Sturgeon cartilage collagen immune peptide rich in hydrophobic amino acids and its application

Abstract

The present invention relates to the field of active peptide technology, and specifically to a heptapeptide with immunity-enhancing activity and its application. In order to enrich the types of factors that enhance immune activity, the present invention provides a collagen peptide component F2‑7 isolated from sturgeon cartilage and a polypeptide HVPLPLP isolated from F2‑7. The present invention first uses bioinformatics methods, such as peptide activity, toxicity prediction tools and molecular docking technology, to verify the immune activity of the synthetic peptide and its mechanism of immunomodulation. On this basis, RAW264.7 macrophages were selected, and the immune activity of the heptapeptide was further evaluated in vitro by detecting immune activity indicators such as cell proliferation ability, phagocytic ability and NO release. It was determined that the heptapeptide is a polypeptide with in vitro immunomodulatory activity, which can be applied to health products, biopharmaceuticals and other fields.

Description

Technical field:

[0002] The present invention relates to the technical field of active peptides, and in particular to a heptapeptide capable of improving immunity and application thereof. Background technology:

[0004] Immunity is crucial for maintaining overall health. It not only participates in defense against infection but is also closely linked to autoimmune diseases and tumors. The immune system is a complex and highly regulated system composed of multiple cells, proteins, and organs that work together to maintain homeostasis and combat pathogens. Maintaining immune homeostasis is crucial for overall health. Bioactive peptides have been shown to enhance immunity. Cai et al. isolated immunomodulatory peptides from trypsin hydrolysates of tuna scraps. The purified peptide, T1, demonstrated the best immune activity enhancement. T1 significantly accelerated TLR2 and TLR4 expression, promoted IKK and p65 phosphorylation, further activated the NF-κB signaling pathway, and promoted the immune response in RAW264.7 cells. These studies demonstrate that peptides are a potentially very effective class of factors for enhancing immune activity.

[0005] NO is an intracellular signaling molecule that not only possesses oxidative capacity but also participates in numerous pathological and physiological processes. It effectively exerts immunomodulatory effects and can serve as a quantitative indicator of macrophage activation. Furthermore, NO, a free radical with antimicrobial activity, can be produced by macrophages in the form of active peptides. Therefore, it has the potential to play an important role in enhancing the body's defenses against bacteria. Cell phagocytosis is also an indicator of cell activation. Activated cells enhance their proliferation and phagocytic abilities, and promote the secretion of nitric oxide and cytokines, making them a key nonspecific immune response.

[0006] The present invention provides a collagen peptide isolated from sturgeon cartilage, and uses RAW264.7 macrophages as an in vitro immune activity evaluation model. The NO secretion amount and neutrophil phagocytosis ability of RAW264.7 macrophages are used as characteristic indicators of immune activity to evaluate the immune activity effect of the peptide segment. Summary of the invention:

[0008] In order to enrich the types of factors that improve immune activity, the present invention provides a collagen peptide component F2-7 isolated from sturgeon cartilage and a polypeptide isolated from F2-7, specifically a heptapeptide with improved immunity. The present invention first uses bioinformatics methods, such as peptide activity, toxicity prediction tools and molecular docking technology, to verify the immune activity of the synthetic peptide and its mechanism of immunomodulation. On this basis, RAW264.7 macrophages were selected, and the immune activity of the heptapeptide was further evaluated in vitro by detecting immune activity indicators such as cell proliferation ability, phagocytic ability and NO release. It was determined that the heptapeptide is a polypeptide with in vitro immunomodulatory activity, which can be used in health products, biopharmaceuticals and other fields.

[0009] One of the technical solutions provided by the present invention is a sturgeon cartilage peptide, which is obtained by using sturgeon cartilage as raw material, and undergoing enzymatic hydrolysis, purification by Sephadex G-15 dextran gel chromatography column, purification by DEAE-52 anion chromatography column, and further purification by reversed-phase high performance liquid chromatography to obtain the F2-7 component;

[0010] Furthermore, the preparation method of the F2-7 component is as follows:

[0011] (1) Enzymatic hydrolysis: Weigh an appropriate amount of sturgeon cartilage → add water and boil for 7 h (so that the final material-liquid ratio is 1:30) → add 0.75% (m / v) alkaline protease and 0.25% (m / v) neutral protease after cooling → adjust the pH to 9.0 → stir with a constant temperature magnetic stirrer (enzymatic hydrolysis at 55 °C for 5.5 h) → inactivate the enzyme at high temperature for 10 min → cool → centrifuge at 10,000 r / min for 10 min → collect the supernatant and vacuum concentrate → freeze-dry for 72 h to obtain sturgeon cartilage enzymatic hydrolysate;

[0012] (2) Purification by Sephadex G-15 dextran gel chromatography column: The sturgeon cartilage hydrolysate was purified by Sephadex G-15 dextran gel chromatography column, eluted with primary water, and the main absorption peak components were collected to obtain sturgeon cartilage peptide component 1;

[0013] (3) Purification by DEAE-52 anion chromatography column: The first sturgeon cartilage peptide component was purified by DEAE-52 cellulose anion exchange chromatography column, and eluted with pure water and 0.1 mol / L sodium chloride solution in sequence, and the main absorption peak was collected to obtain the second sturgeon cartilage peptide component F2;

[0014] (4) Purification by reversed-phase high-performance liquid chromatography: The sturgeon cartilage peptide component F2 was separated and purified by reversed-phase high-performance liquid chromatography, and the eluents were eluent A (0.1% trifluoroacetic acid-water) and eluent B (0.1% trifluoroacetic acid-acetonitrile). From 0 to 3 min, the volume percentage of eluent A was constant at 95%; from 3 to 10 min, the volume percentage of eluent A decreased from 95% to 80%; from 10 to 20 min, the volume percentage of eluent A decreased from 80% to 50%; from 20 to 23 min, the volume percentage of eluent A decreased from 50% to 20%; from 23 to 25 min, the volume percentage of eluent A increased from 20% to 95%; from 25 to 35 min, the volume percentage of eluent A was constant at 95%; the time was 35 min, the flow rate was 1 mL / min, the column temperature was 35 °C, and the column temperature was 280 °C. The detection was performed at dual wavelengths of 200 nm and 220 nm; the eluate with a peak time of 20.8 min to 22.6 min was collected, concentrated under reduced pressure, and freeze-dried to obtain the target component F2-7 of sturgeon cartilage peptide.

[0015] The second technical solution provided by the present invention is a heptapeptide, wherein the heptapeptide is: HVPLPLP, the amino acid sequence is: His-Val-Pro-Leu-Pro-Leu-Pro, and the molecular weight is 771.46Da.

[0016] His represents the corresponding residue of the amino acid whose English name is Histidine and Chinese name is histidine;

[0017] Val represents the corresponding residue of the amino acid whose English name is Valine and Chinese name is valine;

[0018] Pro represents the corresponding residue of the amino acid whose English name is Proline and Chinese name is Proline;

[0019] Leu represents the corresponding residue of an amino acid whose English name is Leucine and whose Chinese name is Leucine.

[0020] Furthermore, the heptapeptide HVPLPLP can be obtained by enzymatic separation of sturgeon cartilage, or by chemical synthesis, preferably by chemical solid phase synthesis.

[0021] The third technical solution provided by the present invention is the use of F2-7 described in the first technical solution or the heptapeptide HVPLPLP described in the second technical solution, especially in immunomodulation, more especially in enhancing immune ability, and more especially in the preparation of health products or medicines that enhance immune activity.

[0022] A fourth technical solution provided by the present invention is a composition comprising F2-7 described in technical solution one and / or the heptapeptide HVPLPLP described in technical solution two, wherein the composition comprises F2-7 and / or the heptapeptide HVPLPLP as the sole active ingredient, and may also comprise other active ingredients and pharmaceutically acceptable excipients;

[0023] The other active ingredients include but are not limited to ingredients with immune-enhancing activity.

[0024] The fifth technical solution provided by the present invention is the use of the composition comprising F2-7 and / or the heptapeptide HVPLPLP as described in the third technical solution, particularly in immunomodulation, and more particularly in enhancing immune capacity.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] This study discloses the heptapeptide HVPLPLP for the first time. Bioinformatics methods are used to determine the biological activity and toxicity of the peptide, and molecular docking technology is used to predict key pathways and key proteins that exert immune effects. The synthetic peptide was also tested for its effects on the proliferation rate of RAW264.7 cells, its ability to phagocytose neutral red, and the release of NO, confirming its ability to enhance immunity and its potential application in the preparation of related health products and pharmaceuticals. At a concentration of 200 μg / mL, the heptapeptide HVPLPLP exhibited the most pronounced effect on cell proliferation, reaching 169.53%. The macrophage phagocytosis rate was 142.47%, and the macrophage NO release was 25.10 μmol / L. Description of the drawings:

[0028] The accompanying drawings are helpful for further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0029] Figure 1 This is a map of the purification of sturgeon cartilage peptide using Sephadex G-15 dextran gel chromatography column;

[0030] Figure 2 The spectrum of F2 was obtained after purification by DEAE-52 cellulose anion exchange chromatography column;

[0031] Figure 3 The spectrum of F2-7 was obtained by further purification of the F2 fraction by reversed-phase high-performance liquid chromatography;

[0032] Figure 4The effects of different components of RP-HPLC on RAW264.7 cell proliferation, phagocytosis, and the release of NO, inflammatory factors TNF-α, IL-1β, and IL-6;

[0033] Compared with the NC group, * P <0.05, ** P <0.01, *** P <0.001; compared with the LPS group, # P <0.05, ## P <0.01, ### P <0.001;

[0034] Figure 5 The effect of sturgeon cartilage peptide F2-7 on RAW264.7 cell morphology;

[0035] Figure 6 The effect of sturgeon cartilage peptide F2-7 on the expression of immune-related proteins (IKK, P-IKK, p65 and P-p65);

[0036] Compared with the NC group, * P <0.05, ** P <0.01, *** P <0.001; compared with the LPS group, # P <0.05, ## P <0.01, ### P <0.001;

[0037] Figure 7 3D diagram of the interaction between His-Val-Pro-Leu-Pro-Leu-Pro and IKK;

[0038] Figure 8 This is the secondary mass spectrum of the His-Val-Pro-Leu-Pro-Leu-Pro peptide;

[0039] Figure 9 The effect of His-Val-Pro-Leu-Pro-Leu-Pro on the proliferation of RAW264.7 cells;

[0040] Compared with NC, * P <0.05,** P <0.01, *** P <0.001; compared with LPS, # P <0.05, ## P <0.01, ### P <0.001;

[0041] Figure 10 The effect of His-Val-Pro-Leu-Pro-Leu-Pro on NO release in RAW264.7 cells;

[0042] Compared with NC, * P <0.05, ** P <0.01, *** P <0.001;

[0043] Figure 11 The effect of His-Val-Pro-Leu-Pro-Leu-Pro on the phagocytosis of RAW264.7 cells;

[0044] Compared with NC, * P <0.05, ** P <0.01, *** P <0.001. Specific implementation method:

[0046] In order to make the purpose, technical solutions and advantages of this application more clear, the following patent is further described in detail with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain this patent and are not intended to limit the present invention.

[0047] This study uses sturgeon cartilage as raw material, enzymatically hydrolyzing the peptide to produce sturgeon cartilage peptide. The peptide is then purified using a Sephadex G-15 column, a DEAE-52 anion column, and reverse-phase high-performance liquid chromatography to obtain the F2-7 fraction. F2-7 was found to promote cell proliferation, phagocytosis, NO release, and the secretion of inflammatory factors. The F2-7 fraction was further evaluated for its in vitro immunoreactivity in RAW264.7 cells, and its peptide sequence was identified using LC-MS / MS.

[0048] This paper discloses for the first time a heptapeptide, HVPLPLP, isolated from sturgeon cartilage. Its amino acid sequence is: His-Val-Pro-Leu-Pro-Leu-Pro, its molecular weight is 771.46 Da, and its hydrophobic amino acids account for 85.71%. Bioinformatics and in vitro experiments confirmed that HVPLPLP is a polypeptide with immunomodulatory effects.

[0049] The heptapeptide HVPLPLP can be obtained by enzymatic separation of sturgeon cartilage, or by chemical synthesis, preferably by chemical solid phase synthesis.

[0050] In certain embodiments of the present invention, molecular docking of the synthetic peptide with IKK, a characteristic protein of the immune regulatory pathway, was performed to preliminarily verify the immune regulatory pathway. The immunomodulatory effect of HVPLPLP was then evaluated by in vitro measuring the effect of the synthetic peptide on RAW264.7 cells and detecting the cell proliferation ability, phagocytic ability and NO release.

[0051] In certain embodiments of the present invention, the bioactivity of the heptapeptide HVPLPLP was predicted using PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ), with a bioactivity prediction score of 0.704132. Bioactivity prediction values ​​range from 0 to 1, with higher scores indicating better bioactivity of the peptide.

[0052] In certain embodiments of the present invention, the potential toxicity of the heptapeptide HVPLPLP was predicted and the hydrophobicity of the peptide was analyzed using ToxinPred online analysis software (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html), and the results showed that the peptide was non-toxic (Non-Toxin).

[0053] In certain embodiments of the present invention, the heptapeptide HVPLPLP was successfully docked with the receptor protein IKK, forming five hydrogen bonds with four residues of the receptor protein: CYS-89, TYR-90, VAL-64, and LEU-63. The hydrogen bond lengths were 2.5 Å, 2.0 Å, 2.7 Å, 2.9 Å, and 2.9 Å, respectively. NF-κB is an important nuclear transcription factor in cells that participates in the body's immune and inflammatory responses and is a key regulatory factor in immune and inflammatory responses. Its transcriptional activity on cell signals is primarily regulated by two other family proteins: the κB inhibitor and the κB kinase (IKK). Therefore, IKK has long been considered to be a dominant upstream player in the NF-κB signaling pathway. The docking results of a peptide with IKK can be used to determine whether the peptide can activate the NF-κB signaling pathway, thereby participating in the body's immune and inflammatory responses.

[0054] In certain embodiments of the present invention, the polypeptide HVPLPLP was obtained by chemical synthesis with a purity greater than 95%, and was subsequently used for in vitro evaluation of immune activity. In certain embodiments of the present invention, the heptapeptide HVPLPLP had the most significant effect on cell proliferation at a concentration of 200 μg / mL, with a proliferation rate of 169.53%. In certain embodiments of the present invention, the heptapeptide at a concentration of 200 μg / mL enhanced the phagocytic ability of macrophages, with a phagocytic rate of 142.47%. In certain embodiments of the present invention, the heptapeptide at a concentration of 200 μg / mL enhanced the NO release of macrophages, with the NO release reaching 25.10 μmol / L.

[0055] The present invention will be further explained below with reference to specific embodiments.

[0056] Example 1 Isolation, purification and immunological activity identification of sturgeon cartilage collagen peptides

[0057] 1. Preparation of Sturgeon Cartilage Collagen Peptide

[0058] Weigh an appropriate amount of cartilage → add water and boil for 7 h (so that the final material-liquid ratio is 1:30) → add 0.75% (m / v) alkaline protease and 0.25% (m / v) neutral protease after cooling → adjust the pH to 9.0 → stir with a constant temperature magnetic stirrer (enzymatic hydrolysis at 55°C for 5.5 h) → inactivate the enzyme at high temperature for 10 min → cool → centrifuge at 10,000 rpm for 10 min → collect the supernatant and vacuum concentrate → freeze-dry for 72 h to obtain sturgeon cartilage hydrolysate.

[0059] 2. Isolation and Purification of Sturgeon Cartilage Collagen Peptides

[0060] 2.1Sephadex G-15 gel chromatography column purification

[0061] 2.1.1 Column loading and balancing: Weigh enough Sephadex-G15 gel and soak it in grade 1 water to fully swell it. Then, use permeabilized grade 1 water to prepare a slurry for later use. Rinse the column with permeabilized grade 1 water and keep a water level of 2 cm at the bottom of the column. Use a glass rod to pour the gel slurry into the column by drainage. No bubbles should be generated during the process. Allow it to slowly settle in the column. After complete settling, connect the two ends of the column and let it stand for 12 hours. Then, turn on the peristaltic pump and use 2 to 3 times the volume of permeabilized grade 1 water to balance the column so that the pressure and elution flow rate at all points in the column remain constant.

[0062] 2.1.2 Sampling and elution: Weigh the lyophilized enzymatic hydrolysate and dissolve it in first-grade water to a concentration of 20 mg / mL. Then filter it with a 0.22 μm microporous filter membrane and apply the sample. After application, elute it with first-grade water and collect it in an automatic collector. Set the flow rate to 1 mL / min and collect each tube for 6 minutes. Use a UV spectrophotometer to measure the absorbance of each tube at 220 nm. Draw an elution curve with the number of eluted tubes as the horizontal axis and the absorbance as the vertical axis (such as Figure 1 As shown), the eluate with peak elution time of 54 min to 162 min (tubes 9-27) was collected, and then the collected elution fractions were concentrated under reduced pressure at 50°C and freeze-dried to obtain sturgeon cartilage peptide fraction 1;

[0063] 2.2 DEAE-52 anion chromatography column purification

[0064] The above-mentioned sturgeon cartilage peptide component 1 was dissolved and diluted with distilled water to a concentration of 20 mg / mL, passed through a 0.22 μm microporous filter membrane and ultrasonically removed small bubbles. The sample was loaded into a DEAE-52 cellulose anion exchange chromatography column and eluted with a sodium chloride concentration gradient of 0.0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L (the time was 240 min, 300 min, 240 min, 240 min, 186 min, and 240 min, respectively). The automatic collector flow rate was set to 1 mL / min, and each tube was collected for 6 minutes. The eluate in each tube was detected one by one using an ultraviolet spectrophotometer. The elution curve is shown in the figure below. Figure 2 As shown, the eluate with a peak time of 300min to 480min (i.e., the 0.1 mol / L sodium chloride elution fraction) was collected, and the eluate containing the sturgeon cartilage peptide F2 fraction was collected in a test tube, concentrated under reduced pressure, and vacuum-freeze-dried to obtain the sturgeon cartilage peptide fraction 2 F2;

[0065] 2.3 Further purification by reversed-phase high-performance liquid chromatography

[0066] The sturgeon cartilage peptide fraction F2 was dissolved and diluted, separated and purified by reverse phase high performance liquid chromatography, and eluted with a gradient elution, the eluents being eluent A (0.1% trifluoroacetic acid-water) and eluent B (0.1% trifluoroacetic acid-acetonitrile). From 0 to 3 min, the volume percentage of eluent A was constant at 95%; from 3 to 10 min, the volume percentage of eluent A decreased from 95% to 80%; from 10 to 20 min, the volume percentage of eluent A decreased from 80% to 50%; from 20 to 23 min, the volume percentage of eluent A decreased from 50% to 20%; from 23 to 25 min, the volume percentage of eluent A increased from 20% to 95%; from 25 to 35 min, the volume percentage of eluent A was constant at 95%; the elution time was 35 min, the flow rate was 1 mL / min, the column temperature was 35 °C, and detection was performed at dual wavelengths of 280 nm and 220 nm. The detection curve is shown in FIG. Figure 3 As shown, fractions F2-1, F2-2, F2-3, F2-4, F2-5, F2-6, F2-7, F2-8, and F2-9 were collected from different elution peaks, and their effects on RAW264.7 cell proliferation, phagocytosis, and the release of NO, inflammatory factors TNF-α, IL-1β, and IL-6 were detected.

[0067] 3. Effects of different components of RP-HPLC on RAW264.7 cell proliferation, phagocytosis, and release of NO, inflammatory factors TNF-α, IL-1β, and IL-6

[0068] 3.1 Cell culture

[0069] RAW264.7 macrophages were cultured in 100 μL of fresh culture medium (high-glucose DMEM containing 10% fetal bovine serum, 1% double-antibody (penicillin / streptomycin) and 1% glutamine) in a constant temperature incubator at 5% CO2 and 37°C for 24 h.

[0070] 3.2 RAW264.7 cell proliferation activity assay

[0071] The proliferation activity of RAW264.7 cells was determined by CCK-8 assay. 4Cells were plated in 96-well plates, with six replicates per group, and each well contained 100 μL of culture medium. The cells were cultured in a 37°C, 5% CO2 incubator for 24 hours. After the cells attached, the supernatant was removed, and 100 μL of fresh culture medium was added. The sample group consisted of 100 μL of fresh culture medium containing 100 μg / mL of each of the reversed-phase HPLC components (F2-1, F2-2, F2-3, F2-4, F2-5, F2-6, F2-7, F2-8, and F2-9). The positive control group consisted of 100 μL of culture medium containing 1 μg / mL lipopolysaccharide (LPS). The negative control group consisted of 100 μL of fresh culture medium added to the cell wells.

[0072] After drug intervention, cells were cultured for 24 hours before removal from the 96-well plate. 10 μL of CCK-8 assay reagent was added to each well and the plate was returned to the incubator for another 1–4 hours. A sterile environment was maintained during the operation, and bubbles were avoided to prevent them from affecting the absorbance. Finally, the absorbance was measured at 450 nm, and cell viability was calculated using the following formula:

[0073]

[0074] A1 is the drug group, which contains the absorbance of cells, CCK-8 and drug solution;

[0075] A2 is the absorbance of the blank group, which contains culture medium and CCK-8 solution but no cells;

[0076] A0 is the absorbance of the negative control group, which contains cells and CCK-8 solution but no drug solution.

[0077] Cell viability refers to cell proliferation activity or cytotoxic activity.

[0078] 3.3 Determination of NO secretion in RAW264.7 cells

[0079] RAW264.7 cells in the logarithmic growth period were homogenized at 5×10 4Cells were plated in 96-well plates, with six replicates per group, and each well was incubated in a 37°C, 5% CO2 incubator for 24 hours. After the cells attached, the supernatant was removed, and 100 μL of fresh culture medium was added. The sample group consisted of 100 μL of fresh culture medium containing 100 μg / mL of each of the reversed-phase HPLC components (F2-1, F2-2, F2-3, F2-4, F2-5, F2-6, F2-7, F2-8, and F2-9). The positive control group consisted of 100 μL of culture medium containing 1 μg / mL lipopolysaccharide (LPS). The negative control group consisted of 100 μL of fresh culture medium added to the cell wells. After 24 h of cell culture, the cell supernatant was collected and centrifuged. 50 μL of cell supernatant was added to each well of a 96-well plate, followed by 50 μL of Griess Reagent I and II. The absorbance was measured at 540 nm, and the NO content in μM (μmol / L) was calculated based on the standard curve.

[0080] 3.4 Detection of phagocytic ability of RAW264.7 cells

[0081] RAW264.7 cells in the logarithmic growth period were homogenized at 5×10 4 Cells were plated in 96-well plates, with six replicates per well. Each well contained 100 μL of culture medium and incubated in a 37°C, 5% CO2 incubator for 24 hours. After cell attachment, the supernatant was removed and 100 μL of fresh culture medium was added. The sample group consisted of 100 μL of fresh culture medium containing 100 μg / mL of each reversed-phase HPLC fraction (F2-1, F2-2, F2-3, F2-4, F2-5, F2-6, F2-7, F2-8, and F2-9). The positive control group consisted of 100 μL of culture medium containing 1 μg / mL lipopolysaccharide (LPS). The negative control group consisted of 100 μL of fresh culture medium added to the cell wells. After 24 hours of culture, the cells were discarded and washed two to three times with PBS. Add 200 μL of medium containing neutral red to each well (medium:neutral red = 10:1), incubate in the incubator for 1-2 h, then discard the medium and wash five times with PBS. Add 200 μL of cell lysis buffer to each well and shake for 15 min. Finally, measure the absorbance at 540 nm and calculate the phagocytic capacity of the cells using the following formula:

[0082]

[0083] A1 is the drug group, which contains the absorbance of cells, neutral red and drug solution;

[0084] A2 is the blank group, containing culture medium and neutral red solution but no cell absorbance;

[0085] A0 is the absorbance of the negative control group, which contains cells and neutral red but no drug solution.

[0086] 3.5 Detection of cytokine secretion

[0087] RAW264.7 cells in the logarithmic growth period were homogenized at 5×10 4 Cells were plated in 96-well plates, with six replicates per group, and each well contained 100 μL of culture medium. The cells were cultured in a 37°C, 5% CO2 incubator for 24 hours. After cell attachment, the supernatant was removed and 100 μL of fresh culture medium was added. The sample group consisted of 100 μL of fresh culture medium containing 100 μg / mL of each reversed-phase HPLC fraction (F2-1, F2-2, F2-3, F2-4, F2-5, F2-6, F2-7, F2-8, and F2-9). The positive control group consisted of 100 μL of culture medium containing 1 μg / mL lipopolysaccharide (LPS). The negative control group consisted of 100 μL of fresh culture medium added to the cell wells. After 24 hours of cell culture, the supernatant was collected and centrifuged. The secretion of inflammatory cytokines (IL-1β, IL-6, and TNF-α) in the supernatant of each cell group was determined according to the ELISA kit instructions.

[0088] The results of immunoassay of each component at 100 μg / mL by reversed-phase high performance liquid chromatography are as follows: Figure 4 As shown, the details are as follows:

[0089] Figure 4 A is the effect of each component on cell proliferation ability, Figure 4 As shown in Figure 3, none of the components were toxic to RAW264.7 cells. In addition, compared with the NC group, the proliferation rates of RAW264.7 cells in the F2-2, F2-4, and F2-7 groups increased by 116.31%, 147.56%, and 191.78%, respectively, with F2-7 showing the best proliferation effect.

[0090] Figure 4 B is the effect of each component on the NO secretion of cells, Figure 4 C is the effect of each component on cell phagocytosis. Figure 4 As shown in Figures B and C, the NO secretion level of the F2-7 fraction was 26.83 μmol / L, and its phagocytic ability was comparable to that of the LPS group.

[0091] Figure 4 The results of A, B, and C showed that the F2-7 component could promote the proliferation of RAW264.7 cells and stimulate the secretion of NO by RAW264.7 cells. It is a good immunomodulator and can activate the immune response ability of cells and enhance their phagocytic ability.

[0092] When stimulated by immunogens or stimuli, the body releases cytokines through transcription and translation. These cytokines are low-molecular-weight, soluble proteins. Their release enhances the defense capabilities of macrophages and serves as a key signaling molecule in the immune response. Tumor necrosis factor-α (TNF-α), primarily produced by activated macrophages or monocytes, promotes T cell proliferation and the secretion of chemokines, interleukin-6 (IL-6), and interleukin-1β (IL-1β) at other sites of infection, making it a key mediator of inflammatory responses in the body. Therefore, cytokines such as TNF-α, IL-6, and IL-1β are key analytical indicators in most in vitro studies of cellular immune activity. Figure 4 D, E, and F show the effects of each component on cytokine levels in RAW264.7 cells after treatment with reversed-phase HPLC. Compared with the NC group, F2-7 significantly promoted the secretion of TNF-α and IL-6 in RAW264.7 cells, and the secretion amounts were close to those in the LPS group, which were 4092.00 pg / mL and 7.58 pg / mL, respectively ( P After F2-7 stimulated RAW264.7 cells, the secretion of IL-1β was even higher than that of the LPS group (334.86 pg / mL).

[0093] In summary, the F2-7 fraction obtained after separation by Sephadex G-15 dextran gel chromatography column, DEAE anion chromatography column and reversed-phase high-performance liquid chromatography has a promoting effect on cell proliferation, phagocytosis, NO release and secretion of cellular inflammatory factors, and has positive significance for the body's protection and enhancement of immune ability. Therefore, we will further explore the immune activity mechanism of F2-7 on the body and conduct more in-depth research on it.

[0094] Therefore, collecting Figure 3 The eluate with a peak time of 20.8min to 22.6min was concentrated under reduced pressure and freeze-dried in vacuum to obtain the sturgeon cartilage peptide target component F2-7, which was used for further in vitro immune activity evaluation of RAW264.7 cells and LC-MS / MS identification of the peptide sequence.

[0095] Example 2 Evaluation of the in vitro immune activity of F2-7 fraction on RAW264.7 cells

[0096] 1. Configuration and grouping of F2-7 samples

[0097] RAW264.7 cells in the logarithmic growth period were homogenized at 5×10 4Cells were plated in 96-well plates, with six replicates per group, and each well was incubated in a 37°C, 5% CO2 incubator for 24 hours. After the cells attached, the supernatant was removed and 100 μL of fresh culture medium was added. The sample group received 100 μL of fresh F2-7 culture medium containing 50, 100, or 200 μg / mL of LPS. The LPS-positive control group received 100 μL of culture medium containing 1 μg / mL of LPS. The negative control group received 100 μL of fresh culture medium added to the cell wells.

[0098] 2. Effects of F2-7 components on cell morphology

[0099] The experimental grouping and operation were the same as step 1. After 24 hours of cell culture, the cells were collected and the cell growth status was observed under an optical microscope. Figure 5 As shown, cells in the NC blank group had clear boundaries and were uniformly round or oval in size. In the LPS group, cells became larger, with numerous vacuoles and long pseudopodia, and their morphology became spindle-shaped or irregular. Related studies have shown that the presence of numerous and long pseudopodia in macrophages indicates successful activation, and these pseudopodia can enhance cell adhesion and phagocytosis. In this experiment, after stimulation with different concentrations of F2-7, RAW264.7 cells showed morphological changes compared to normal NC cells, exhibiting spindle-shaped, dendritic structures. Furthermore, the number of pseudopodia increased with increasing concentration, indicating that F2-7 can activate RAW264.7 cells, enhance their adhesion and phagocytosis, and confer a certain degree of immune activity.

[0100] 3. Study on the immune activity mechanism of sturgeon cartilage collagen peptide F2-7

[0101] Western blot analysis was performed according to standard laboratory procedures for Western blot analysis. Following step 1, cells were cultured, harvested, and washed once with PBS. Total protein was extracted using RIPA lysis buffer and the protein concentration was determined using a BCA assay. SDS-PAGE electrophoresis, transfer to a membrane, blocking, incubation, and development were performed to complete the assay. The following primary antibodies were used: IKK (Cat. No. ab32041), P-IKK (Cat. No. 2697), p65 (Cat. No. 10745-1-AP), and P-p65 (Cat. No. ab76302). Incubation with the primary antibody was performed overnight at 4°C, followed by incubation with the corresponding secondary antibody for 2 h at room temperature. Protein bands were obtained and grayscale values ​​were calculated using Image J.

[0102] The NF-κB signaling pathway is present in nearly all animal cell types and is a key regulator and major transcription factor for the expression of multiple cytokines. It participates in cellular responses to stimuli and plays a crucial role in the immune response to infection. To elucidate the mechanism of F2-7's immunoreactivity against RAW264.7 cells, Western blotting was used to investigate whether F2-7 could induce the expression of IKK and p65, proteins involved in the NF-κB signaling pathway, and their phosphorylation in RAW264.7 macrophages. Cells were stimulated with low, medium, and high doses of F2-7 for 24 hours. LPS was used as a positive control, and immunohistochemistry was used to measure the protein expression levels of IKK, p-IKK, p65, and p-p65. Actin β was used as an internal control.

[0103] The results are as follows Figure 6 As shown, compared with the NC blank group, the protein expression levels of IKK, P-IKK, p65, and P-p65 in the LPS-positive control group were significantly upregulated, indicating that RAW264.7 cells were activated. Treatment of RAW264.7 cells with different concentrations of the F2-7 component significantly upregulated the protein expression levels of IKK, P-IKK, p65, and P-p65 compared with the NC blank group, with the highest effect at an F2-7 concentration of 200 μg / mL, demonstrating a concentration-dependent effect. Western blotting further demonstrated that the immunity-enhancing effect of the F2-7 component was mediated by activation of the NF-κB pathway.

[0104] Example 3 LC-MS / MS Identification and Bioinformatics Prediction of Peptides

[0105] 1. LC-MS / MS identification of peptide sequences in fraction F2-7

[0106] In this study, the F2-7 component was subjected to LC-MS / MS analysis and de novo sequencing. PEAKSStudio 8.5 software was used for data processing and retrieval analysis. Based on the Acipenseriformes species protein database, 164 peptides were obtained with a molecular weight distribution ranging from 600 to 2000 Da. Their amino acid sequences were analyzed, and finally 8 peptides with high credibility were obtained, including HVPLPLP.

[0107] 2. Use PeptideRanker to predict the biological activity of synthetic peptides

[0108] The bioactivity of the peptide HVPLPLP was predicted using PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ). Enter "HVPLPLP" in the search box and view the search results. The result, representing the predicted bioactivity score, is 0.704132. A bioactivity score greater than 0.5 indicates strong bioactivity. The predicted bioactivity value ranges from 0 to 1, with higher scores indicating greater bioactivity.

[0109] 3. Predicting the potential toxicity of synthetic peptides using ToxinPred

[0110] The peptide HVPLPLP was analyzed for potential toxicity and hydrophobicity using the ToxinPred online analysis software (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html). After entering the URL, clicking "Design Peptide," and entering "HVPLPLP" in the search box, the results indicated that the peptide HVPLPLP was non-toxic (Non-Toxin). The hydrophobic amino acid ratio was 85.71%.

[0111] 4. Molecular docking

[0112] (1) Pretreatment of docking ligand

[0113] The 2D structure of HVPLPLP was drawn using software, then converted into a 3D structure and saved in PDB format for subsequent molecular docking.

[0114] (2) Receptor protein pretreatment

[0115] In the PubChem protein database, search for the 3D structure of the desired receptor protein IKK. The screening criteria are: mouse Mus musculus, resolution less than 2.0 Å, download and save the target protein in PDB format, upload the obtained target protein to PyMOL, delete the water molecules (remove solvent) operation, and then remove the ligand small molecule on the protein structure (remove organic), and finally save it in PDB format for subsequent molecular docking operations.

[0116] (3) Docking of target peptide and receptor protein

[0117] Open the receptor protein and ligand molecule in Auto Dock. After the box appears, select the range of the pocket according to the box size so that the box can completely wrap the pocket. Select flexible ligand docking as the calculation mode and the maximum output conformation as the other settings. The docking product is automatically generated. The smaller the negative value of the binding energy of the output result, the stronger the predicted binding force between the receptor and the ligand, and the stronger its ligand activity.

[0118] The molecular docking of the heptapeptide HVPLPLP with the receptor protein IKK was successful. Figure 7 As shown, HVPLPLP binds to the four residues CYS-89, TYR-90, VAL-64 and LEU-63 of the receptor protein, forming a total of five hydrogen bonds with hydrogen bond lengths of 2.5 Å, 2.0 Å, 2.7 Å, 2.9 Å and 2.9 Å, respectively.

[0119] Example 4 Solid Phase Synthesis of HVPLPLP

[0120] Solid-phase synthesis of polypeptides is a common technique in the art. Those skilled in the art can synthesize the peptides themselves or entrust a chemical synthesis company to synthesize the peptides based on the amino acid sequence. This application entrusts a chemical synthesis company to synthesize HVPLPLP. After purification, the final product is obtained with a purity of more than 95%, and the structure is identified by MS (e.g. Figure 8 ), and obtain HVPLPLP polypeptide, which is used to determine the activity of subsequent polypeptides.

[0121] Example 5 Evaluation of the efficacy of the synthetic peptide HVPLPLP in improving immunity

[0122] 1. Cell Culture

[0123] Cell culture was the same as in 3.1 of Example 1;

[0124] 2. Experimental Methods

[0125] 2.1 RAW264.7 cell proliferation activity assay

[0126] The proliferation activity of RAW264.7 cells was measured using the CCK-8 assay. The experimental procedures were the same as those in Section 3.2 of Example 1, except for the differences in samples. The sample group consisted of 100 μL of fresh culture medium containing different concentrations of the heptapeptide HVPLPLP (100, 200, and 400 μg / mL). The positive control group consisted of 100 μL of culture medium containing 1 μg / mL lipopolysaccharide. The negative control group consisted of 100 μL of fresh culture medium added to the cell wells.

[0127] Cell viability refers to cell proliferation activity or cytotoxic activity. The effect of the synthetic peptide His-Val-Pro-Leu-Pro-Leu-Pro on the proliferation rate of RAW264.7 cells is as follows: Figure 9 As shown.*** P Indicates that the differences between each group and NC are extremely significant ( P <0.001), the same below. Figure 9 Results showed that all concentrations of the synthetic peptide promoted the proliferation of RAW264.7 cells. At concentrations of 100, 200, and 400 μg / mL, the cell proliferation rate increased with increasing peptide concentration and then stabilized. The most pronounced effect on cell proliferation was observed at 200 μg / mL, with a proliferation rate of 169.53%, significantly higher than the 126.71% proliferation rate in the LPS group. At 400 μg / mL, the proliferation rate reached 168.54%. Therefore, a concentration of 200 μg / mL of the synthetic peptide His-Val-Pro-Leu-Pro-Leu-Pro was selected for further investigation of its immunomodulatory mechanism.

[0128] 2.2 Determination of NO secretion in RAW264.7 cells

[0129] Except for the different sample configurations, the other experimental methods were the same as those in 3.3 of Example 1. The sample group consisted of 100 μL of fresh culture medium containing 200 μg / mL heptapeptide, the positive control group consisted of 100 μL of culture medium containing 1 μg / mL lipopolysaccharide, and the negative control group consisted of 100 μL of fresh culture medium added to the cell wells.

[0130] like Figure 10 The figure shows the effect of synthetic peptide HVPLPLP on NO secretion in RAW264.7 cells. Compared with the NC blank group, the synthetic peptide has a stimulating effect on RAW264.7 cells, which is extremely significant ( P <0.001) promoted the secretion of NO by cells, and the release amount was 25.10 μM, which had no significant difference with the LPS positive group (33.64 μM).

[0131] 2.3 Detection of phagocytic ability of RAW264.7 cells

[0132] Except for the different sample configurations, the other experimental methods were the same as those in 3.4 of Example 1. The sample group consisted of 100 μL of fresh culture medium containing 200 μg / mL heptapeptide, the positive control group consisted of 100 μL of culture medium containing 1 μg / mL lipopolysaccharide, and the negative control group consisted of 100 μL of fresh culture medium added to the cell wells.

[0133] Effects of synthetic peptides on the phagocytic ability of cells with neutral red Figure 11 As shown, compared with the NC blank group, the synthetic peptide HVPLPLP group and LPS group significantly improved the phagocytic ability of macrophages ( P<0.001), among which the phagocytic ability of HVPLPLP group was 142.47%, and the phagocytic ability of LPS group was 167.76%, indicating that the synthetic peptide HVPLPLP can activate macrophages, enhance the phagocytic ability of cells, and improve the immune response.

[0134] 2.4 Conclusion

[0135] In summary, the bioactivity of the synthetic peptide HVPLPLP was predicted using PeptideRanker, resulting in a predicted score of 0.704132, indicating good bioactivity. ToxinPred also predicted the synthetic peptide to be non-toxic and suitable for use in additives.

[0136] This application uses IKK protein as the receptor protein for molecular docking to explore the binding ability of HVPLPLP synthetic peptide to it in order to determine whether the active peptide can activate the NF-κB signaling pathway. The molecular docking of the heptapeptide HVPLPLP with the receptor protein IKK was successful, indicating that the HVPLPLP synthetic peptide may exert its immunomodulatory effect by activating the NF-κB signaling pathway. The results are as follows Figure 7 As shown, it binds to four residues of the receptor protein, CYS-89, TYR-90, VAL-64, and LEU-63, forming a total of five hydrogen bonds with lengths of 2.5 Å, 2.0 Å, 2.7 Å, 2.9 Å, and 2.9 Å, respectively. The binding forces between synthetic peptide ligands and IKK receptors are primarily ionic, hydrogen, van der Waals, and covalent. Hydrogen bonds are the strongest intermolecular forces and play a crucial role in molecular interactions. Generally speaking, shorter hydrogen bond lengths indicate a tighter binding between small peptides and amino acid residues, and thus a higher activity. Therefore, the synthetic peptide HVPLPLP exhibits excellent immunogenicity.

[0137] The heptapeptide HVPLPLP of the present invention has a hydrophobic amino acid ratio of 85.71%. Studies have shown that the proportion of hydrophobic amino acids affects the biological activity of the peptide segment. The higher the proportion of hydrophobic amino acids, the stronger the immunological activity of the peptide segment. Therefore, it can be inferred that HVPLPLP has better immunological activity.

[0138] The results of the efficacy evaluation of the synthetic peptide in improving immunity showed that the synthetic peptide HVPLPLP can increase the proliferation rate of RAW264.7 cells, the ability to phagocytize neutral red, and the release of NO, and the synthetic peptide has the ability to improve immunity.

[0139] Molecular docking results indicate that HVPLPLP exerts its immunomodulatory effects by activating the NF-κB signaling pathway. In vitro cellular immune activity further confirmed that HVPLPLP can induce macrophage proliferation, enhance phagocytic capacity, and increase NO secretion, demonstrating its immunomodulatory effects. In summary, HVPLPLP binds to IKK, activating the NF-κB pathway and exerting immunomodulatory effects, enhancing immune responses. This suggests that HVPLPLP has the potential to be developed as a food-derived bioactive peptide.

[0140] The above-described embodiments merely illustrate the implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that, without departing from the concept of this patent, a person skilled in the art would be able to make various variations, combinations, and improvements to the above-described embodiments, all of which fall within the scope of protection of this patent. Therefore, the scope of protection of this patent shall be determined by the claims.

Claims

1. A heptapeptide, characterized in that The heptapeptide is: HVPLPLP, and the amino acid sequence is: His-Val-Pro-Leu-Pro-Leu-Pro.

2. A composition, characterized in that The composition comprises the heptapeptide HVPLPLP according to claim 1.

3. The composition according to claim 2, wherein The preparation method is as follows: The F2-7 fraction was obtained by enzymatic hydrolysis of sturgeon cartilage, purification by Sephadex G-15 dextran gel chromatography, purification by DEAE-52 anion chromatography, and further purification by reverse-phase high-performance liquid chromatography. The preparation method of the F2-7 component is as follows: (1) Enzymatic hydrolysis: Weigh sturgeon cartilage → add water and boil → add alkaline protease and neutral protease after cooling → adjust pH to 9.0 → stir with a constant temperature magnetic stirrer, enzymatic hydrolysis → high temperature enzyme inactivation → cooling → centrifugation → vacuum concentration of the supernatant → freeze drying to obtain sturgeon cartilage enzymatic hydrolysate; (2) Purification by Sephadex G-15 dextran gel chromatography column: The sturgeon cartilage hydrolysate was purified by Sephadex G-15 dextran gel chromatography column, eluted with primary water, and the main absorption peak components were collected to obtain sturgeon cartilage peptide component 1; (3) Purification by DEAE-52 anion chromatography column: The first sturgeon cartilage peptide component was purified by DEAE-52 cellulose anion exchange chromatography column, and eluted with pure water and 0.1 mol / L sodium chloride solution in sequence, and the main absorption peak was collected to obtain the second sturgeon cartilage peptide component F2; (4) Purification by reversed-phase high-performance liquid chromatography: The sturgeon cartilage peptide component F2 was separated and purified by reversed-phase high-performance liquid chromatography, and the eluents were eluent A and eluent B, respectively. From 0 to 3 min, the volume percentage of eluent A was constant at 95%; from 3 to 10 min, the volume percentage of eluent A decreased from 95% to 80%; from 10 to 20 min, the volume percentage of eluent A decreased from 80% to 50%; from 20 to 23 min, the volume percentage of eluent A decreased from 50% to 20%; from 23 to 25 min, the volume percentage of eluent A increased from 20% to 95%; from 25 to 35 min, the volume percentage of eluent A was constant at 95%; the elution time was 35 min, the flow rate was 1 mL / min, the column temperature was 35 °C, and the peak values ​​were 280 nm and 220 nm. The detection was performed at dual wavelengths under 400 nm; the eluate with a peak time of 20.8 min to 22.6 min was collected, concentrated under reduced pressure, and freeze-dried in vacuum to obtain the sturgeon cartilage peptide target component F2-7, which contained the heptapeptide HVPLPLP; The eluent A is 0.1% trifluoroacetic acid-water; The eluent B is 0.1% trifluoroacetic acid-acetonitrile.

4. Use of the heptapeptide HVPLPLP according to claim 1 or the composition according to claim 2, characterized in that: The invention is used in the preparation of health care products for enhancing immune activity.

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