Pearl oyster active peptide with anti-inflammatory effect and application thereof

Abstract

The present invention discloses a pearl oyster active peptide with anti-inflammatory effect and its application, and relates to the technical field of active peptides. The amino acid sequence of the pearl oyster active peptide is FPGA. The pearl oyster active peptide with anti-inflammatory effect provided by the present invention can regulate the release of cellular inflammatory mediators, specifically, can reduce the release amount of NO, reduce the secretion of pro-inflammatory cytokines, and can also promote the secretion of anti-inflammatory cytokines, showing excellent anti-inflammatory activity, and can be used in the preparation of medicines, functional foods or cosmetics.

Description

Technical Field

[0001] The invention belongs to the technical field of active peptides, and more specifically, relates to a pearl oyster active peptide with anti-inflammatory effect and application thereof. Background Art

[0002] Inflammation is what we usually call "inflammation". It is a basic pathological process that occurs when biological tissues are stimulated by certain damaging factors such as trauma, infection, etc., which is mainly a defensive response. Existing commonly used anti-inflammatory drugs such as non-steroidal anti-inflammatory drugs, while exerting therapeutic effects, also produce some harmful reactions that are unrelated to the purpose of medication, namely adverse drug reactions. It is of great significance to find active ingredients with anti-inflammatory effects and few adverse reactions.

[0003] Pearls are natural raw materials for both medicine and food. They have the functions of regulating endocrine, clearing away heat and detoxifying, and promoting skin repair. The matrix protein obtained by hydrolysis of pearls also has multiple functions such as anti-oxidation, anti-inflammatory and anti-apoptosis. However, as a precious medicinal material, pearls have complex processing technology, low yield and high price. Pinctada martensii, also known as Pinctada martensii, has high yield and rich nutritious meat, which contains rich protein. However, the comprehensive utilization rate of by-products such as shell meat after pearl extraction is low, and most of them are discarded, resulting in waste of resources and environmental pollution.

[0004] The prior art discloses a method for preparing and applying a novel small molecule immunoactive peptide from marine shellfish. The patent points out that the polypeptide mixture obtained by enzymatic hydrolysis of marine shellfish (including pearl oysters) has antioxidant and other activities, but it does not study the active ingredients at the level of peptide sequences, let alone any research on the anti-inflammatory activity of active peptides derived from pearl oysters. Summary of the invention

[0005] The purpose of the present invention is to overcome the defect of insufficient research on the anti-inflammatory active components of pearl oyster meat, and provide a pearl oyster active peptide with anti-inflammatory effect. The active peptide can inhibit the activity of cyclooxygenase-2, regulate the secretion of inflammatory mediators by cells, including reducing the release of NO, reducing the secretion of pro-inflammatory cytokines, and promoting the secretion of anti-inflammatory cytokines, has anti-inflammatory effect, can be used to prepare anti-inflammatory additives, and is widely used in the fields of medicine, functional food or cosmetics.

[0006] Another object of the present invention is to provide a use of the pearl oyster active peptide in the preparation of an anti-inflammatory additive.

[0007] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0008] A pearl shell active peptide with anti-inflammatory effect, the active peptide having a sequence as shown in SEQ ID NO: 1.

[0009] The inventors of the present invention extracted a pearl oyster active peptide with anti-inflammatory effect from pearl oysters. The sequence of the pearl oyster active peptide is FPGA (Phe-Pro-Gly-Ala) and the molecular weight is 390.1903Da. The pearl oyster active peptide can occupy the key active site of cyclooxygenase-2, inhibit the activity of cyclooxygenase-2, and can also regulate the secretion of inflammatory mediators by cells, including reducing the release of NO, reducing the secretion of pro-inflammatory cytokines and promoting the secretion of anti-inflammatory cytokines. It has anti-inflammatory effect, can be used to prepare anti-inflammatory additives, and is widely used in the fields of medicine, functional food or cosmetics.

[0010] The present invention specifically protects the use of the pearl oyster active peptide in the preparation of an anti-inflammatory additive.

[0011] Specifically, the anti-inflammatory additive is used to prepare functional food.

[0012] Specifically, the anti-inflammatory additive is used to prepare cosmetics.

[0013] The active peptide provided by the invention is derived from pearl oyster meat, can regulate cytokines, has anti-inflammatory efficacy, and can play an anti-inflammatory role when used in cosmetics.

[0014] Specifically, the anti-inflammatory additive is used for preparing medicines.

[0015] Specifically, the drug is a drug that inhibits the activity of cyclooxygenase-2.

[0016] Prostaglandin endoperoxide synthase, commonly known as cyclooxygenase (COX), is mainly composed of cyclooxygenase-1 and cyclooxygenase-2 subtypes. It is a key enzyme in the biosynthetic pathway that induces the production of prostaglandins (PGs) and regulates inflammatory responses. Prostaglandins play an important role in inflammation. By inhibiting the activity of cyclooxygenase, especially the activity of cyclooxygenase-2, the expression of proinflammatory cytokines in the downstream of the inflammatory pathway can be reduced, interfering with the pathogenesis of inflammatory diseases, and effectively alleviating the symptoms of inflammatory diseases.

[0017] Cyclooxygenase-2 has three important active regions. The first region is the hydrophobic pocket, which is composed of Tyr 385, Trp 387, Phe 518, Ala 201, Tyr 248 and Leu 352 amino acid residues; the second region is the entrance of the active site, which is composed of hydrophilic amino acid residues Arg 120, Glu 524, Tyr 355; the third region is the side pocket composed of His 90, Arg 513 and Val523. The amino acid residues His 90, Arg 120, Gln 192, Val 349, Leu 352, Ser 353, Tyr 355, Leu 359, Tyr 385, Trp 387, Arg 513, Ala516, Phe518, Val523 523, Gly 526, Ala 527 and Leu 531 associated with the A chain of the cyclooxygenase-2 protein (the main region of the cyclooxygenase-2 active site) are all involved in the protein-ligand complementation activity. The pearl oyster active peptide (FPGA) provided by the present invention can form hydrogen bonds with the residues Thr206, His 207, and Tyr 385 of the cyclooxygenase-2 protein, and form hydrophobic bonds with Val 291 and Leu 294, wherein Tyr 385 is a characteristic amino acid residue in the hydrophobic pocket of cyclooxygenase-2. Therefore, the pearl oyster active peptide of the present invention can occupy the key area of ​​the active site of cyclooxygenase-2, inhibit the activity of cyclooxygenase-2, and thus inhibit the inflammatory response.

[0018] Specifically, the drug is a drug that reduces the amount of NO released from cells.

[0019] NO is an important intracellular and intercellular signaling molecule that participates in regulating a variety of physiological and pathological processes. Under normal conditions, NO can regulate physiological functions such as neurotransmission, vasodilation and immune response. However, excessive NO in cells is not conducive to relieving inflammatory responses and can damage cells and lead to cell apoptosis. The pearl oyster active peptide of the present invention can control the secretion of NO by cells, reduce the level of NO, and inhibit inflammatory responses.

[0020] Specifically, the drug is a drug that reduces the secretion of pro-inflammatory factors.

[0021] Specifically, the drug is a drug that promotes the secretion of anti-inflammatory factors.

[0022] Cellular inflammatory factors are important inflammatory mediators in the immune system. According to their roles in inflammatory responses, they can be divided into pro-inflammatory factors (TNF-α, IL-2, IL-6, IL-8, IL-1β and IFN-γ) and anti-inflammatory factors (IL-4, IL-10 and TGBβ). The pearl shell active peptides of the present invention can significantly reduce the secretion of pro-inflammatory cytokines TNF-α, IL-6 and IL-1β, while increasing the secretion of the anti-inflammatory cytokine IL-10.

[0023] Specifically, the anti-inflammatory additive is added in an amount of 0.0625 to 2.0 mg / mL in functional foods, cosmetics or medicines.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The pearl oyster active peptide with anti-inflammatory effect provided by the present invention can occupy the key active site of cyclooxygenase-2, inhibit the activity of cyclooxygenase-2, and can also regulate the secretion of inflammatory mediators by cells, including reducing the release amount of NO, reducing the secretion of pro-inflammatory cytokines, and promoting the secretion of anti-inflammatory cytokines. It has anti-inflammatory effect, can be used to prepare anti-inflammatory additives, and is widely used in the fields of medicine, functional food or cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the effect of pearl oyster meat hydrolysate, <3kDa fraction and ≥3kDa fraction on NO release from mouse macrophage RAW264.7;

[0027] Figure 2 This is the absorption wavelength spectrum of the component <3kDa separated and purified by RP-HPLC at 280nm;

[0028] Figure 3 This is a graph showing the effects of components F1, F2 and F3 on the survival rate of RAW264.7 macrophages induced by LPS;

[0029] Figure 4 This is a graph showing the effects of components F1, F2 and F3 on NO release from RAW264.7 macrophages induced by LPS;

[0030] Figure 5 It is the absorption wavelength spectrum of component F1 at 280nm.

[0031] Figure 6 is the total electron flow diagram of component F1.

[0032] Figure 7 3D (A) and 2D (B) images of the molecular docking model for the interaction between cyclooxygenase-2 and pearl oyster active peptides.

[0033] Figure 8 This is a graph showing the effect of pearl oyster active peptides on the survival rate of RAW264.7 cells.

[0034] Fig. 9 This is a graph showing the effects of different concentrations of pearl oyster active peptides on the NO release of RAW264.7 macrophages.

[0035] Fig.10 This is a graph showing the effect of pearl oyster active peptides on the release of TNF-α factor from RAW264.7 macrophages.

[0036] Fig.11 This is a graph showing the effect of pearl oyster active peptides on the release of IL-6 factor from RAW264.7 macrophages.

[0037] Fig.12 This is a graph showing the effect of pearl oyster active peptides on the release of IL-1β factor from RAW264.7 macrophages.

[0038] Fig.13 This is a graph showing the effect of pearl oyster active peptides on the release of IL-10 factor from RAW264.7 macrophages. DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with examples. These examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually carried out according to conventional conditions in the art or according to the conditions recommended by the manufacturer; the raw materials, reagents, etc. used, unless otherwise specified, are all raw materials and reagents that can be obtained from commercial channels such as conventional markets. Any non-substantial changes and substitutions made by those skilled in the art on the basis of the present invention belong to the scope of protection claimed by the present invention.

[0040] The specific process of evaluating the cell anti-inflammatory activity of each embodiment of the present invention is as follows:

[0041] Cell culture: Mouse RAW264.7 macrophages (purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences) were cultured in a 5% CO2, 37°C cell culture incubator using DMEM medium (purchased from Gibco) containing 10% FBS (purchased from Gibco, inactivated at 56°C for 30 min), and the cells were passaged when they grew to the logarithmic growth phase. When the cell plating rate was greater than 80% of the culture flask area, the original culture medium was aspirated, 2 mL of culture medium was aspirated with a pipette to blow the cells until they were completely detached, the cells were counted, the concentration of the cell suspension was adjusted according to the counting results, the plates were inoculated, and the cells were cultured in a constant temperature incubator.

[0042] Cytotoxicity test: Mouse RAW264.7 macrophages were used to evaluate the cytotoxicity of the samples. The concentration of RAW264.7 macrophages was adjusted to 1×10 4 / mL, 100 μL of cells were inoculated in a 96-well plate (6 replicates for each group) per well, incubated overnight, the old culture medium was discarded, a control group and a sample group were set up, 100 μL of complete culture medium containing the sample was added to the sample group, and an equal amount of complete culture medium was added to the blank group. After continued cultivation for 24 hours, the old culture medium was discarded, 100 μL of MTT solution (purchased from Sigma, 0.5 mg / mL) was added to each well and incubated in an incubator for 4 hours, and finally the MTT solution was discarded, 150 μL of DMSO (purchased from Sigma) was added to each well to dissolve the solid matter, and the crystals were fully dissolved by shaking for 10 minutes. The absorbance of each well at 490 nm was measured using an enzyme marker; the cell viability was expressed as a percentage of the control group. Compared with the control group, the sample concentration that reduced the cell viability by more than 10% was considered to be cytotoxic.

[0043] Determination of NO release: The concentration of RAW264.7 macrophages was adjusted to 5×10 4 / mL, 500μL per well was inoculated in a 24-well plate (3 replicates were set for each group). Incubate overnight at 37°C, 5% CO2. Dexamethasone (DEX, 1μg / mL, purchased from Sigma) was selected as the treatment control for the positive control group. 500μL of samples of different concentrations were added to the sample group, and the blank group and model group were added with an equal amount of complete medium (cell culture medium). After continuing to culture for 24h, the supernatant was aspirated. 500μL LPS (purchased from Sigma, 1μg / mL) was added to the model group, sample group and positive control group to stimulate the cells for 24h, and the blank group was added with an equal amount of complete medium. The cell culture supernatant was collected, and the NO content in the culture supernatant was measured by the Griess method (NO detection kit was purchased from Beyotime Biotechnology), which was the NO release amount.

[0044] Example 1 Enzymatic hydrolysis, ultrafiltration and separation and purification of pearl oyster meat

[0045] (1) Enzymatic hydrolysis: The pearl oyster meat was enzymatically hydrolyzed using a neutral protease, and the specific process was as follows: the pearl oyster meat (Pinctada martensii meat, purchased from Beihai Black Pearl Marine Biotechnology Co., Ltd.) was washed and drained, and crushed into a pearl oyster meat homogenate, deionized water was added at a solid-liquid ratio (mass ratio) of 1:1, and the pH was adjusted to 7.0; neutral protease (10 U / g) (purchased from Guangxi Nanning Pangbo Bioengineering Co., Ltd.) was added at an enzyme-substrate ratio of 0.31%, and enzymatic hydrolysis was performed at 46.3°C for 84 min; the enzyme was inactivated at 90°C for 10 min, then cooled to room temperature, centrifuged at 4000 r / min for 10 min, the supernatant was collected, and freeze-dried to obtain the pearl oyster meat enzymatic hydrolysate, which was stored at -20°C for subsequent analysis.

[0046] (2) Ultrafiltration: The pearl oyster meat hydrolysate was separated using an ultrafiltration tube with a molecular weight cutoff of 3 kDa. The mixture was centrifuged at 4000 r / min for 30 min to collect the components with molecular weights <3 kDa and ≥3 kDa. The components were freeze-dried in vacuum and stored at -20°C.

[0047] Pearl oyster meat hydrolysate, <3 kDa fraction and ≥3 kDa fraction were used as samples to evaluate their cellular anti-inflammatory activity.

[0048] The results of the evaluation of the cellular anti-inflammatory activity of pearl oyster meat hydrolysate, <3 kDa fraction and ≥3 kDa fraction are as follows Figure 1 As shown, Figure 1 In the data, ### indicates P<0.001 compared with the blank group; * and *** indicate P<0.05, P<0.01 and P<0.001 compared with the model group, respectively. Figure 1 It can be seen that compared with the model group, the NO release of the positive control group (DEX, 1μg / ml) decreased by 44.84%; at a concentration of 2.0mg / mL, pearl oyster meat hydrolysate, ≥3kDa components and <3kDa components all had a significant inhibitory effect on the release of NO (P<0.001), and their NO release decreased by 70.00±0.69%, 62.60±1.98%, and 74.08±1.83% (P<0.001), respectively. It can be seen that the <3kDa component has the strongest anti-inflammatory activity, and it is separated and purified in the next step.

[0049] (3) Separation and purification: Components <3 kDa were separated and purified using high performance liquid chromatography (RP-HPLC). The specific process is as follows:

[0050] First, the components <3 kDa were filtered through a 0.45 μm filter membrane and then separated using a C18 self-assembled chromatographic column (10 μm, 10 mm×250 mm) preparative reversed-phase high performance liquid chromatography; wherein the chromatographic conditions were: mobile phase A: 0.1% trifluoroacetic acid aqueous solution; mobile phase B: 0.1% trifluoroacetic acid methanol solution; injection volume: 3 mL; elution flow rate: 10 mL / min; detection wavelength: 280 nm; elution program is as shown in Table 1:

[0051] Table 1. Elution program

[0052] time Mobile phase 0-45min 5~10% mobile phase B 45-65min 10~20% mobile phase B 65-75min 20~50% mobile phase B 75-80min 50~95% mobile phase B 80-85min 95~5% mobile phase B

[0053] Collect each elution peak, use rotary evaporation to concentrate, and score components F1, F2 and F3. The HPLC chromatogram is as follows: Figure 2 As shown, the cell anti-inflammatory activity was evaluated after concentration and freeze drying. Components F1, F2 and F3 were used as samples, and the cell anti-inflammatory activity of components F1, F2 and F3 was evaluated according to the above method. The results are shown in Figure 3 and Figure 4 shown.

[0054] Figure 3 This is a graph showing the effects of components F1, F2 and F3 on the survival rate of RAW264.7 macrophages induced by LPS; Figure 4 Figure 2 is the effect of components F1, F2 and F3 on the NO release of RAW264.7 macrophages induced by LPS; ### indicates P<0.001 compared with the blank group; ** and *** indicate P<0.01 and P<0.001 compared with the model group, respectively; Figure 3 It can be seen that components F1, F2 and F3 have no toxic side effects on RAW264.7 macrophages within the range of 0-2.0 mg / mL. Figure 4 It can be seen that the NO release of RAW264.7 macrophages induced by LPS in the model group increased significantly (P<0.001). As the concentrations of components F1, F2 and F3 gradually increased, their inhibitory effects on NO release of RAW264.7 macrophages gradually increased. Compared with the NO release of RAW264.7 cells in the model group, at a concentration of 2.0 mg / mL, the NO release of RAW264.7 macrophages in the components F1, F2 and F3 treatment groups decreased by 58.98%, 50.76% and 53.45%, respectively (P<0.001). It can be seen that component F1 has the strongest inhibitory effect on NO secretion by RAW264.7 cells, and component F1 was selected for further separation and purification.

[0055] Component F1 was subjected to secondary separation, wherein the chromatographic conditions were: C18 column (5 μm, 4.6 mm×200 mm); mobile phase A: 0.1% trifluoroacetic acid aqueous solution; mobile phase B: 0.1% trifluoroacetic acid methanol solution; injection volume: 1 mL; elution flow rate: 10 mL / min; detection wavelength: 280 nm; the elution program is as shown in Table 2:

[0056] Table 2. Elution program

[0057] time Mobile phase 0-45min 5~10% mobile phase B 45-50min 10~20% mobile phase B 50-55min 20~95% mobile phase B 55-70min 95~5% mobile phase B

[0058] The F1 component is further separated and purified to have only one absorption peak, such as Figure 5 Therefore, component F1 was used for subsequent peptide composition and amino acid sequence analysis.

[0059] Example 2 Structural identification, molecular docking screening and synthesis of pearl oyster active peptides

[0060] 2.1 Structural identification: Component F1 was dissolved in deionized water with 0.1% formic acid, desalted in a C18 desalting column (AcclaimPepMap 100, 75 μm×2 cm), and then analyzed for peptide composition and amino acid sequence by LC-MS / MS with an online nanospray ion source. Chromatographic conditions: C18 column (Acclaim PepMap, 75 μm×25 cm); column flow rate controlled at 300 nL / min, column temperature at 40°C, injection volume: 3 μL; electrospray voltage 2 kV. Mobile phase A is 0.1% formic acid aqueous solution, and mobile phase B is 0.1% formic acid in acetonitrile solution; elution program: 0-3min, 2-6% mobile phase B; 3-42min, 6-20% mobile phase B; 42-47min, 20-35% mobile phase B; 47-48min, 35-100% mobile phase B; 48-60min, 100% mobile phase B. The mass spectrometer was operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition, and the mass spectrometry parameters were set as follows: ① primary mass spectrometry scan range (m / z): 350-1800; resolution: 70000; automatic gain control target: 3e6; maximum injection time: 50ms; scan charge: 2-6; ② HCD-MS / MS, resolution: 17500; isolation window: 2m / z; automatic gain control target: 1e5; maximum injection time: 45ms; collision energy: 28; dynamic exclusion time: 30s. Tandem mass spectra were analyzed using De Novo software (PEAKS Studio X+). The uniprot-Mytilus coruscus database was searched through PEAKS DB, and None enzyme digestion was set. Search parameters: fragment ion mass tolerance: 0.02Da; parent ion mass tolerance: 7ppm; maximum missed cuts: 2; variable modification: Oxidation (M) 15.99, Deamidation (NQ) 0.98; protein card value: -10lgP ≥ 0, at least 1 unique peptide; peptide card value: -10lgP ≥ 15. The identification results are as follows Figure 6 As shown, more than 260 peptides with different molecular masses were identified from component F1. The identified peptides were all short peptides with 3 to 10 amino acid residues, and the molecular weights ranged from 349.167 to 1102.58.

[0061] 2.2 Molecular docking screening: First, the 3D structure of cyclooxygenase-2 (COX-2, PDB ID: 1CX2) was preprocessed and optimized, and output as a mol2 format file for subsequent molecular docking. The 3D structure of cyclooxygenase-2 was downloaded from the Protein Data Bank (PDB) (https: / / www1.rcsb.org / ); PyMol software was used to remove the repeated chains, water molecules and original ligands in the receptor protein molecule and save them as PDB format files; then Autodock Tools 1.5.6 software was used to add polar hydrogen atoms to the receptor protein, calculate the gasteiger charge and set the rotation bonds, and save them as PDBQT format files; the 2D structure of the ligand was drawn by Marvin Sketch software, optimized for energy minimization, and the 3D structure was constructed and saved as PDBQT format files; AutoDock Vina 1.1.2 was used to perform molecular simulation flexible docking on the above receptors and ligands. The docking results were expressed as binding energy values, and the docking parameters were as follows: the following amino acid residues in the receptor protein were set to be able to flexibly transform conformations, His 90, Arg 120, Gln 192, Val 349, Leu 352, Ser 353, Tyr 355, Tyr 385, Trp 387, Arg 513, Ala 516, Phe 518, Val 523, Gly 526, Ala 527, Leu351. The center position of the grid box was set to: center_x = 28.684, center_y = 28.603, center_z = 9.285, the box size was 40 × 40 × 40, and the other parameters were all default values; Finally, the molecular docking results were visualized and analyzed using Discovery Studio 4.5 software.

[0062] By setting the confidence level (-10lgP) ≥ 36.0, a total of 70 peptides were screened for molecular simulation docking experiments to study their effects on the interaction with cyclooxygenase-2. Each peptide was subjected to molecular docking simulation analysis with the cyclooxygenase-2 receptor protein. Each peptide was repeated 10 times, and the binding energy results were averaged. The peptides with lower binding energy with the cyclooxygenase-2 receptor protein in molecular docking were selected to further analyze their anti-inflammatory mechanism.

[0063] Among them, the pearl oyster active peptide (FPGA, Phe-Pro-Gly-Ala) has a lower binding energy, as shown in Table 3, indicating that the pearl oyster active peptide can spontaneously and stably bind to the key active site in the cyclooxygenase-2 receptor protein.

[0064] Table 3. Molecular simulation docking results of pearl oyster active peptides and cyclooxygenase-2 receptor protein

[0065]

[0066] Table 4. Binding energy and interaction sites of cyclooxygenase-2 and active peptides based on molecular docking simulation

[0067]

[0068] Figure 7 The 3D (A) and 2D (B) diagrams of the molecular docking model for the interaction between cyclooxygenase-2 and pearl oyster active peptides. Figure 7 As shown in Table 4, the pearl oyster active peptide (FPGA) can form hydrogen bonds with the residues Thr 206, His 207, and Tyr 385 of the cyclooxygenase-2 protein, and form hydrophobic bonds with Val 291 and Leu 294, among which the pearl oyster active peptide forms a hydrogen bond with the characteristic amino acid residue Tyr 385 in the hydrophobic pocket of cyclooxygenase-2. It can be seen that the pearl oyster active peptide binds to different amino acid residues in the active site of cyclooxygenase-2, and the interaction in different active regions can affect the activity of cyclooxygenase-2 protein, inhibit its catalytic activity, and induce anti-inflammatory function.

[0069] 2.3 Synthesis: Pearl oyster active peptides were synthesized by solid phase synthesis. The pearl oyster active peptides were synthesized by Jie Peptide Biotechnology Co., Ltd. with a purity of more than 95% and were used for further research on their anti-inflammatory activity.

[0070] Example 3

[0071] Pearl oyster active peptides were used as samples to evaluate their cellular anti-inflammatory activity and determine the NO release from RAW264.7 macrophages. At the same time, the content of cellular inflammatory factors (TNF-α, IL-6, IL-1β and IL-10) in the culture supernatant was measured by quantitative enzyme-linked immunosorbent assay (ELISA) (the kit was purchased from Xinbosheng Biotechnology Co., Ltd.).

[0072] Cytotoxicity test evaluation:

[0073] The cytotoxicity of pearl oyster active peptide (FPGA) on RAW264.7 macrophages at concentrations of 0.0625-2.0 mg / mL was evaluated by MTT method. Figure 8 As shown, after treating RAW264.7 macrophages for 24 hours, the active peptide did not produce significant cytotoxicity and contributed to the proliferation of RAW264.7 macrophages. Therefore, the pearl shell active peptide did not produce cytotoxicity even at a high concentration of 2.0 mg / mL. The NO release amount can be determined in the concentration range of 1 to 200 μg / mL in the future.

[0074] Determination of NO release from RAW264.7 macrophages:

[0075] Depend on Fig. 9 The results of the cell test showed that the NO release of RAW264.7 macrophages in the model group increased significantly after LPS stimulation (P<0.001). Compared with the model group, the positive control group could significantly inhibit the NO release of RAW264.7 macrophages, and the NO release decreased by 40.85±5.55% (P<0.001); the pearl shell active peptide treatment group could significantly inhibit the NO release of RAW264.7 macrophages at different concentrations, and all were concentration-dependent; at a concentration of 200μg / mL, the NO release of the active peptide treatment group decreased by 36.47±1.97% (P<0.001). The experimental results show that active peptides can effectively inhibit the NO release of RAW264.7 macrophages induced by LPS.

[0076] Determination of cytokines in RAW264.7 macrophages:

[0077] Fig.10 This is the effect of pearl oyster active peptide on the release of TNF-α factor in RAW264.7 macrophages; Fig.11 This is the effect of pearl oyster active peptide on the release of IL-6 factor in RAW264.7 macrophages; Fig.12 This is the effect of pearl oyster active peptide on the release of IL-1β factor in RAW264.7 macrophages; Fig.13 This is a graph showing the effect of pearl oyster active peptides on the release of IL-10 factor in RAW264.7 macrophages. Figure 10 to Figure 13 It can be seen that compared with the blank control group, under LPS stimulation, the expression of inflammatory factors TNF-α ( Fig.10 )、IL-6( Fig.11 ), IL-1β( Fig.12 ) and IL-10( Fig.13) release significantly increased. Compared with the model group, the positive control group can significantly reduce the release of proinflammatory factors TNF-α, IL-6, and IL-1β, that is, DEX treatment can significantly inhibit the secretion of TNF-α, IL-6, and IL-1β inflammatory mediators by RAW264.7 macrophages stimulated by LPS (P<0.001), indicating that the cell inflammation model used in the present invention can be used as an effective evaluation method for anti-inflammatory substances. The active peptide treatment group can not only significantly reduce the secretion of proinflammatory cytokines TNF-α, IL-6, and IL-1β by RAW264.7 macrophages, but also show dose-dependent effects at concentrations of 1 to 200 μg / mL. When the concentration reaches 200 μg / mL, it can also significantly increase the secretion of anti-inflammatory factor IL-10. The effect on the secretion of cellular inflammatory factors at the highest concentration of 200 μg / mL is shown in Table 5.

[0078] Table 5. Effects of each sample group on inflammatory factors of RAW264.7 macrophages (P<0.001)

[0079]

[0080] Through Examples 2 to 3, it is shown that the pearl oyster active peptide of the present invention can bind to the active site of cyclooxygenase-2, inhibit the activity of cyclooxygenase-2, and at the same time reduce the levels of NO and pro-inflammatory factors, promote the secretion of anti-inflammatory factors, thereby effectively regulating the secretion of inflammatory factors of RAW264.7 macrophages, and has excellent anti-inflammatory activity.

[0081] Finally, it should be pointed out that the above embodiments are only representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content of the present invention by a person skilled in the art should be considered as the protection scope of the claims of the present invention.

Claims

1. An application of pearl oyster active peptide in the preparation of anti-inflammatory additives, characterized in that: The active peptide is the sequence shown in SEQ ID NO: 1.

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