Anti-inflammatory polypeptide nanoparticles and preparation method thereof

Abstract

The invention discloses anti-inflammatory polypeptide nanoparticles and a preparation method thereof, and belongs to the technical field of bioactive peptides. The anti-inflammatory polypeptide nano-particles are nano-particles obtained by coating polypeptide with an anti-inflammatory effect with chitosan and sodium tripolyphosphate, the polypeptide with the anti-inflammatory effect contains bioactive peptides of which the amino acid sequences are YAADQ, FPYTT and YVTYA. The phycocyanin anti-inflammatory peptide is natural in source, the performance of the phycocyanin anti-inflammatory peptide can be remarkably improved through the nanometer embedding technology, and compared with traditional drugs, the phycocyanin anti-inflammatory peptide has lower toxic and side effects and potential cost advantages. The polypeptide nanoparticles prepared by the invention can effectively prevent the phycocyanin anti-inflammatory peptide from being degraded in the digestive tract, have a remarkable treatment effect on mice with ulcerative colitis, and have an important application prospect in preparation of ulcerative colitis prevention and treatment products.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide technology, specifically relating to polypeptide nanoparticles for anti-inflammatory purposes and their preparation methods. Background Technology

[0002] Ulcerative colitis (UC) is a chronic, nonspecific inflammatory bowel disease that primarily affects the mucosa and submucosa of the colon and rectum. Clinical manifestations include recurrent diarrhea, mucus and bloody stools, abdominal pain, and systemic symptoms.

[0003] Current clinical treatment of UC mainly relies on drugs such as aminosalicylic acid preparations, glucocorticoids, immunosuppressants, and biologics. Although these therapies can relieve symptoms, they have significant limitations: aminosalicylic acid preparations have limited efficacy in moderate to severe patients, and long-term use may cause gastrointestinal adverse reactions; while glucocorticoids can rapidly control inflammation, they can lead to systemic side effects such as osteoporosis and increased risk of infection; biologics (such as anti-TNF-α monoclonal antibodies) have strong targeting, but are expensive and some patients are prone to developing drug resistance. Therefore, developing safe, effective, and low-cost novel treatment strategies has become an urgent need for UC research.

[0004] Phycocyanin (PC) is a natural pigment protein extracted from spirulina, possessing various biological activities such as antioxidant, anti-inflammatory, and immunomodulatory effects. Studies have shown that its enzymatic hydrolysis products—phycocyanin peptides (PCPs)—not only retain the functional properties of the parent protein but may also exhibit stronger biological activity and higher absorption efficiency due to their reduced molecular weight. They show significant therapeutic potential in animal models of ulcerative colitis and hepatitis. However, the clinical application of phycocyanin peptides faces challenges: natural peptides are easily degraded by the gastrointestinal environment, resulting in poor bioavailability. Developing nanodelivery technologies suitable for phycocyanin peptides can protect them from damage by gastric acid and digestive enzymes while enhancing the absorption efficiency of small intestinal epithelial cells, thereby significantly improving the in vivo stability and targeting of the peptides. Summary of the Invention

[0005] In view of the problems existing in the prior art, the purpose of this invention is to provide peptide nanoparticles for anti-inflammatory purposes and a method for preparing the same.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A bioactive peptide with anti-inflammatory effects, wherein the amino acid sequence of the bioactive peptide is shown in SEQ ID NO:1~SEQ ID NO:3.

[0007] The above-mentioned bioactive peptides with anti-inflammatory effects are used in the preparation of products with anti-inflammatory effects.

[0008] A bioactive peptide preparation with anti-inflammatory effects, wherein the active ingredient is at least one of the bioactive peptides YAADQ, FPYTT, and YVTYA.

[0009] Based on the above scheme, the concentration of the active ingredient is 1 mg / mL.

[0010] Anti-inflammatory peptide nanoparticles are obtained by coating peptides with anti-inflammatory effects with chitosan and sodium tripolyphosphate. The anti-inflammatory polypeptide contains bioactive peptides with the amino acid sequences YAADQ, FPYTT, and YVTYA.

[0011] Based on the above scheme, the method for preparing the peptide with anti-inflammatory effect is as follows: Distilled water was added to phycocyanin at a ratio of 1:20 to dissolve it, and the mixture was sonicated at 450 W for 30 min. Then, 2000 U / g of alkaline protease was added and the mixture was enzymatically hydrolyzed at pH 10.5 and 50℃ for 6 h. After the enzymatic hydrolysis was completed, the hydrolysate was boiled at 100℃ for 10 min to terminate the reaction. The hydrolysate was then centrifuged at 6000 r / min and 4℃ for 15 min, and the supernatant was retained and freeze-dried to obtain the final product.

[0012] Based on the above scheme, the concentration of the peptide with anti-inflammatory effect is 1 mg / mL.

[0013] The preparation method of the above-mentioned anti-inflammatory peptide nanoparticles includes the following steps: (1) Chitosan with a molecular weight of 30kDa~200kDa was dissolved in an acetic acid solution with a mass fraction of 1% w / v and stirred overnight to obtain a chitosan solution with a concentration of 0.2 mg / mL~2 mg / mL; (2) Adjust the pH of the chitosan solution to 2-6 with 1 mol / L NaOH, and add a polypeptide with anti-inflammatory effect at a concentration of 1 mg / mL at a uniform rate. The amount of polypeptide added is 5% of the volume of the chitosan solution. (3) Then, under magnetic stirring, a sodium tripolyphosphate solution with a concentration of 0.8 mg / mL is added dropwise at a mass ratio of chitosan to sodium tripolyphosphate of 7:1 to 3:1. The pH is adjusted before and after to keep it consistent. The mixture is then magnetically stirred for 30 min at room temperature to obtain the final product.

[0014] Based on the above scheme, in step (1), the molecular weight of chitosan is 50 kDa and the concentration of chitosan solution is 0.5 mg / mL.

[0015] Based on the above scheme, the pH value in step (2) is adjusted to 3; in step (3), the mass ratio of chitosan to sodium tripolyphosphate is 4:1. Advantages of the technical solution of this invention: This invention employs protease to enzymatically hydrolyze phycocyanin, and separates and purifies peptides of different molecular weights using techniques such as ultrafiltration, gel chromatography, and reverse high-performance liquid chromatography (RP-HPLC). Furthermore, an anti-inflammatory peptide with significant NO-inhibiting activity was screened using a lipopolysaccharide (LPS)-induced macrophage (RAW264.7) inflammation model. However, natural bioactive peptides are easily degraded by gastrointestinal enzymes and have low bioavailability, limiting their clinical application.

[0016] This invention utilizes chitosan and sodium tripolyphosphate to prepare nanoparticles for encapsulating phycocyanin anti-inflammatory peptides. Phycocyanin anti-inflammatory peptides are naturally derived, and nano-encapsulation technology can significantly enhance their performance, offering lower toxicity and potential cost advantages compared to traditional drugs. The phycocyanin anti-inflammatory peptide nanoparticles prepared in this invention effectively prevent the degradation of phycocyanin anti-inflammatory peptides in the digestive tract, significantly reduce fecal bleeding in mice with ulcerative colitis, slow weight loss, significantly reduce serum levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β, and increase the level of the anti-inflammatory cytokine IL-10. This has significant application prospects in the preparation of products for the prevention and treatment of ulcerative colitis. Furthermore, the industrialization of the phycocyanin anti-inflammatory peptide nano-encapsulation system will promote the development of related industries such as algal resource development and nanomaterial preparation, creating new economic growth points and achieving a win-win situation for both social and economic benefits. Attached Figure Description

[0017] Figure 1 Effects of ultrafiltration components of different molecular weights on NO production in LPS-stimulated RAW264.7 cells; Figure 2 Superdex™ Increase 10 / 300 GL gel chromatograms of components with a molecular weight <3 kDa; Figure 3 Effects of three purified components on NO production in LPS-stimulated RAW264.7 cells; Figure 4 This is the second-order mass spectrum of YAADQ; Figure 5 The mass spectrum of FPYTT is shown. Figure 6 This is the secondary mass spectrum of YVTYA; Figure 7 Effects of synthetic peptides on the viability of RAW264.7 cells; Figure 8 Effects of synthetic peptides on NO release in RAW264.7 cells; Figure 9 The effects of protease type and ultrasonic treatment on the degree of hydrolysis of phycocyanin; Figure 10 Effects of phycocyanin hydrolysates obtained from different proteases on the viability of RAW264.7 cells; Figure 11 Effects of phycocyanin hydrolysates obtained from different proteases on LPS-induced RAW264.7 cytotoxicity; Figure 12 Effects of phycocyanin hydrolysates obtained from different proteases on LPS-induced NO release from RAW264.7; Figure 13 The effect of chitosan molecular weight on nanoparticle size and PDI value; Figure 14 The effect of chitosan concentration on nanoparticle size, PDI value and potential; Figure 15 Effect of chitosan to sodium tripolyphosphate mass ratio on nanoparticles; Figure 16 The effect of chitosan solution pH on nanoparticles; Figure 17 Effects of chitosan-sodium tripolyphosphate nanoparticles prepared by different methods on the encapsulation efficiency and embedding ability of phycocyanin peptides (where a is the molecular weight of chitosan, b is the concentration of chitosan, c is the mass ratio of chitosan to sodium tripolyphosphate, and d is the pH of the chitosan solution). Figure 18 The effect of PCP-NPs on NO production in LPS-stimulated RAW264.7 cells; Figure 19 Before and after encapsulation, the results of in vitro gastrointestinal simulated digestion of chitosan-sodium tripolyphosphate nanoparticles and phycocyanin peptides (where SGF represents simulated gastric juice and SIF represents simulated intestinal juice). Figure 20 Blood in the stool of mice in each treatment group; Figure 21 Daily weight changes of mice in each group; Figure 22 Daily assessment of DAI scores for each group; Figure 23 Colon length in each group of mice; Figure 24 The serum levels of IL-1β (A), IL-6 (B), TNF-α (C), and IL-10 (D) in mice of each group. Detailed Implementation

[0018] The terminology used in this invention, unless otherwise specified, generally has the meanings commonly understood by those skilled in the art. The invention is further described in detail below with reference to specific embodiments and data. The following embodiments are merely illustrative and are not intended to limit the scope of the invention in any way.

[0019] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the experimental materials, reagents, and chemicals used in the following embodiments can be obtained through general channels.

[0020] In the following embodiments, 1. Determination of degree of hydrolysis (DH) The degree of hydrolysis of phycocyanin was determined using the o-phthalaldehyde (OPA) method. The specific procedure is as follows: 80 mg of OPA was dissolved in 2 mL of anhydrous ethanol, 200 μL of β-mercaptoethanol, 5 mL of 10% SDS (sodium dodecyl sulfate) (w / v), and 92.8 mL of 0.1 M sodium tetraborate to prepare a 100 mL OPA reagent solution. 4 mL of each of the phycocyanin hydrolysates (alkaline protease, papain, trypsin, pepsin, neutral protease, and acidic protease hydrolysates) was mixed with 4 mL of OPA reagent, incubated at room temperature for 2 min, and the absorbance was measured at 340 nm.

[0021] Phycocyanin was placed in 6M HCl and reacted at 4℃ for 24h. The number of free amino acids was determined using a serine standard curve, which was taken as the number of free amino acids produced by the complete hydrolysis of phycocyanin.

[0022] The degree of hydrolysis of phycocyanin is calculated using the following formula: Degree of hydrolysis (%) = [(NH2)] t -(NH2) t0 ] / (NH2) T In the formula: (NH2) t This indicates the amount of free amino acids in the enzymatic hydrolysis product at time t; (NH2) t0 Indicates the amount of free amino acids that have not undergone enzymatic hydrolysis; (NH2) T This indicates the number of free amino acids after complete hydrolysis.

[0023] 2. Culture of RAW264.7 cells Mouse macrophages RAW264.7 were cultured in DMEM high-glucose complete medium (supplemented with 10% fetal bovine serum and 0.1 mg / mL streptomycin and 0.1 mg / mL penicillin). Cells were cultured in a constant temperature incubator at 37°C with 95% humidity and 5% carbon dioxide until the logarithmic growth phase was reached for experimental use.

[0024] 3. Measurement of cell viability Cell viability was determined using CCK-8 assay. Cells in logarithmic growth phase were detached by trypsin digestion and diluted to a density of 5 × 10⁶ cells / mL using DEME high-glucose complete medium. 4 Cells were seeded at a density of 100 μL / mL into 96-well plates. After standing for about 5 minutes to allow cells to settle, the culture flasks were placed in a cell culture incubator overnight. After cell attachment (12–24 hours), the old culture medium was discarded, and the 96-well plates were gently washed twice with PBS buffer at 37°C. 100 μL of phycocyanin hydrolysate or phycocyanin peptides at different concentrations (0, 1, 5, 10, 15, 20 mg / mL) were added, and the plates were cultured for 24 h. After 24 h, 10 μL of CCK-8 solution was added to each well, and the plates were incubated again for 1 h. The absorbance at 450 nm (OD450) was measured, and cell viability was calculated using the following formula: Cell viability (%) = [A (加药) -A (空白) ] / [A (0加药) -A (空白) ]×100 In the formula: A （加药） : Absorbance of pores containing cells, CCK-8 solution, and drug solution; A （空白） : Absorbance of pores containing culture medium and CCK-8 solution but without cells; A （0加药） : Absorbance of pores containing cells and CCK-8 solution but no drug solution.

[0025] 4. Determination of nitric oxide (NO) content NO content was detected using the Griess method. Cells in the logarithmic growth phase were subjected to a reaction at a concentration of 5 × 10⁻⁶ cells / mL. 4Cells were seeded at a density of 100 μL per well in 96-well plates. The plates were incubated overnight. After cell attachment (usually 12–24 h), the old culture medium was discarded. Then, 100 μL of different concentrations of LPS and phycocyanin digest (or phycocyanin peptide) were added to each well and incubated for the specified time. After incubation, the cell culture supernatant was collected, and the NO content was determined using a NO detection kit. In short, Griess Reagent I and II were first brought to room temperature. A NO standard gradient was prepared using appropriate dilutions according to the sample type. 50 μL of the standard and the treated sample were added to each well of the 96-well plate. Then, 50 μL of Griess Reagent I and II were added sequentially, mixed, and the absorbance was measured at 540 nm. A standard curve (0–100 µmol / L) was plotted using sodium nitrite (NaNO2) standards, and the NO content was calculated based on the standard curve.

[0026] 5. Methods for establishing cellular inflammation models Lipopolysaccharide (LPS) can activate immune cells such as macrophages and induce the release of inflammatory factors (such as TNF-α, IL-6, and IL-1β), making it a classic method for establishing cellular inflammation models. Logarithmically growing RAW264.7 cells were seeded in 96-well plates and cultured at 37°C with 5% CO2 for 12–24 hours until adherence. The old culture medium was discarded, and 1 μg / mL LPS was added, followed by further culturing for 12–24 hours to obtain the cellular inflammation model group (LPS treatment only).

[0027] The phycocyanin used in the following examples was purchased from Zhejiang Binmei Biotechnology Co., Ltd.

[0028] Example 1 The method for extracting bioactive peptides with anti-inflammatory effects from phycocyanin includes the following steps: (1) Distilled water was added to phycocyanin at a ratio of 1:20 (w / v) to dissolve it, and the mixture was sonicated at 450 W for 30 min. Then, 2000 U / g of alkaline protease was added and the mixture was enzymatically hydrolyzed at pH 10.5 and 50℃ for 6 h. After the enzymatic hydrolysis was completed, the hydrolysate was boiled at 100℃ for 10 min to terminate the reaction. Then, the hydrolysate was centrifuged at 6000 r / min and 4℃ for 15 min, and the supernatant was retained.

[0029] (2) The prepared phycocyanin peptides (PCP) were separated by ultrafiltration. The phycocyanin peptides were fractionated sequentially using ultrafiltration centrifuge tubes with molecular weight cutoffs of 10 kDa and 3 kDa (15 mL per ultrafiltration cycle). The ultrafiltration conditions were 6000 g / min, centrifugation at 4℃ for 20 min. Three fractions were obtained by ultrafiltration separation of PCP (PCP-Ⅰ: <3 kDa, PCP-Ⅱ: 3-10 kDa, and PCP-Ⅲ: >10 kDa). Each fraction was freeze-dried and its anti-inflammatory activity was analyzed. The fraction with the highest activity was freeze-dried again and stored for further purification.

[0030] The effects of ultrafiltration components on NO production in LPS-stimulated RAW264.7 cells are as follows: Figure 1 As shown, compared with the control group, LPS-stimulated RAW264.7 macrophages exhibited a significant increase in NO release, indicating the successful establishment of the LPS-induced inflammation model. Compared with the model group, the phycocyanin peptides of the three components—PCPs-I (MW < 3kDa), PCPs-II (3 < MW < 10 kDa), and PCPs-III (MW > 10 kDa)—all reduced NO release. Among them, PCPs-I (MW < 3kDa) showed the strongest anti-inflammatory activity, with an 81.9% reduction in anti-inflammatory indicators compared to the model group (p < 0.05). The smaller the molecular weight of the ultrafiltration component, the less NO was produced, indicating stronger anti-inflammatory activity. Therefore, PCPs-I was selected for further purification.

[0031] (3) The <3 kDa fraction was further purified on a Superdex™ Increase 10 / 300 GL gel permeation column (10 × 300 mm). The column was eluted with deionized water (pH 10), and the fractions were collected at a flow rate of 1 mL / min. Three fractions were detected at 280 nm. Figure 2 Further investigation was conducted to examine the effects of the three components on NO production in LPS-stimulated RAW264.7 cells, and the results are as follows: Figure 3 As shown, compared with the control group, the NO release of RAW264.7 macrophages stimulated by LPS was significantly increased ( Figure 3 The presence of the model in the data indicates that the LPS-induced inflammation model was successfully established. Compared with the model group, the phycocyanin peptides of the three components (F1, F2, and F3) all reduced NO release, indicating that they all have certain anti-inflammatory effects. Among them, the anti-inflammatory index of component F3 was significantly reduced by 84.0% compared with the model group, making it the most effective component in exerting anti-inflammatory activity. Therefore, component F3 was selected for mass spectrometry identification.

[0032] (4) The peptide sequences in fraction F3, which has high anti-inflammatory potential, were identified using LC-MS / MS. The LC conditions were: C18 column (75 μm × 150 mm, 3 μm), flow rate 300 nL / min. Mobile phase A consisted of 0.1% formic acid and 2% ACN; mobile phase B consisted of 0.1% formic acid and 80% ACN; elution was performed with 6–9% B for 8 min, 9–14% B for 14 min, 14–30% B for 36 min, 30–40% B for 15 min, 40–95% B for 3 min, and 95% B hold for 5 min. The mass spectrometry conditions were: MS / MS scan range (m / z) 100–1500, AGC target: 3e6; resolution: 70000, etc. Mass spectrometry data were retrieved from the Uniport database using MaxQuant software, yielding eight peptides.

[0033] (5) Bioinformatics analysis was performed on the identified peptide sequences to determine their potential anti-inflammatory potential: ① Sequences with <10 amino acid residues were screened; ② The BIOPEP database was used to check whether the peptide sequences had been validated; ③ PeptideRanker was used to predict the potential biological activity of the identified peptide sequences, and peptides with a prediction score > 0.5 were screened; ④ PepDraw was used to analyze the physicochemical properties of the screened peptides; ⑤ Bioinformaticstool was used to predict whether the peptide sequences had potential toxicity, and non-toxic peptides were screened. After screening, three peptide sequences, YAADQ, FPYTT, and YVTYA, were finally obtained.

[0034] (6) The three polypeptide sequences YAADQ, FPYTT, and YVTYA were chemically synthesized, and their mass spectra are shown below. Figures 4-6 As shown.

[0035] Example 2 A bioactive peptide preparation with anti-inflammatory effects, wherein the active ingredient is at least one of the bioactive peptides YAADQ, FPYTT, and YVTYA.

[0036] SEQ ID NO:1: YAADQ; SEQ ID NO:2: FPYTT; SEQ ID NO:3: YVTYA.

[0037] Using water as a solvent, single peptides YAADQ, FPYTT, and YVTYA, and complex peptides YAADQ+FPYTT (hereinafter referred to as YAA+FPY), YAADQ+YVTYA (hereinafter referred to as YAA+YVT), and FPYTT+YVTYA (hereinafter referred to as FPY+YVT) were prepared into solutions with a concentration of 1 mg / mL. In the complex peptides, the mass ratio of the two peptides was 1:1, and the concentration of each peptide was 1 mg / mL. The effects of the single peptides YAADQ, FPYTT, and YVTYA, and the complex peptides YAA+FPY, YAA+YVT, and FPY+YVT on the viability of RAW264.7 cells were determined. The results are as follows: Figure 7 As shown, compared with the Control group, the YAADQ, FPYTT, and YVTYA polypeptide sequences can significantly improve cell viability. In addition, the compound peptides YAA+FPY, YAA+YVT, and FPY+YVT all have a proliferative effect on the growth of RAW264.7 cells.

[0038] The effects of single peptides YAADQ, FPYTT, YVTYA and complex peptides YAA+FPY, YAA+YVT, FPY+YVT on NO release in RAW264.7 cells were determined, and the results are as follows: Figure 8 As shown, compared with the control group, the release of NO from cells increased to varying degrees after treatment with LPS and peptides. The Model group, induced by LPS (1 μg / mL), showed a significant increase in NO release, indicating the successful establishment of an LPS-induced inflammation model. Compared with the Model group, YAADQ, FPYTT, and YVTYA significantly inhibited NO production. The complex peptides YAA+FPY, YAA+YVT, and FPY+YVT all inhibited NO release, reducing NO release by 85%, 83.8%, and 83.0% respectively compared to the model group. These results indicate that phycocyanin peptides possess good anti-inflammatory activity; phycocyanin, after enzymatic hydrolysis, produces anti-inflammatory peptides that can inhibit cellular inflammation. Example 3 The effects of protease type and ultrasonic treatment on the degree of hydrolysis and anti-inflammatory effect of phycocyanin. Distilled water was added to phycocyanin at a ratio of 1:20 (w / v) to dissolve it. The phycocyanin solution was divided into two major groups, each containing six subgroups. The first group underwent sonication for cell disruption at 450 W for 30 min before enzymatic hydrolysis; the second group did not undergo sonication. Alkaline protease, papain, trypsin, pepsin, neutral protease, and acidic protease were added to each of the six subgroups within each major group to hydrolyze the phycocyanin. The hydrolysis was performed at the optimal temperature and pH for each protease (Table 1) for 6 h. After the hydrolysis reaction, the hydrolysate was boiled at 100℃ for 10 min to terminate the reaction. Then, the hydrolysate was centrifuged at 6000 r / min and 4℃ for 15 min, and the supernatant was retained.

[0039] Table 1. Optimal reaction conditions for six proteases The effects of protease type and ultrasonic treatment on the degree of hydrolysis of phycocyanin were determined, and the results are as follows: Figure 9 As shown, the degree of hydrolysis of phycocyanin hydrolysates treated with ultrasound-assisted cell disruption was compared with that without ultrasound assistance. The results showed that the degree of hydrolysis of phycocyanin with ultrasound assistance was better than that without ultrasound assistance, and the neutral and alkaline proteases involved in ultrasound assistance had the best hydrolysis degrees, at 27.57% and 25.76%, respectively.

[0040] The effects of phycocyanin hydrolysates obtained from different proteases on the viability of RAW264.7 cells were determined, and the results are as follows: Figure 10 As shown, RAW264.7 macrophages were treated with different concentrations of PCPs (0, 0.5, 1, 5, 10, 15, 20 mg / mL) obtained by enzymatic hydrolysis with different proteases, and cell viability was measured after 24 h. Results showed that all PCPs at concentrations of 0.5–1 mg / mL had no toxic side effects on RAW264.7 macrophages. Ne-PCPs at concentrations of 0.5–5 mg / mL were non-toxic to RAW264.7 macrophages and promoted cell proliferation. Furthermore, except for Ne-PCPs, other PCPs showed significant toxicity to RAW264.7 macrophages within the concentration range of 5–20 mg / mL with increasing concentration of the hydrolysate (p<0.05). Therefore, a PCP concentration of 1 mg / mL was selected for practical applications.

[0041] The effects of phycocyanin hydrolysates obtained from different proteases on LPS-induced cytotoxicity of RAW264.7 cells were determined, and the results are as follows: Figure 11As shown, LPS had no effect on the survival rate of mouse RAW264.7 macrophages compared to the control group (0 mg / mL). Furthermore, within the concentration range of 0.5–5 mg / mL, different PCPs showed no toxicity to LPS-induced RAW264.7 macrophages, indicating that the concentration range of 1–5 mg / mL is a safe range. However, within the concentration range of 10–20 mg / mL, different PCPs exhibited significant toxicity to RAW264.7 macrophages with increasing concentration of the enzymatic hydrolysate (p<0.05).

[0042] The effect of different phycocyanin hydrolysates obtained from enzymatic digestion on LPS-induced NO release in RAW264.7 cells was determined. Logarithmically growing RAW264.7 cells were seeded in 96-well plates and cultured at 37°C with 5% CO2 for 12–24 hours. The old culture medium was discarded, and cells were pre-incubated with different concentrations of phycocyanin hydrolysates for 2 hours, followed by stimulation with 1 μg / mL LPS for 12–24 hours. Cell viability and NO content in the cell supernatant were then measured. A cell inflammation model group (LPS treatment only) was used as a control. Results are shown below. Figure 12 As shown, compared with the blank group (0 mg / mL), the release of NO from RAW264.7 macrophages stimulated by LPS was significantly increased (p<0.05), indicating that the LPS-induced inflammation model was successfully established. Compared with the model group (LPS), phycocyanin peptides at concentrations of 0.5-20 mg / mL all reduced NO release, indicating that they all have certain anti-inflammatory effects. In summary, Ne-PCPs have a high degree of hydrolysis and exhibit excellent in vitro anti-inflammatory activity in inhibiting NO production.

[0043] Example 4 The preparation method of peptide nanoparticles for anti-inflammatory purposes is as follows: (1) Chitosan with a molecular weight of 50 kDa was dissolved in acetic acid solution with a mass fraction of 1% w / v and stirred overnight to obtain a chitosan solution with a concentration of 0.5 mg / mL.

[0044] (2) Adjust the pH of the chitosan solution to 3 with 1 mol / L NaOH, and add phycocyanin peptide with a concentration of 1 mg / mL at a uniform rate. The phycocyanin peptide is the phycocyanin hydrolysis product prepared by the method in step (1) of Example 1. The amount of phycocyanin peptide added is 5% of the volume of the chitosan solution.

[0045] (3) Then, under magnetic stirring, a sodium tripolyphosphate solution with a concentration of 0.8 mg / mL was added dropwise at a mass ratio of chitosan to sodium tripolyphosphate of 4:1. The pH was adjusted before and after to keep it consistent. The mixture was then magnetically stirred for 30 min at room temperature to obtain a suspension of phycocyanin peptide nanoparticles (PCP-NPs).

[0046] Effect of chitosan molecular weight on nanoparticle size and PDI value Based on Example 4, chitosan nanoparticles with different molecular weights (30 kDa, 50 kDa, 100 kDa, and 200 kDa) were prepared. The chitosan concentration was 1 mg / mL, the mass ratio of chitosan to sodium tripolyphosphate was 5:1, and the pH of the chitosan solution was 4. The chitosan-sodium tripolyphosphate composite PCP nanoparticles prepared using chitosan of different molecular weights were characterized by dynamic light scattering. Detection was performed using a DTS0012 and DTS1070 sample cell in a nanoparticle size and potential analyzer. The results are as follows: Figure 13 As shown, smaller nanoparticles are more easily absorbed by the human body through the small intestinal epithelial cells. PDI is an indicator of particle uniformity, ranging from 0 to 1. The closer the PDI value is to 0, the more homogeneous the system. Among them, the nanoparticles prepared from chitosan with a molecular weight of 50 kDa have a particle size of 157.2 nm and a PDI of 0.27, with the smallest particle size and PDI, thus the most homogeneous system.

[0047] Effects of chitosan concentration on nanoparticle size, PDI value, and potential Based on Example 4, chitosan solutions of different concentrations (0.2 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, and 2 mg / mL) were prepared. The pH was adjusted to 4 with 1 mol / L NaOH, and 0.8 mg / mL of sodium tripolyphosphate was added at a chitosan to sodium tripolyphosphate mass ratio of 5:1 to prepare nanoparticles. The chitosan-sodium tripolyphosphate composite PCP nanoparticles prepared using chitosan of different molecular weights were characterized by dynamic light scattering. The nanoparticles were analyzed using DTS0012 and DTS1070 sample cells in a nanoparticle size and potential analyzer. The results are as follows: Figure 14 As shown, with the chitosan concentration increasing from 0.5 mg / mL to 2 mg / mL, the particle size of the chitosan-sodium tripolyphosphate nanoparticles increased from 230.27 nm to 417.06 nm. With the chitosan concentration increasing from 0.2 mg / mL to 2 mg / mL, the potential increased from 32.3 mV to 45.6 mV. At a chitosan concentration of 0.5 mg / mL, the PDI value was 0.254, indicating that the system was homogeneous and stable at this point.

[0048] Effect of chitosan to sodium tripolyphosphate mass ratio on nanoparticles Based on Example 4, the mass ratio of chitosan to sodium tripolyphosphate was set to 7:1~3:1 (7:1, 6:1, 5:1, 4:1, 3:1), the chitosan concentration was 0.5 mg / mL, and the pH was adjusted to 4 with 1 mol / L NaOH. The particle size, PDI value, and potential of the nanoparticles were measured. The results are as follows: Figure 15 As shown, when the ratio of chitosan to sodium tripolyphosphate is lower than 3:1 or higher than 7:1, the turbidity of the system increases. Furthermore, when the ratio of chitosan to sodium tripolyphosphate increases to 7:1 or is lower than 3:1, a milky white precipitate appears in the system, indicating the formation of large particles. When the ratio of chitosan to sodium tripolyphosphate is 4:1, the particle size is as low as 142.1 nm, and the PDI is 0.274, indicating that the system is homogeneous and stable at this point. Therefore, a chitosan to sodium tripolyphosphate ratio of 4:1 is used as the condition for the following experiments.

[0049] Effect of pH of chitosan solution on nanoparticles Based on Example 4, the chitosan concentration was 0.5 mg / mL, and the pH was adjusted to 2, 3, 4, 5, and 6 using 1 mol / L NaOH. The mass ratio of chitosan to sodium tripolyphosphate was 4:1. Nanoparticles were prepared, and the particle size, PDI value, and potential of the nanoparticles were measured. The results are as follows: Figure 16 As shown, the mechanism of action for preparing chitosan-sodium tripolyphosphate nanoparticles is electrostatic interaction. Therefore, the pH value of the system is particularly important for the formation of nanoparticles and their subsequent encapsulation of food-derived bioactive peptides. When the pH value of the solution increases from 3 to 6, the charge of the nanoparticles decreases from 41.8 mV to 26.6 mV. With the increase of the solution pH value, the -NH4+ on the chitosan molecular chain... 3+ As deprotonation gradually occurs, the charge on the chitosan-sodium tripolyphosphate nanoparticles gradually decreases, leading to a decline in the electrostatic repulsion between the particles. Therefore, the optimal pH value for the chitosan solution is 3.

[0050] Effects of chitosan-sodium tripolyphosphate nanoparticles prepared by different methods on the encapsulation efficiency and embedding ability of phycocyanin peptides The effects of chitosan of different molecular weights, chitosan solution concentration, chitosan to sodium tripolyphosphate mass ratio, and chitosan solution pH on the encapsulation efficiency and embedding capacity of phycocyanin peptides were determined. The determination methods are as follows: Chitosan-sodium tripolyphosphate (PCP) nanoparticle suspensions prepared under different conditions were injected into ultrafiltration centrifuge tubes (10 kDa) and then centrifuged at 4 °C and 4000 r / min for 20 min. Unencapsulated phycocyanin peptides were ultrafiltered and centrifuged into the filtrate. The filtrate was appropriately diluted, and the absorbance was measured at 280 nm. The content of unencapsulated phycocyanin peptides was calculated using a standard curve. Encapsulation efficiency (EE) and encapsulation capacity (EC) were calculated using the following formulas: EE (%) = (M0-m) / M0 × 100% EC (%) = (M0-m) / M × 100% In the formula, M0 is the total mass of phycocyanin peptides (mg), m is the mass of unencapsulated free peptides calculated by the standard curve (mg), and M is the total mass of nanoparticles (mg).

[0051] The results are as follows Figure 17 As shown, the encapsulation efficiency of the nanoparticles was highest at a chitosan molecular weight of 50 kDa, reaching 77.33%. Therefore, chitosan with a molecular weight of 50 kDa was selected for subsequent experiments. The encapsulation efficiency and encapsulation capacity of the nanoparticles showed a trend of first increasing and then decreasing with increasing chitosan concentration, reaching the highest encapsulation efficiency of 85.3% at a chitosan concentration of 0.5 mg / mL. The encapsulation efficiency and encapsulation capacity showed a trend of first increasing and then decreasing with decreasing mass ratio of chitosan to sodium tripolyphosphate, reaching the maximum encapsulation efficiency of 89.3% at a mass ratio of 4:1. At pH 3, the encapsulation efficiency was 91.3%.

[0052] In vitro anti-inflammatory activity of phycocyanin peptide nanoparticle complex The in vitro anti-inflammatory activity of the phycocyanin peptide nanoparticles prepared by the method in Example 4 was detected as follows: Logarithmically growing RAW264.7 cells were seeded in 96-well plates and cultured at 37°C in a 5% CO2 incubator for 12–24 hours. The old culture medium in the wells was discarded, and 100 μL of the phycocyanin peptide nanoparticles prepared by the method in Example 4 (concentration 1 mg / mL) was added for pre-incubation for 2 h. Then, 1 μg / mL LPS was added for stimulation for 12–24 h. After the stimulation, cell viability and the NO content in the cell supernatant were measured. A control group (without phycocyanin peptide nanoparticles and LPS) was set up and compared with the LPS-treated model group.

[0053] The results are as follows Figure 18As shown, compared with the control group, the NO release of RAW264.7 macrophages stimulated by LPS was significantly increased, indicating that the LPS-induced inflammation model was successfully established. The anti-inflammatory activity of the phycocyanin peptide nanoparticle group (PCP-NPs) was significantly reduced by 68.1% compared with the model group, indicating that it has a strong anti-inflammatory effect.

[0054] In vitro simulated digestion test of phycocyanin peptide nanoparticle complex The phycocyanin peptides prepared by the method in step (1) of Example 1 and the phycocyanin peptide nanoparticles prepared by the method in Example 4 were respectively added to a solution containing pepsin, the pH was adjusted to 2, and the mixture was magnetically stirred at 37°C for 2 h. Subsequently, the pH was adjusted to 7.5, and trypsin was added for enzymatic hydrolysis for 2 h. The absorbance of the samples digested by pepsin and trypsin was measured at 280 nm, and the retention rate was calculated using a standard curve and the following formula.

[0055] Retention rate % = Amount of undegraded peptides (mg) / Total peptides (mg) × 100% The retention rate of phycocyanin peptides in simulated digestive fluid before and after nanoparticle encapsulation was investigated using an in vitro gastrointestinal simulated digestion method, thereby evaluating the effect of chitosan-sodium tripolyphosphate nanoparticles on improving the bioavailability of phycocyanin peptides. Results are as follows: Figure 19 As shown, phycocyanin peptides were all degraded after in vitro gastrointestinal simulated digestion. However, after being encapsulated by chitosan-sodium tripolyphosphate nanoparticles, the retention rate of phycocyanin peptides in the gastrointestinal simulated digestion fluid was improved by 61.3% and 43.2%, respectively.

[0056] The therapeutic effect of phycocyanin peptide nanoparticle complex on ulcerative colitis in mice. C57BL / 6J mice (purchased from Shandong Pengyue Experimental Animal Technology Co., Ltd.) were housed in an SPF environment with independent ventilation, a 12-hour light-dark cycle, and a relative humidity of 55%±5%. Food and water were provided freely. After one week of acclimatization, the mice were separated into individual cages. The cages were disinfected weekly, and the bedding was changed three times a week. The experimental animals were divided into 5 groups, with 6 mice in each group. Different drugs were administered via gavage at fixed times each day. The experimental groups are as follows: Control group: Free access to sterile distilled water for 10 days, while also receiving sterile distilled water by gavage; Model group (DSS group): Ulcerative colitis model was established by free drinking of 3% DSS (sodium dextran sulfate) solution for 10 days, while sterile distilled water was administered by gavage. DSS+PCPs group (PCPs group): Ulcerative colitis model was constructed by free drinking of 3% DSS solution for 10 days. At the same time, phycocyanin peptides (PCPs) prepared in step (1) of Example 1 were administered by gavage at a dose of 100 mg / kg. PCPs were dissolved in water and administered by gavage. DSS+Drug Group (Positive Control Group): The ulcerative colitis model was established by free drinking of 3% DSS solution for 10 days. At the same time, mesalazine enteric-coated tablets were administered by gavage at a dose of 100 mg / kg, which was dissolved in water beforehand. DSS+chitosan-sodium tripolyphosphate composite PCPs nanoparticle group (PCP-NPs group): Ulcerative colitis model was established by free drinking of 3% DSS solution for 10 days, and at the same time, phycocyanin peptide nanoparticles (PCP-NPs) prepared by the method in Example 4 were administered by gavage at a dose of 100 mg / kg, which was dissolved in water beforehand.

[0057] Starting from day 0, the mice's diet, activity, fur and other conditions were monitored and recorded regularly. The mice's weight was recorded, the mice's fecal condition was observed, and their disease activity index (DAI) was scored based on their weight, bloody stool and fecal morphology (Table 2).

[0058] Table 2 DAI Scoring Table The fecal bleeding of mice in each treatment group was observed, and the results were as follows: Figure 20 As shown, during the experiment, the stool of the control group mice was normal and granular. Compared with the control group, the model group mice experienced severe diarrhea, watery and bloody stools, and anal bleeding after DSS treatment. The positive control group had mild diarrhea and loose stools. The PCPs group had watery and bloody stools and slight anal bleeding. The PCP-NPs group had mild diarrhea, loose stools, and slight anal bleeding.

[0059] The daily weight changes of mice in each group were measured, and the results are as follows: Figure 21 As shown, the weight of mice in the normal control group showed a slow growth trend during the experiment. Compared with the control group, the weight of mice in the DSS model group began to decrease on the fifth day after DSS modeling. As the experiment progressed, the weight of mice in the DSS group, drug group, PCPs group, and PCP-NPs group showed an overall decreasing trend, while the drug group, PCPs group, and PCP-NPs group could slow down the DSS-induced weight loss.

[0060] Based on the mice's mental state, rectal bleeding, and fecal consistency, daily assessments of the DAI scores for each group were obtained as follows: Figure 22As shown in the figure. During the experiment, the mice in the control group were in good spirits, active, and their stools were normal and granular. Compared with the control group, the mice in the DSS group began to show lethargy, mild diarrhea, and loose stools on the fifth day of DSS treatment. By the eighth day, the mice in the DSS group were listless, curled up, and showed varying degrees of watery and bloody stools and anal bleeding. Compared with the DSS group, the drug group, PCPs group, and PCP-NPs group could alleviate the above symptoms induced by DSS. Therefore, the comprehensive evaluation of the mouse disease activity index (DAI) showed that the PCPs group and PCP-NPs group could alleviate the decrease in DAI score caused by DSS.

[0061] Mice were sacrificed after cervical dislocation. The colon and rectum were dissected, and the colonic tissue characteristics and intestinal contents were observed. The colonic length was measured and calculated using a ruler. Four segments of each colonic tissue were harvested. Segments 2 and 3 were preserved in 4% formalin, while segments 1 and 4 were preserved in sterile EP tubes, flash-frozen in liquid nitrogen, and then stored at -80°C. Colonic shortening is a typical symptom of ulcerative colitis. The colonic length of mice in each treatment group was measured. The results are as follows: Figure 23 As shown, compared with the control group, DSS caused a shortening of the colon, bloodstains in the colon and cecum, and increased viscosity of intestinal contents. Interventions in the drug group, PCPs group, and PCP-NPs group significantly alleviated the shortening of the colon and could protect against DSS-induced colon shortening.

[0062] Mice in each group were anesthetized with ether, and blood was collected from their eyes. The EP tubes containing the collected blood were placed on ice and kept at 4°C in the dark for at least 2 hours. After centrifugation at 4000 r / min for 10 min, the serum was collected and stored at -80°C. The levels of pro-inflammatory cytokines TNF-α, IL-6, IL-1β, and the anti-inflammatory cytokine IL-10 in mouse serum were measured using ELISA. The levels of the inflammatory factors interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in serum were detected using the corresponding enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions. Results are as follows: Figure 24The results showed that, compared with the control group, the levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β were significantly increased in the DSS group (p<0.05), while the level of anti-inflammatory cytokine (IL-10) was significantly decreased, indicating that DSS induces inflammation in mice. After treatment with PCPs and PCP-NPs, the levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β in the serum of colitis mice decreased, while the level of the anti-inflammatory cytokine IL-10 increased, indicating that PCP-NPs can alleviate the inflammatory state in colitis mice.

[0063] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A bioactive peptide with anti-inflammatory effects, characterized in that, The amino acid sequences of the bioactive peptides are shown in SEQ ID NO:1~SEQ ID NO:

3.

2. The use of the bioactive peptide with anti-inflammatory effects as described in claim 1 in the preparation of products with anti-inflammatory effects.

3. A bioactive peptide preparation with anti-inflammatory effects, characterized in that, The active ingredient is at least one of the bioactive peptides of YAADQ, FPYTT, and YVTYA.

4. The bioactive peptide preparation with anti-inflammatory effects according to claim 3, characterized in that, The concentration of the active ingredient is 1 mg / mL.

5. Anti-inflammatory polypeptide nanoparticles, characterized in that, Nanoparticles obtained by coating anti-inflammatory peptides with chitosan and sodium tripolyphosphate; The anti-inflammatory polypeptide contains bioactive peptides with the amino acid sequences YAADQ, FPYTT, and YVTYA.

6. The polypeptide nanoparticles for anti-inflammatory purposes according to claim 5, characterized in that, The method for preparing the peptide with anti-inflammatory effects is as follows: Distilled water was added to phycocyanin at a ratio of 1:20 to dissolve it, and the mixture was sonicated at 450 W for 30 min. Then, 2000 U / g of alkaline protease was added and the mixture was enzymatically hydrolyzed at pH 10.5 and 50℃ for 6 h. After the enzymatic hydrolysis was completed, the hydrolysate was boiled at 100℃ for 10 min to terminate the reaction. The hydrolysate was then centrifuged at 6000 r / min and 4℃ for 15 min, and the supernatant was retained and freeze-dried to obtain the final product.

7. The polypeptide nanoparticles for anti-inflammatory purposes according to claim 5 or 6, characterized in that, The concentration of the peptide with anti-inflammatory effects was 1 mg / mL.

8. The method for preparing the anti-inflammatory polypeptide nanoparticles according to claim 7, characterized in that, The steps are as follows: (1) Chitosan with a molecular weight of 30kDa~200kDa was dissolved in an acetic acid solution with a mass fraction of 1% w / v and stirred overnight to obtain a chitosan solution with a concentration of 0.2 mg / mL~2 mg / mL; (2) Adjust the pH of the chitosan solution to 2-6 with 1 mol / L NaOH, and add a polypeptide with anti-inflammatory effect at a concentration of 1 mg / mL at a uniform rate. The amount of polypeptide added is 5% of the volume of the chitosan solution. (3) Then, under magnetic stirring, a sodium tripolyphosphate solution with a concentration of 0.8 mg / mL is added dropwise at a mass ratio of chitosan to sodium tripolyphosphate of 7:1 to 3:

1. The pH is adjusted before and after to keep it consistent. The mixture is then magnetically stirred for 30 min at room temperature to obtain the final product.

9. The method for preparing anti-inflammatory polypeptide nanoparticles according to claim 8, characterized in that, In step (1), the molecular weight of chitosan is 50 kDa and the concentration of the chitosan solution is 0.5 mg / mL.

10. The method for preparing anti-inflammatory polypeptide nanoparticles according to claim 8, characterized in that, In step (2), the pH value is adjusted to 3; in step (3), the mass ratio of chitosan to sodium tripolyphosphate is 4:1.

Images