic environment (pH 3.0), the proliferation and phagocytosis rates decreased to 47.95% and 56.43%, respectively; while at alkaline condition (pH 11.0), the activity decrease was more significant, at 43.61% and 53.21%, respectively. Although the activity decreased under acidic and alkaline conditions, this component still exhibited relatively stable immunostimulatory activity within the pH range of 3.0 to 11.0, indicating that it has a certain degree of acid-base stability. Figure 4 The results showed that after complete gastrointestinal digestion, the F3 component exhibited significantly higher activity in promoting RAW264.7 cell proliferation and phagocytosis compared to the untreated group and the group treated only with gastric digestion, with proliferation and phagocytosis rates reaching 51.07% and 49.57%, respectively (p < 0.05). These results indicate that the F3 component is resistant to gastric acid and enzymatic hydrolysis, maintaining good activity even after complete gastrointestinal digestion. In conclusion, the F3 component maintains good stability under conditions ranging from -20 to 100℃, pH 3.0 to 11.0, and simulated gastrointestinal digestion, demonstrating significant potential for development.

[0035] (5) LC-MS / MS identification of peptide sequences The peptide sequence of fraction F3 was identified using liquid chromatography-mass spectrometry (LC-MS / MS). The sample was quantified after treatment with 0.1% trifluoroacetic acid and desalted using a C18 solid-phase extraction column. The desalted sample was then separated using an Easy nLC nano-liquid chromatography system with 0.1% formic acid aqueous solution (A) and 0.1% formic acid acetonitrile solution (B) as the mobile phase. The separated peptides were then analyzed using a Q-Exactive HF-X mass spectrometer in positive ion mode and data-dependent acquisition to obtain secondary mass spectrometry information. The raw data were retrieved and analyzed using MaxQuant software for sequence identification. Results are as follows: Figure 5 As shown, the F3 component contains 2821 polypeptide sequences, which are rich in short-chain, low-molecular-weight, hydrophobic amino acid-rich peptides, consistent with the structural characteristics of most typical immunologically active peptides.

[0036] (6) Prediction of peptide bioactivity To further explore the bioactive peptides identified by LC-MS / MS, bioinformatics methods were used for screening and prediction. The bioactivity of 2821 peptides identified by LC-MS / MS was predicted using the Peptide Ranker website (http: / / distilldeep.ucd.ie / PeptideRanker / ), with a scoring range of 0 to 1; higher scores indicated stronger potential bioactivity. A PeptideRanker scoring threshold of 0.8 was set; peptides exceeding this threshold were considered to have potential bioactivity. 348 peptides with scores > 0.8 were identified.

[0037] (7) Prediction of physicochemical properties of polypeptides For 348 peptides with a score >0.8, their water solubility, isoelectric point, toxicity, and the proportion of hydrophobic amino acids were predicted using the BIOPEP database and the PEPTIDE 2.0 online platform. Ultimately, 15 peptide sequences were selected, and the results are shown in Table 1. Table 1 shows that none of the 15 peptides exhibited toxicity, while possessing good water solubility. Their isoelectric points (pI) ranged from 4.0 to 12.0, and hydrophobic amino acids were predominantly present in these 15 peptide sequences. These prediction results theoretically confirm the good development potential of the selected peptides, particularly their common characteristics of non-toxicity and high water solubility.

[0038] Table 1. Physicochemical properties of peptides predicted by computer simulation. The 15 selected peptides with good water solubility and no toxicity were searched in the BIOPEP database. FFR peptides were included in the database, while the remaining 14 were new peptides not included in the database.

[0039] (8) Prediction of ADMET peptide properties ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) analysis is a commonly used method for evaluating in vivo behavior and safety in drug screening. To systematically assess the drug potential of food-derived bioactive peptides, the pharmacokinetic (ADMET) properties of 14 novel peptides screened were predicted. The absorption, distribution, metabolism, and toxicity characteristics of these peptides were predicted using the ADMETlab3.0 online platform (https: / / admetlab3.scbdd.com / server / evaluationCal). Absorption indicators included human gastrointestinal absorption (HIA) and Caco-2 cell permeability; distribution indicators included blood-brain barrier penetration (BBB) ​​and plasma protein binding (PPB); metabolic indicators included cytochrome P450 / 2D6 inhibition; and toxicity indicators included hepatotoxicity (DILI) and carcinogenicity. The prediction results are shown in Table 2. Most of the ultimately screened peptides showed good intestinal absorption potential (HIA+). Their Caco-2 permeability prediction values ​​were negative, indicating that the peptides could not penetrate Caco-2 cells. Regarding distribution and metabolism, all peptides showed poor transmissibility across the blood-brain barrier (BBB-), indicating a low risk of central nervous system side effects. Furthermore, most peptides did not exhibit inhibitory activity against cytochrome P450 / 2D6, and their plasma protein binding rates were all below 90%, suggesting a low risk of drug-drug interactions and minimal impact on the metabolism and binding efficiency of other substances. In addition, none of the candidate peptides showed hepatotoxicity or carcinogenicity, consistent with previous ToxinPre toxicity predictions, indicating good biocompatibility.

[0040] Table 2. ADMET analysis of peptides (9) Molecular docking The binding energies of 14 novel peptides to TLR2 and TLR4 / MD2 receptors were evaluated and screened using molecular docking methods. X-ray crystal structures of the TLR2 receptor protein (PDB ID: 6NIG) and the LPS-containing TLR4 / MD2 receptor protein (PDB ID: 3FXI) were obtained from the RCSB PDB database (https: / / www.rcsb.org / ). The receptor protein and peptide structures were preprocessed using PyMOL software, including removal of water of crystallization and unnecessary small molecules, addition of hydrogen atoms, and structural normalization. Molecular docking analysis was performed using AutoDock Vina software. By setting appropriate docking parameters, the binding energies of the peptides and receptors were simulated, and the binding energies were evaluated based on the docking scores. The results are shown in Table 3.

[0041] Table 3. Binding energies of peptides to receptors TLR2 and TLR4 / MD2 molecules. Based on the combined ADMET prediction results and the principle of optimal binding energy, camel placental immunoactive peptides capable of forming stable complexes with receptors were screened. SEQ ID NO: 1 is WFR, and its mass spectrum is as follows: Figure 6 As shown; SEQ ID NO: 2 is RFGPA, and its mass spectrum is as follows: Figure 7 As shown; SEQ ID NO: 3 is FSGDPNWFPK, and its mass spectrum is as follows: Figure 8 As shown; SEQ ID NO: 4 is FHGDPRLPYG, and its mass spectrum is as follows: Figure 9 As shown; SEQ ID NO: 5 is FGYDGDFYR, and its mass spectrum is as follows: Figure 10 As shown.

[0042] The SEQ ID NO for the remaining peptide is: SEQ ID NO: 6 is FFR; SEQ ID NO: 7 is FMR; SEQ ID NO: 8 is YRF; SEQ ID NO: 9 is NRF; SEQ ID NO: 10 is RFLR; SEQ ID NO: 11 is GPRL; SEQ ID NO: 12 is DLRF; SEQ ID NO: 13 is LQRF; SEQ ID NO: 14 is PRL; SEQ ID NO: 15 is ASGPVGPAGPRGPAGPSGPA.

[0043] Example 2: The immunomodulatory activity of the camel placental immunomodulatory peptides obtained through screening was evaluated. Each peptide was synthesized using a solid-phase synthesis method and named C1 (WFR), C2 (RFGPA), C3 (FSGDPNWFPK), C4 (FHGDPRLPYG), and C5 (FGYDGDFYR), respectively.

[0044] (1) Effect of camel placental immunomodulatory peptides on the proliferation rate of RAW264.7 cells The effect of camel placental immunomodulatory peptides on the proliferation of RAW264.7 cells was determined using the MTT assay. RAW264.7 cells in the logarithmic growth phase were divided into groups of 1.5 × 10⁻⁶ cells. 4Seeds were inoculated at a density of 1 sample per well in 96-well plates and cultured at 37 ℃ and 5% CO2 for 24 h. After discarding the culture medium, 100 μL / well of different concentrations of camel placental immunoactive peptide sample solutions (final concentrations of 25, 50, 100, and 200 μg / mL), as well as blank control and positive control LPS (1.5 μg / mL), were added to each well. After incubation for 24 h, 10 μL of MTT (5 mg / mL) was added to each well and incubated in the dark for 4 h. The supernatant was discarded, and 150 μL of DMSO was added. The plates were shaken in the dark for 10 min to dissolve the formazan, and the absorbance was measured at 490 nm. The results are as follows: Figure 11 As shown.

[0045] Cell proliferation rate (%) = (Experimental group OD - Blank group OD) / Blank group OD × 100% (2) Effect of camel placental immunomodulatory peptides on phagocytic rate of RAW264.7 cells The effect of camel placental immunomodulatory peptides on the phagocytic function of RAW264.7 cells was evaluated using the neutral red uptake method. RAW264.7 cells in the logarithmic growth phase were divided into groups of 1.5 × 10⁻⁶ cells. 4 Camel placenta immunoactive peptide sample solutions of different concentrations (25, 50, 100, 200 μg / mL) and LPS (1.5 μg / mL) for the blank control and positive control were seeded at a density of 100 μL / well in 96-well plates and cultured at 37 ℃ and 5% CO2 for 24 h. After incubation for 24 h, the supernatant was discarded, and 100 μL of neutral red staining solution was added to each well. The plates were incubated at 37 ℃ for 30 min. The staining solution was discarded, and the plates were washed 1-2 times with PBS. 150 μL of neutral red lysis buffer was added to each well, and the plates were lysed at room temperature with shaking for 10 min. The absorbance was measured at 540 nm to evaluate phagocytic activity. The results are as follows: Figure 12 As shown.

[0046] Neutral red phagocytosis rate (%) = (Experimental group OD - Blank group OD) / Blank group OD × 100% (3) Effects of camel placental immunomodulatory peptides on NO secretion in RAW264.7 cells The Griess reaction was used to detect nitrite levels in cell supernatants to assess the effects of different synthetic peptides on NO release from macrophages. RAW264.7 cells in logarithmic growth phase were used at a concentration of 3.5 × 10⁻⁶ cells / cell supernatant. 5Seeds were planted at a density of 1 sample per well in 24-well plates, with 500 μL of culture medium added to each well. The plates were incubated at 37 °C and 5% CO2 for 24 h. The original culture medium was then discarded, and solutions of five synthetic peptides at concentrations of 25, 50, 100, and 200 μg / mL were added for co-incubation. A blank control and a positive control (LPS, 1.5 μg / mL) were also included. After another 24 h of incubation, 50 μL of supernatant was transferred from each well to a new 96-well plate. Then, 50 μL each of Griess Reagent I and Griess Reagent II, equilibrated at room temperature, were added sequentially, mixed, and incubated at room temperature for 10 min. Finally, the absorbance was measured at 540 nm. A standard curve was plotted using sodium nitrite standard solution, and the NO concentration in each sample was calculated based on the curve. Results are as follows: Figure 13 As shown.

[0047] Depend on Figure 11 It was found that each synthetic peptide exhibited varying degrees of promoting effect at different concentrations. The cell proliferation rate of all peptide-treated groups was lower than that of the LPS-positive control group, but significantly higher than that of the blank group, indicating that all five peptides possess certain immunostimulatory activities. Among them, peptide WFR showed the most significant promoting effect on cell proliferation at a concentration of 50 μg / mL, reaching 93.83%, and exhibited a concentration-dependent trend (p < 0.05). Peptides FSGDPNWFPK and FGYDGDFYR also significantly increased cell proliferation in the concentration range of 50-100 μg / mL, at 82.49% and 78.22%, respectively (p < 0.05). In contrast, FHGDPRLPYG and RFGPA showed weaker proliferation-promoting effects, but still exhibited certain proliferative effects at moderate concentrations. At a high concentration of 200 μg / mL, the cell proliferation rate decreased, indicating that excessively high concentrations may inhibit cell proliferation. In summary, all five peptides can promote the proliferation of RAW264.7 cells within a certain concentration range, with WFR, FSGDPNWFPK, and FGYDGDFYR showing the most significant immune-enhancing effects. Figure 12 The phagocytic rate of cells in the LPS-treated group was significantly higher than that in the blank control group (p < 0.05), validating the effectiveness of the experimental model. Compared with the blank control group, all synthetic peptide treatment groups significantly enhanced phagocytic capacity (p < 0.05) in a dose-dependent manner. Among them, WFR at a concentration of 50 μg / mL showed the most significant promoting effect on the phagocytic function of RAW264.7 cells, with a phagocytic rate of 86.14% (p < 0.05). In contrast, peptides FHGDPRLPYG, FSGDPNWFPK, FGYDGDFYR, and RFGPA only promoted phagocytic activity at moderate concentrations, and the effect was lower than that of the WFR-treated group. Figure 13It was found that, compared with the blank control group, LPS treatment significantly increased the release level of NO from cells (p < 0.05), indicating that the cells were effectively activated. After treatment with each synthetic peptide, the NO release from macrophages increased to varying degrees, but the effect showed significant differences based on concentration and peptide. Among them, the WFR peptide had the most significant promoting effect on NO release, reaching 16.85 μM, and increased NO levels within a concentration range of 50-200 μg / mL, exhibiting a certain dose-dependent effect. In contrast, while the FHGDPRLPYG, FSGDPNWFPK, FGYDGDFYR, and RFGPA peptides increased NO release at certain concentrations, their promoting effect was weaker than that of WFR. Overall, compared with the blank control group, all five synthetic peptides could induce NO production in macrophages to some extent. In conclusion, all five synthetic peptides could promote RAW264.7 cell proliferation, enhance phagocytic function, and induce NO release to varying degrees, and all possessed certain immunomodulatory activities.

[0048] The number of devices and processing scale described herein are for simplification of the present invention. Applications, modifications, and variations of the camel placental immunoactive peptides, their preparation methods, and applications of the present invention will be readily apparent to those skilled in the art.

[0049] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. Camel placental immunomodulatory peptides, characterized in that, Its amino acid sequence is shown in SEQ ID NO: 1, 2, 3, 4 or 5.

2. The use of the camel placental immunomodulatory peptide as described in claim 1 in the preparation of drugs, functional foods or health products that help enhance immunity.

3. A camel placental immunomodulatory peptide composition, characterized in that, It contains one or more of the camel placental immunoactive peptides as described in claim 1.

4. The use of the camel placenta immunomodulatory peptide composition as described in claim 3 in the preparation of medicines, functional foods or health products that help enhance immunity.

5. The method for preparing camel placental immunoactive peptides as described in claim 1, characterized in that, Includes the following steps: S1. After crushing the camel placenta, it is degreased and freeze-dried to obtain camel placenta powder; S2. Suspend camel placenta powder in phosphate buffer, add protease for enzymatic hydrolysis, and obtain hydrolysate. S3. After filtering or centrifuging the enzymatic hydrolysate, take the supernatant. Separate the supernatant through an ultrafiltration membrane to retain solutions with a molecular weight of less than 3kDa. Then freeze-dry the supernatant to obtain active peptide powder. S4. Prepare the active peptide powder into a sample peptide solution and identify the peptide sequence of the sample peptide solution using liquid chromatography-mass spectrometry. S5. The bioactivity of the polypeptide sequences obtained in S4 is scored, and peptides with scores >0.8 are screened out. Then, the toxicity, water solubility and pharmacokinetic properties of the peptides are predicted, and non-toxic and water-soluble active peptides are screened out. S6. The binding energy between the active peptide and the receptor was evaluated using molecular docking. Based on the binding energy and the predicted pharmacokinetic properties, five active peptides were screened out, which were then obtained as camel placental immunoactive peptides.

6. The method for preparing camel placental immunoactive peptides as described in claim 5, characterized in that, The protease is papain, and one or more of neutral protease, alkaline protease, flavor protease or trypsin, and the mass ratio of papain to other proteases is 1:1 to 3:

1.

7. The method for preparing camel placental immunoactive peptides as described in claim 6, characterized in that, The enzymatic hydrolysis conditions are: enzyme addition of 3000 U / g to 7000 U / g, hydrolysis time of 2 h to 6 h, and material-to-liquid ratio of 1:1 to 1:3.

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