Rana chensinensis meat protein source active peptide and its application in preparing immunomodulator

Abstract

The application discloses frog meat protein source active peptides and application thereof in preparation of immunomodulators, and belongs to the technical field of active peptides. Two novel immunomodulatory peptides GIHETTYNS and IVRDIKEK are identified from the hydrolysate of Rana spinosa meat, which can significantly improve the proliferation activity of macrophage RAW264.7, accelerate the cell cycle, promote the production of NO and cytokines, and improve the level of active oxygen. The results of inflammation reaction experiments show that the active peptides can relieve inflammation. The results of molecular docking show that the active peptides are combined with the key region of TLR4 / MD-2 through hydrogen bond and hydrophobic interaction. In addition, bioinformatics analysis shows that the active peptides have good safety and stability. In summary, the active peptides are expected to become new raw materials of natural immunomodulators and anti-inflammatory agents, and promote the high-value utilization of Rana spinosa.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide technology, specifically relating to bioactive peptides derived from frog meat protein and their application in the preparation of immunomodulators. Background Technology

[0002] The spiny-breasted frog (Quasipaa spinosa), also known as the giant spiny frog or stone frog, is a valuable resource in traditional Chinese medicine and food due to its high protein content. According to the *Compendium of Materia Medica* and *Chinese Materia Medica of Medicinal Animals*, it has nourishing and strengthening effects, enhancing immunity, memory, and preventing cardiovascular diseases (Li Jian et al. 2023, Zheng Chaoce 2011). Widely distributed in Jiangsu and Zhejiang provinces of China, its farming scale has expanded significantly in recent years (Hu Wenfang et al. 2017, Chen Dunxue et al. 2019, Mei Yiyun et al. 2015). Therefore, how to deeply process the spiny-breasted frog and increase its added value has become an urgent problem to be solved.

[0003] Immunity is one of the important mechanisms for the human body to resist disease and maintain health, and weakened immunity is closely related to many diseases. Weakened immunity makes one more susceptible to infection by various pathogens such as bacteria, viruses, fungi, and parasites, increasing the risk of influenza, colds, pneumonia, and sexually transmitted diseases. Natural bioactive peptides that enhance immunity have become a research hotspot for immunomodulators. For example, hydrolysates or peptides of immunomodulatory proteins obtained from natural sources have been widely reported (Chalamaiah et al. 2018, Rivero-Pino et al. 2023). For instance, enzymatically prepared coix gluten hydrolysate can promote the proliferation of mouse spleen cells and stimulate RAW264.7 cells to secrete NO at low concentrations (Li Ling-Ling et al. 2017a). Defatted wheat germ globulin hydrolysate can promote the proliferation, phagocytic function, and secretion of TNF-α, IL-6, and NO by immune cells (Wu et al. 2016). An immunomodulatory peptide with the sequence DHAV was identified from silkworm pupa protein hydrolysate. This peptide exhibited good splenocyte proliferation activity and stimulated the expression of immune-related genes (IL-6, IL-12, NF-κB, etc.) (Li Zhiyong et al. 2020). The peptides DNSIAMESMK and LLQLGSGR identified from oyster hydrolysate increased macrophage proliferation rate and phagocytic capacity, and promoted cytokine and NO production (Li W. et al. 2019a). These findings indicate that protein hydrolysates are an important source of immunomodulatory peptides, and further research on these peptides is of great significance.

[0004] The innate immune system is the host's first line of defense against pathogens (Akar-Ghibril 2022). It activates host defense signaling pathways through pattern recognition receptors (PAMPs) to control the expression of various immune response genes (Medzhitov and Janeway 2000). Toll-like receptors (TLRs) are members of the pattern recognition receptor family, mediating the recognition of pathogen-associated molecular patterns and playing a crucial role in the innate immune system (McCusker et al. 2018). The TLR4 receptor is one of the most studied receptors in the toll-like receptor family, exhibiting high selectivity and sensitivity to invading pathogens. The TLR4 receptor can recognize PAMPs from fungi, viruses, and mycoplasma, such as those mediated by lipopolysaccharide (LPS) or bacterial endotoxins, thus mediating inflammatory responses (Ain et al. 2020, Zhang Yongsheng et al. 2022a). Immunomodulatory substances play an indispensable role in resisting pathogen invasion and participating in inflammatory responses.

[0005] To combat autoimmune diseases, immunodeficiency, malignant tumors, and weakened immunity, drugs that regulate human immune function are used clinically. However, their cost and adverse reactions (such as abdominal pain, vomiting, and decreased appetite) hinder prevention and long-term use (del Palacio et al. 2016, Zhang P. et al. 2023b). Naturally derived peptides have the advantages of strong biological activity and high safety (Feng et al. 2022), and naturally derived immunomodulatory peptides for enhancing immunity and inhibiting excessive inflammatory responses hold promise as new sources for related foods and medicines (Kim Jae Hwan et al. 2022). Summary of the Invention

[0006] To address these issues, the primary objective of this invention is to provide active peptides derived from frog meat protein.

[0007] Another object of the present invention is to provide the application of the above-mentioned frog meat protein source active peptides.

[0008] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0009] Frog meat protein-derived bioactive peptides, which have any of the following amino acid sequences:

[0010] (1)Gly-Ile-His-Glu-Thr-Thr-Tyr-Asn-Ser(GIHETTYNS);

[0011] (2)Ile-Val-Arg-Asp-Ile-Lys-Glu-Lys(IVRDIKEK).

[0012] Furthermore, the active peptides are prepared by enzymatic hydrolysis of frog meat, solid-phase synthesis, or genetic engineering techniques.

[0013] Frog meat enzymatic hydrolysate containing the above-mentioned active peptides.

[0014] Solid-phase synthetic products containing the above-mentioned active peptides.

[0015] A nucleic acid that encodes the aforementioned active peptide.

[0016] A biomaterial comprising the aforementioned nucleic acid, wherein the biomaterial is recombinant DNA, an expression cassette, a transposon, a vector, or a host cell.

[0017] The application of the above-mentioned active peptides, frog meat enzymatic hydrolysates, solid-phase synthetic products, nucleic acids or biological materials in the preparation of immunomodulators and / or anti-inflammatory agents.

[0018] The application of the above-mentioned active peptides, frog meat enzymatic hydrolysate, solid-phase synthetic products, nucleic acids or biological materials in the preparation of antibacterial and / or anti-infective agents.

[0019] The application of the above-mentioned active peptides, frog meat enzymatic hydrolysate, solid-phase synthetic products, nucleic acids or biological materials in the preparation of macrophage proliferation promoters.

[0020] Furthermore, the active peptide is one of an oral preparation or a topical preparation.

[0021] The present invention has the following advantages and effects compared with the prior art:

[0022] This invention purifies and identifies novel bioactive peptides from the hydrolysate of spiny-breasted frog meat, and explores their activity and mechanism of action. Results show that two novel immunomodulatory peptides, GIHETTYNS (1020.4512 Da) and IVRDIKEK (999.6077 Da), were identified from the hydrolysate. These bioactive peptides significantly enhance the proliferation activity of RAW264.7 macrophages and accelerate the cell cycle, promote the production of NO and cytokines (IL-6, TNF-α), and increase reactive oxygen species (ROS) levels. Inflammation response experiments indicate that these bioactive peptides can alleviate inflammatory responses. Molecular docking results show that these bioactive peptides bind to key regions of TLR4 / MD-2 via hydrogen bonds and hydrophobic interactions, with action sites at LYS A:458, ARG A:434, and ARG D:90. Furthermore, bioinformatics analysis indicates that these bioactive peptides possess good safety and stability. In conclusion, these bioactive peptides hold promise as new raw materials for natural immunomodulators and anti-inflammatory agents, promoting the high-value utilization of spiny-breasted frogs. Attached Figure Description

[0023] Figure 1 Figure 1 shows the results of a study on the effects of frog meat protein-derived bioactive peptides on the proliferation activity and cell cycle distribution of RAW264.7 cells. In the figure, A represents cell proliferation activity, B represents the percentage of cell cycle distribution in G0 / G1, S, and G2 / M phases, and C represents the flow cytometry cell cycle distribution.

[0024] Figure 2 Figure 1 shows the results of a study on the effects of frog meat protein-derived bioactive peptides on NO and cytokine release in RAW264.7 cells; where A represents cellular NO, B represents cytokine IL-6, and C represents cytokine TNF-α.

[0025] Figure 3 The effect of frog meat protein-derived bioactive peptides on ROS levels in RAW264.7 cells is shown in the flow cytometry diagram, where A represents the ROS content equivalent to DCF and B represents the ROS content equivalent to the fluorescence intensity of DCF.

[0026] Figure 4 The study investigated the effects of active peptides derived from frog meat protein on NO release induced by LPS in RAW264.7 cells. In Figure A, the effect of active peptides on NO release in RAW264.7 cells was observed 12 h after LPS induction, and in Figure B, the effect of active peptides on NO release in RAW264.7 cells was observed 24 h after LPS induction.

[0027] Figure 5The image shows the docking results of the frog meat protein source active peptide GIHETTYNS with TLR4 / MD-2 molecules; where A is a 3D image of the complex after docking, B is a 3D image of the interaction between the peptide and TLR4 / MD-2, and C is a 2D image of the interaction site between the peptide and TLR4 / MD-2.

[0028] Figure 6 The image shows the docking results of the frog meat protein source active peptide IVRDIKEK with TLR4 / MD-2 molecules; where A is a 3D image of the docked complex, B is a 3D image of the interaction between the peptide and TLR4 / MD-2, and C is a 2D image of the interaction site between the peptide and TLR4 / MD-2. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto.

[0030] Example 1: Synthesis of bioactive peptides derived from frog meat protein

[0031] The bioactive peptides screened from the hydrolysate of spiny-breasted frog meat have the amino acid sequences GIHETTYNS and IVRDIKEK. They were synthesized by Nanjing Jietai Biotechnology Co., Ltd. HPLC analysis showed that the purity of the synthesized peptides was ≥98%.

[0032] Example 2: Immunomodulatory Activity Analysis of Frog Meat Protein-Derived Bioactive Peptides

[0033] 2.1 Cell Culture

[0034] RAW 264.7 macrophages were cultured in DMEM containing 20% ​​FBS and incubated in an incubator at 37°C and 5% CO2. When the cells reached nearly 80% growth, they were passaged by repeated pipetting. Cells in the logarithmic growth phase were selected for experiments.

[0035] 2.2 Cell proliferation activity

[0036] RAW264.7 macrophages were used at a concentration of 5 × 10⁻⁶. 4 Cells were seeded at a density of 10 cells / well in 96-well plates and cultured for 24 h. Then, a peptide solution or positive control LPS (10 μg / mL) was added for 24 h of intervention. The blank control group received only culture medium. The supernatant was discarded, and 20 μL of 5 mg / mL MTT solution was added to each well. The plates were incubated at 37°C in a 5% CO2 cell culture incubator for 4 h. The supernatant was discarded, and 100 μL of DMSO was added to each well. The plates were incubated at room temperature for 20 min, and the absorbance was measured at OD490 nm using a microplate reader (Perkin Elmer, USA) after shaking for 5 s. Cell proliferation activity was determined based on the absorbance value.

[0037] 2.3 NO release, cytokine secretion, and cell cycle

[0038] NO and cytokines were measured according to the method of Yu and Yang et al. (Yang Q. et al. 2020a, Yu Y. et al. 2021), with slight modifications. RAW264.7 macrophages were sampled at 5 × 10⁻⁶ cells. 4 Cells were seeded at a density of 10 cells / well in 24-well plates and cultured for 48 h. The supernatant was discarded, and the cells were treated with either a peptide solution (50-300 μg / mL) or a positive control LPS (10 μg / mL) for 24 h. The blank control group received only culture medium. The supernatant was used to determine the NO release, IL-6 secretion, and TNF-α secretion levels of RAW264.7 cells using NO assay kits and IL-6 and TNF-α ELISA kits, following the kit instructions. After discarding the supernatant, each well was washed with pre-chilled PBS and centrifuged (800 rpm, 3 min) to collect the cells. After discarding the supernatant, 70% pre-chilled ethanol was added, and the cells were fixed at 4°C for 24 h. Subsequently, cell cycle analysis was performed using a cell cycle assay kit (Beckman, USA) (Yang Q. et al. 2020a).

[0039] 2.4 Determination of Reactive Oxygen Species (ROS) Content

[0040] The determination of reactive oxygen species was based on a slight modification of the method described by Zhang et al. (Zhang Z. et al. 2022b). The cell density was adjusted to 1 × 10⁻⁶. 5 Cells were seeded per well in 24-well plates and incubated for 24 hours. The culture medium was then discarded, and cells were treated with different concentrations of peptide solution (50-300 μg / mL) or positive control LPS (10 μg / mL) for 24 hours. The blank control group received only culture medium. The supernatant was discarded, and the cells were washed three times with PBS. Subsequently, the cells were analyzed using a reactive oxygen species (ROS) detection kit, and fluorescence intensity was measured by flow cytometry.

[0041] 2.5 Statistical Analysis

[0042] Data processing and graphing were performed using GraphPad Prism 8 (GraphPad Software, Inc., San Diego, California, USA), and data analysis was conducted using IBM SPSS Statistic 21 (Version 21.0, IBM Co., New York, USA). Different letters indicate significant differences (p < 0.05).

[0043] 2.6 Results and Analysis

[0044] (1) Effects of immunologically active peptides on the proliferation activity and cell cycle of RAW264.7 cells

[0045] like Figure 1 As shown in Figure A, within the concentration range of 50-300 μg / mL, treatment with GIHETTYNS and IVRDIKEK significantly enhanced cell proliferation activity (p<0.05). Cell proliferation after GIHETTYNS treatment was concentration-dependent, exhibiting the best cell proliferation activity (135.61%) at 300 μg / mL, which was 1.35 times that of the blank control group and 0.91 times that of the positive control. IVRDIKEK also showed good cell proliferation activity at very low concentrations (50 μg / mL) (128.26%), significantly increasing by 28.26% compared to the blank control group (p<0.05). Similar macrophage proliferation-promoting activities have been previously reported, such as tilapia peptide (1.250 mg / mL, proliferation rate 112.51%) (Liu Kuan-Yu et al. 2023b), and our results show superiority over tilapia peptide at low concentrations.

[0046] The preparation for cell division between two consecutive cell divisions is called the cell cycle. Key phases of the cell cycle include G1, S, G2, and M phases. Cell cycle regulation plays a crucial role in cell proliferation, growth, and repair; therefore, we investigated the cell cycle distribution in RAW264.7 cells. Figure 1 Figures B and C show the cell cycle distribution after treatment with GIHETTYNS and IVRDIKEK. Compared with the blank control group, the proportion of RAW264.7 cells in G0 / G1 phase decreased, while the proportion in S phase increased to varying degrees. Specifically, LPS treatment reduced G0 / G1 phase by 21.6% and increased S phase by 18.7%, respectively. At the same concentration (300 μg / mL), GIHETTYNS and IVRDIKEK reduced G0 / G1 phase by 15.17% and 16.20% respectively compared to the blank control group, while increasing S phase by 45.24% and 41.44%, respectively. This indicates that treatment with immunomodulatory peptides shortened the DNA synthesis preparation period of RAW264.7 cells and accelerated the DNA synthesis process, thereby accelerating the cell cycle and promoting RAW264.7 cell proliferation. This result is also consistent with... Figure 1 The result for A is consistent.

[0047] (2) Effects of bioactive peptides on NO and cytokine secretion in RAW264.7 cells

[0048] Nitric oxide (NO) is a key regulator of immune cell function, participating in the body's inflammatory and immune regulatory processes, and has the function of killing bacteria, viruses, and tumor cells. Figure 2As observed in Figure A, NO release from RAW264.7 cells after treatment with GIHETTYNS and IVRDIKEK showed a concentration-dependent effect, increasing from a minimum of 8.11 pg / mL to 20.57 pg / mL, reaching a maximum at 300 μg / mL. The effect of the GIHETTYNS group was particularly significant (p<0.05). Compared to the blank control group (2.30 pg / mL), NO release at concentrations of 10, 50, 100, and 200 μg / mL was 2.9, 3.24, 3.14, and 3.33 times that of the blank control group, respectively. Furthermore, at the high concentration (300 μg / mL), the NO release was close to that of the positive control (20.57 pg / mL). NO plays an important role in immune regulation. It has been reported that Atlantic cod peptides and soybean peptides can significantly upregulate the expression of the NO precursor iNOS mRNA in RAW264.7 cells and induce NO production (Yuan et al. 2023). This indicates that GIHETTYNS, IADRMQKE, and IVRDIKEK can significantly promote NO production in RAW264.7 cells.

[0049] Cytokines are regulatory proteins that modulate the immune system and inflammation. Among them, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) can enhance macrophage activation and antigen presentation in the immune response and regulate immunity through different mechanisms. Figure 2 As shown in Figures B and C, compared with the blank control group, LPS significantly stimulated RAW264.7 cells to secrete IL-6 (383.01 pg / mL) and TNF-α (879.96 pg / mL) (p<0.05). After treatment with GIHETTYNS and IVRDIKEK, the secretion of TNF-α showed a trend of first increasing and then decreasing. The TNF-α secretion by IVRDIKEK reached its maximum at 100 μg / mL, which was 263.62% higher than that of the blank control group. Some reports indicate that soybean extract and low molecular weight peptides from thick-shelled mussels (Mytilus coruscus) can significantly stimulate RAW264.7 cells to secrete factors such as IL-6, IL-10, and IL-1β. These peptides also have the characteristic of promoting NO production. This suggests that cytokines, similar to NO, are also important mediators of the immunomodulatory effects of peptides.

[0050] (3) Effects of bioactive peptides on reactive oxygen species levels in RAW264.7 cells

[0051] Reactive oxygen species (ROS) are oxidizing reactive substances produced by living cells. ROS are key components in the host's fight against invading viruses and important messengers of innate and adaptive immunity. ROS may participate in immune responses through different signaling pathways, thereby inducing changes in cytokines such as IL-1β, TNF-α, and IFN-β. Therefore, we measured the production of ROS in RAW264.7 cells to explore its impact on immune regulation. DCFH-DA itself does not show any fluorescence but can penetrate the cell membrane, while intracellular ROS can oxidize DCFH-DA to DCF, which then exhibits fluorescence. Therefore, the DCFH-DA method can quantitatively detect changes in intracellular ROS. Figure 3 As shown, the intracellular ROS levels after treatment with GIHETTYNS, IVRDIKEK, and LPS were significantly higher than those in the blank control group (p<0.05). Specifically, GIHETTYNS treatment significantly increased ROS levels by 24.19%, 96.24%, 110.83%, 137.38%, and 44.23% compared to the blank control group, respectively. ROS may act as a signaling molecule in RAW264.7 cells, activating some immune signaling pathways and triggering immune responses. The NF-κB pathway is one of the most frequently reported ROS-induced pathways. NF-κB is also an important pathway related to immune regulation; it can be activated by both classical and non-classical pathways, participating in and regulating immune and inflammatory responses. Currently, the NF-κB pathway is involved in the reported mechanisms of action of various immunomodulators, suggesting a close relationship between ROS and immune regulation. Our results indicate that GIHETTYNS and IVRDIKEK can significantly stimulate RAW24.7 cells to produce reactive oxygen species, thereby participating in the immune response.

[0052] Example 3: Molecular docking and visualization analysis

[0053] 3.1 Immunomodulatory molecular mechanisms of bioactive peptides

[0054] In this invention, GIHETTYNS and IVRDIKEK exhibit good cellular immunomodulatory effects, but their molecular mechanisms remain unclear. Therefore, we explored the molecular mechanisms of immunomodulatory peptides by predicting the interaction between the peptides and the TLR4 / MD-2 receptor through molecular docking. Figure 4As shown, the two peptides enter the hydrophobic cavity of TLR4 / MD-2 in a specific conformation and interact with specific amino acid residues of TLR4 / MD-2. The large hydrophobic cavity of the TLR4 / MD-2 receptor allows the peptides to bind stably within it. GIHETTYNS and IVRDIKEK form covalent and non-covalent interactions with TLR4 / MD-2, including hydrogen bonds, hydrophobic interactions, electrostatic interactions, and van der Waals forces. Among these, hydrogen bonds are the dominant interaction force, primarily binding to groups such as -OH, -C=O, -NH2, -NH, and -CO on the peptides. Hydrogen bonds significantly contribute to the stability of the docking complex, promoting the formation of a stable structure between the peptide and the receptor.

[0055] The TLR4 / MD-2 receptor forms 17 hydrogen bonds with GIHETTYNS, with the main interaction sites being LYS A:458, LYSD:91, VAL D:93, LYSD:89, GLU A:437, ARG A:434, ARG D:90, and PHE B:406. Besides hydrogen bonding, some amino acid residues also form hydrophobic and electrostatic interactions with GIHETTYNS. For example, ALA B:382, HISD:96, and ARG B:337 form hydrophobic interactions with the imidazole ring, C16, and C2 groups on GIHETTYNS (including AlkylHydrophobic and Mixed Pi / Alkyl Hydrophobic), while GLU D:92 and ARG B:380 form hydrophobic interactions with -CH and O groups, respectively. - Ions form electrostatic interactions. IVRDIKEK forms 9 hydrogen bonds and 3 alkyl hydrophobic interactions with the TLR4 / MD-2 acceptor. These include 5 conventional hydrogen bonds and 4 unconventional hydrogen bonds. The main sites for conventional hydrogen bond formation are ASP A:460, ARG434, GLUA:437, and GLU D:92. The sites for hydrophobic interactions are LYS A:458, VAL A:435, and ARG D:90.

[0056] Hydrogen bonding and hydrophobic interactions are the key interactions in coordination compounds. We statistically... Figure 5 and Figure 6The hydrogen bonds and hydrophobic interactions, along with their binding sites, are shown in Table 1. Table 1 reveals that GIHETTYNS and IVRDIKEK share the same binding sites on TLR4 / MD-2: LYS A:458, ARG A:434, and ARG D:90. This indicates that all three immunomodulatory peptides are located in key regions of the TLR4 / MD-2 receptor and exhibit good affinity, which may be crucial sites for the activity of these peptides. Previous reports have shown that immunomodulatory peptides from gelatin hydrolysates and Litopenaeus vannamei bind to TLR4 / MD-2 via hydrogen bonds and hydrophobic interactions, consistent with our results. This suggests that hydrogen bonds and hydrophobic interactions enhance the binding stability of peptides to TLR4 / MD-2, thereby improving the immunomodulatory effect.

[0057] Table 1. Interaction sites between immunomodulatory peptides and TLR4 / MD-2

[0058]

[0059]

[0060] Generally, the activity of immunomodulatory peptides is related to various properties, such as molecular weight, amino acid composition, and hydrophobicity. Peptides containing amino acid residues such as leucine, lysine, histidine, and glycine have been reported to exhibit good immunomodulatory activity. Furthermore, the presence of basic or hydrophobic amino acids at the terminal ligase is also an indicator of immunomodulatory activity. For example, immunomodulatory peptides derived from barley contain 42.20% hydrophobic amino acids and 10.86% basic amino acids. The presence of these amino acids was also observed in the novel peptides we identified. These amino acids all form hydrogen bonds or hydrophobic interactions with the TLR4 / MD-2 receptor. For instance, in GIHETTYNS, glycine forms hydrogen bonds with VAL D:94 and GLU D:92, while isoleucine forms hydrophobic interactions with HISD:96 and ARG B:337. In IVRDIKEK, valine forms hydrogen bonds with ARG A:434 and LYS A:433, and hydrophobic interactions with LYS A:458 and VAL A:435.

[0061] Example 4: Anti-inflammatory activity analysis of frog meat protein-derived bioactive peptides

[0062] 4.1 Establishment of LPS-induced inflammation model in RAW264.7 cells

[0063] Adjust the RAW264.7 cell concentration to 2×10⁻⁶. 5Cells were seeded per well in 24-well plates. After incubation for 24 hours, the culture medium was discarded, and different concentrations of peptide solutions were added. The blank control group and the model group were incubated with culture medium only. After incubation for 12 hours, 50 μL of 10 μg / mL LPS solution (final concentration 1 μg / mL) was added to the model group and the sample group, while the blank control group was incubated with culture medium only. Incubation continued for 12 hours and 24 hours. The cell culture supernatant was collected and relevant indicators were measured.

[0064] 4.2 Determination of NO release in LPS-induced RAW264.7 cells under inflammation

[0065] The supernatant was collected and the NO release of RAW264.7 cells was measured using a NO kit after 12 h and 24 h of LPS induction, respectively, to investigate the effect of peptides on inflammatory response.

[0066] Immune cell proliferation is a crucial manifestation of the immune response; macrophages can respond to infection or inflammation through proliferation. Therefore, we further constructed an inflammation model to investigate the effects of GIHETTYNS and IVRDIKEK on NO secretion. Figure 4 As shown, after treatment with GIHETTYNS and IVRDIKEK, the NO release from RAW264.7 cells showed a decreasing trend with increasing LPS induction time. Specifically, after 24 h of LPS induction, IVRDIKEK at 1 μg / mL and 1.25 μg / mL reduced NO release by 32.40% and 21.04%, respectively, compared to the model group. Our results indicate that GIHETTYNS and IVRDIKEK at low concentrations can significantly alleviate inflammatory states, thereby participating in the immune response.

[0067] Example 5: Physicochemical properties of frog meat protein-derived bioactive peptides

[0068] Small molecule peptides possess a variety of functional active groups and exhibit superior biological activity compared to whole proteins; however, their safety and stability remain significant concerns in food processing. Therefore, we further analyzed the physicochemical properties of immunomodulatory peptides using bioinformatics. As shown in Table 2, both immunomodulatory peptides were non-toxic, indicating high safety and suitability for food applications. Furthermore, both peptides exhibited good hydrophobicity, with hydrophobic forces of +14.99 Kcal / mol and +19.88 Kcal / mol, respectively, consistent with molecular docking results. In addition, both peptides had instability coefficients less than 40 and pI values ​​of 5.24 and 8.59, respectively, indicating stability and preservation of biological activity during processing and digestion. This provides some reference for the application of peptides in food processing.

[0069] Table 2 Physicochemical properties of immunomodulatory peptides analyzed by bioinformatics

[0070]

[0071] Instability Index a Peptides with an index less than 40 are considered stable, while peptides with an index greater than 40 are considered unstable.

[0072] This study obtained novel immunomodulatory peptides GIHETTYNS and IVRDIKEK from the hydrolysate of spiny-breasted frog meat. These peptides promoted the proliferation of RAW264.7 cells and accelerated the cell cycle, promoted the production of NO and cytokines (IL-6, TNF-α), and increased ROS levels, exhibiting good cellular immune activity. Inflammation experiments showed that GIHETTYNS and IVRDIKEK could alleviate LPS-induced inflammatory responses in RAW264.7 cells and reduce NO release. Molecular docking results indicated that these peptides could bind to key regions of TLR4 / MD-2, thereby exerting immunomodulatory activity. Bioinformatics analysis revealed that these peptides possess safety and stability. These results suggest that the spiny-breasted frog meat immunomodulatory peptides GIHETTYNS and IVRDIKEK could serve as a good source of natural immunomodulators and anti-inflammatory agents.

[0073] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of a frog meat protein-derived bioactive peptide in the preparation of an anti-inflammatory agent, characterized in that: The amino acid sequence of the frog meat protein source active peptide is: Ile-Val-Arg-Asp-Ile-Lys-Glu-Lys.

2. The application according to claim 1, characterized in that: The active peptide is one of an oral preparation or a topical preparation.

3. The application of a solid-phase synthesized product in the preparation of anti-inflammatory agents, characterized in that: The solid-phase synthesis product is a frog meat protein-derived bioactive peptide; the amino acid sequence of the frog meat protein-derived bioactive peptide is as follows: Ile-Val-Arg-Asp-Ile-Lys-Glu-Lys.

4. The application of a nucleic acid in the preparation of an anti-inflammatory agent, characterized in that: The nucleic acid encoding the frog meat protein source active peptide; the amino acid sequence of the frog meat protein source active peptide is as follows: Ile-Val-Arg-Asp-Ile-Lys-Glu-Lys.

5. The application of a biomaterial in the preparation of an anti-inflammatory agent, characterized in that: The biological material is an expression cassette, transposon, vector, or host cell containing nucleic acid encoding a frog meat protein-derived bioactive peptide; the amino acid sequence of the frog meat protein-derived bioactive peptide is as follows: Ile-Val-Arg-Asp-Ile-Lys-Glu-Lys.

Images