A composition containing fish oil and polypeptides and its use in anti-inflammation

Abstract

The application belongs to the technical field of biology, and specifically discloses a kind of composition containing fish oil and polypeptide and its use in anti-inflammation.The core active ingredient of the anti-inflammatory composition is fish oil and anti-inflammatory polypeptide P1, P2 and P3 from deep-sea fish, and the amino acid sequences are SEQ ID NO.1:QPMPANCNSTG, SEQ ID NO.2:VQWMMNHF and SEQ ID NO.3:TETSQKE.Three polypeptides are naturally safe and have no cytotoxicity, can significantly down-regulate the NO production of inflammatory cells, and strongly inhibit the secretion of core pro-inflammatory factors.The application scientifically compounds fish oil and anti-inflammatory polypeptide from deep-sea fish, and the two produce significant synergistic anti-inflammatory effect after compounding, and the anti-inflammatory effect is much better than single fish oil or single polypeptide, which can be widely applied to the preparation of anti-inflammatory drugs, anti-inflammatory health foods, anti-inflammatory functional dietary supplements and skin anti-inflammatory repair cosmetics, and has broad industrialization popularization and clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a composition containing fish oil and polypeptides and its use in anti-inflammatory purposes. Background Technology

[0002] Inflammation is a physiological defense response of the body's immune system to harmful external stimuli, pathogen infection, tissue damage, and oxidative stress. A moderate inflammatory response helps the body resist external damage and repair damaged tissues. However, excessive activation of the immune system leading to chronic and persistent inflammation results in the accumulation of large amounts of inflammatory mediators and pro-inflammatory cytokines, continuously damaging normal tissues and organs. Clinical studies have shown that chronic low-grade inflammation is a common core pathological cause of many chronic diseases, including arthritis, cardiovascular and metabolic diseases, intestinal inflammation, sensitive and red skin, and age-related inflammatory lesions.

[0003] Currently, commonly used anti-inflammatory agents in clinical practice are mainly chemically synthesized drugs such as nonsteroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids. These drugs have a rapid onset of anti-inflammatory effects, but long-term continuous use or topical application can easily cause various toxic side effects and adverse reactions, such as gastrointestinal mucosal damage, metabolic burden on liver and kidney function, immune dysfunction, and skin barrier damage. They are not suitable for long-term daily health care or long-term conditioning use by the general population. With the increasing awareness of public health protection, natural, non-toxic, mild, safe, and suitable for long-term use, naturally derived anti-inflammatory active products have become the mainstream of market research and consumption. The research and industrialization of food-derived natural anti-inflammatory active substances has become a research hotspot in the fields of biomedicine and health food.

[0004] Fish oil is a widely used natural active ingredient in the health supplement industry, with its core active ingredient being... Unsaturated fatty acids, primarily including EPA and DHA, possess basic functions such as mild anti-inflammatory effects, regulation of vascular status, and improvement of metabolism. However, fish oil alone has a limited anti-inflammatory target and limited intensity, making it insufficient for potent anti-inflammatory effects when used alone. Furthermore, high-dose supplementation can lead to lipid metabolism burden and gastrointestinal discomfort. Bioactive peptides, on the other hand, are small-molecule short peptides obtained from natural proteins through targeted enzymatic hydrolysis, separation, and purification. They possess numerous advantages, including small molecular weight, easy absorption by the human body, specific biological activity, low cytotoxicity, and good biocompatibility. Among these, deep-sea fish-derived bioactive peptides have been extensively studied and proven to precisely regulate inflammatory signaling pathways, significantly downregulate the release of NO inflammatory mediators, and effectively inhibit... It secretes core pro-inflammatory factors, exhibits clear anti-inflammatory activity, and has extremely high safety.

[0005] In existing technologies, fish oil anti-inflammatory products and single-fish-derived anti-inflammatory peptide products have been developed and marketed. However, there is currently no related technology or product development for the scientific combination of fish oil with multiple high-activity anti-inflammatory peptides from deep-sea fish. Based on the deficiencies of existing technologies, this invention innovatively prepares a composition by combining high-purity fish oil with anti-inflammatory peptides from deep-sea whitebait. Utilizing the different anti-inflammatory targets of fish oil and peptides, a synergistic effect is achieved, significantly enhancing the overall anti-inflammatory activity, reducing the dosage of single raw materials, and avoiding the side effects of chemical anti-inflammatory drugs and the weak anti-inflammatory effects of single natural raw materials. This composition combines the advantages of potent anti-inflammatory effects, safety and non-toxicity, and suitability for long-term use, possessing extremely high industrial application value. Summary of the Invention

[0006] To solve the aforementioned problems, the present invention adopts the following technical solution: This invention provides an anti-inflammatory polypeptide derived from deep-sea fish, wherein the anti-inflammatory polypeptide is selected from polypeptide P1, polypeptide P2 and polypeptide P3.

[0007] Furthermore, the amino acid sequence of the polypeptide P1 is SEQ ID NO.1: QPMPANCNSTG.

[0008] Furthermore, the amino acid sequence of the polypeptide P2 is SEQ ID NO.2: VQWMMNHF.

[0009] Furthermore, the amino acid sequence of the polypeptide P3 is SEQ ID NO.3: TETSQKE.

[0010] Furthermore, the anti-inflammatory peptide is derived from whitebait.

[0011] The present invention also provides an anti-inflammatory composition comprising deep-sea fish-derived anti-inflammatory peptides and fish oil.

[0012] Furthermore, in the fish oil The total purity of unsaturated fatty acids is ≥85%.

[0013] Furthermore, the composition also includes pharmaceutically, culinary, or cosmetically acceptable conventional excipients.

[0014] The present invention also provides the use of the aforementioned anti-inflammatory peptides or anti-inflammatory compositions in the preparation of anti-inflammatory products.

[0015] Furthermore, the product can be any one of the following: medicine, health product, food, and cosmetic.

[0016] The present invention also provides a medicine containing the aforementioned anti-inflammatory polypeptide or anti-inflammatory composition.

[0017] Furthermore, the dosage form of the drug includes capsules, tablets, granules, oral liquids, gels, or creams.

[0018] Compared with the prior art, the present invention has the following advantages and beneficial effects: First, this invention protects three deep-sea fish-derived anti-inflammatory peptides, P1, P2, and P3. These peptides are naturally sourced, highly pure, non-cytotoxic, and biocompatible. Screening has determined that P1 has the best anti-inflammatory effect, making it suitable for long-term anti-inflammatory conditioning use by various populations, avoiding the side effects of chemical anti-inflammatory drugs. Second, the combination of fish oil and deep-sea fish-derived peptide P1 forms a synergistic anti-inflammatory effect, far superior to either peptide P1 or fish oil alone, significantly enhancing anti-inflammatory activity, reducing the dosage of raw materials, and minimizing metabolic burden. Third, the composition of this invention has a simple preparation process, readily available raw materials, and low industrialization difficulty, allowing for flexible preparation into various dosage forms such as pharmaceuticals, health products, and cosmetics. Fourth, the composition of this invention can simultaneously target and inhibit NO inflammatory mediators and... It contains core pro-inflammatory factors and has comprehensive anti-inflammatory targets, demonstrating good intervention effects on both acute and chronic inflammation, and has broad application prospects. Example

[0019] The present invention will be further illustrated below with reference to specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the following embodiments can be obtained from conventional commercial sources or by existing known methods. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0020] Experimental materials: Fresh deep-sea white mullet (purchased directly from deep-sea fisheries via cold chain, with low-temperature quick-freezing treatment throughout the process); fish oil (KinOmega 5530, Total purity ≥85% was purchased from Sichuan Xinmeijia Biomedical Co., Ltd.; trypsin (enzyme activity ≥4000U / g, 64006737) was purchased from Sinopharm Chemical Reagent Co., Ltd.; DMEM high glucose cell culture medium and fetal bovine serum were purchased from Gibco, USA; LPS lipopolysaccharide and MTT cell proliferation and toxicity assay kits were purchased from Beijing Solarbio Science & Technology Co., Ltd.; Griess reagent nitric oxide assay kit, Enzyme-linked immunosorbent assay kit, Enzyme-linked immunosorbent assay (ELISA) kits were purchased from Shanghai Enzyme-Link Biotechnology Co., Ltd.; dexamethasone positive control drug (pharmaceutical standard, purity ≥98%) was purchased from the National Institutes for Food and Drug Control.

[0021] Instruments and equipment: The high-speed refrigerated centrifuge is manufactured by Anhui Zhongke Zhongjia Scientific Instruments Co., Ltd. The vacuum freeze dryer is manufactured by Beijing Songyuan Huaxing Technology Development Co., Ltd. The dextran gel chromatography separation system (with matching chromatography column and automatic collector) is manufactured by GE Healthcare. The reversed-phase high-performance liquid chromatograph was manufactured by Agilent Technologies, Inc., USA. The liquid chromatography-mass spectrometry system is manufactured by Thermo Fisher Scientific, Inc., USA; the Multiskan FC fully automated microplate reader is also manufactured by Thermo Fisher Scientific, Inc., USA. The carbon dioxide cell incubator is manufactured by Shanghai Yuejin Medical Instrument Co., Ltd. The ultrapure water preparation machine is manufactured by Merck Millipore GmbH, Germany; the FA2004 electronic analytical balance is manufactured by Shanghai Liangping Instrument Co., Ltd.; and the high-speed tissue homogenizer is manufactured by Ningbo Xinzhi Biotechnology Co., Ltd.

[0022] Data statistics: Data analysis was performed using GraphPad Prism 8.0 statistical software. Quantitative data were expressed as mean ± standard error (mean ± SEM). Independent samples t-tests were used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. p < 0.05 or p < 0.05 was considered statistically significant. Example 1

[0023] This embodiment provides a method for preparing anti-inflammatory peptides derived from deep-sea fish, including the following steps: S1. Raw material pretreatment: Select fresh deep-sea white mullet, remove the fish head, fish viscera, fish bones and fish skin by-products, and keep only pure fish meat. Wash to remove surface impurities and blood, add ultrapure water at a ratio of 2-6 times the weight of fish meat, and use a high-speed tissue homogenizer to homogenize the fish meat to obtain uniform homogenized fish meat material. S2. Digestion and enzymatic hydrolysis: Adjust the pH of the homogenized material to 7.5, add trypsin (enzyme activity ≥4000U / g, purchased from Sinopharm Chemical Reagent Co., Ltd., 64006737) at a ratio of 2% of the total mass of the homogenized material, mix well and place in a constant temperature water bath at 37℃ for 8 hours for enzymatic hydrolysis; after the enzymatic hydrolysis is completed, place the hydrolyzed material in a boiling water bath for 10 minutes to terminate the enzymatic hydrolysis reaction, and let it cool naturally to room temperature to obtain white cuttlefish enzymatic hydrolysate; S3. Centrifugation and crude peptide preparation: The prepared enzymatic hydrolysate was placed in a high-speed refrigerated centrifuge and centrifuged at 4℃ and 8000rpm for 20min. After centrifugation, the supernatant was collected and freeze-dried using a vacuum freeze dryer to obtain white cuttlefish crude peptide powder, which was stored at low temperature and protected from light for later use. S4. Ultrafiltration fractionation: Prepare a uniform aqueous solution of white cuttlefish crude peptide powder and separate it using an ultrafiltration membrane to retain small molecule ultrafiltration components with a molecular weight of less than 1500 Da. After freeze-drying the active ultrafiltration components, store them in a sealed container at -18℃. S5. Gel Chromatography Purification: The ultrafiltration active component was prepared into an aqueous solution with a concentration of 10 mg / mL. After filtration through a 0.22 μm aqueous microporous membrane, it was separated by Sephadex G-15 dextran gel chromatography. The chromatographic conditions were set as follows: sample loading volume 4 mL, elution flow rate 1 mL / min, detection wavelength 220 nm, and double-distilled water as the eluent. After chromatographic separation, the three components F1, F2, and F3 were collected and freeze-dried for later use. S6. High-performance liquid chromatography (HPLC) for fine purification: The three active components were separated and purified separately using an Agilent 1260 RP-HPLC reversed-phase HPLC system. Mobile phase A was an aqueous solution containing 0.1% TFA, and mobile phase B was an acetonitrile solution containing 0.1% TFA. The gradient elution program was as follows: 0 min 2% B, 0-18 min 2%-10% B, 18-30 min 10%-30% B, 30-45 min 30%-50% B, 45-50 min 50%-70% B, and 50-60 min 70%-10% B. The chromatographic column was a Hypersil GOLD C18 analytical column, with an injection volume of 10 μL and a flow rate of 1 mL / min. The target eluted components were collected separately. S7. Mass Spectrometry Identification and Synthetic Purification: The purified components were sequenced using an LThermo Q Exactive LC-MS / MS liquid chromatography-mass spectrometry system, confirming the presence of three anti-inflammatory peptides: peptide P1 (SEQ ID NO.1: QPMPANCNSTG, molecular weight 1119.24 Da), peptide P2 (SEQ ID NO.2: VQWMMNHF, molecular weight 1092.31 Da), and peptide P3 (SEQ ID NO.3: TETSQKE, molecular weight 821.84 Da). The three peptides were synthesized artificially using a solid-phase synthesis method. After purification, the purity of all peptides was ≥98%, meeting the requirements for subsequent activity experiments. Example 2

[0024] (1) Test bacteria: Escherichia coli (ATCC 13706, Beijing BioBio Biotechnology Co., Ltd.) and Staphylococcus aureus (ATCC 25923, Beijing BioBio Biotechnology Co., Ltd.) were selected as inflammation-related pathogens. The strains were activated by slant passage on LB solid medium and continuously activated in a 37℃ constant temperature bacterial incubator for 18h. Single colonies after activation were picked and inoculated into LB liquid proliferation medium and amplified by shaking in a constant temperature shaker at 37℃ and 180rpm for 12h to prepare the bacterial stock solution. The bacterial stock solution was serially diluted with sterile physiological saline to adjust the absorbance OD600 value of the bacterial suspension to 0.6±0.02, corresponding to a bacterial cell concentration of approximately spare.

[0025] (2) Sample preparation: Weigh out solid powders of polypeptide P1, polypeptide P2 and polypeptide P3 respectively, and dissolve them thoroughly with sterile physiological saline to prepare a 100 μg / mL sterile working solution of polypeptide; the positive control is prepared with gentamicin sulfate standard (SG8690, Beijing Solarbio Science & Technology Co., Ltd.) to prepare a 100 μg / mL sterile working solution of positive drug; all samples and positive drugs are sterilized by filtration through a 0.22 μm filter membrane for later use.

[0026] (3) Experimental methods: LB solid plate culture medium was prepared in advance. After cooling and solidification at room temperature, 0.1 mL of the corresponding test bacterial suspension prepared above was evenly spread on each plate. After even spreading, the plate was allowed to stand and dry for later use. Sterile Oxford cups (6 mm outer diameter, 4 mm inner diameter) were placed vertically and stably on the surface of the solid culture medium plate with bacterial suspension. The plates were pressed together to ensure no gaps. 200 μL of the corresponding polypeptide or positive control sample was accurately added to each Oxford cup. An equal volume of sterile physiological saline was added to the blank control group. After the samples were added, all plates were placed in a 4℃ refrigerator for low-temperature pre-diffusion for 2 h to allow each sample to diffuse evenly into the culture medium. Then, the plates were transferred to a 37℃ constant temperature bacterial incubator and incubated upside down for 24 h. After the incubation, the diameter of the transparent inhibition zone was measured, and the data of each group of parallel samples were recorded and the average value and standard deviation were calculated.

[0027] Table 1. Antibacterial effect of the polypeptides of the present invention.

[0028] As shown in Table 1, the antibacterial experiment results indicate that the blank control group did not produce any clear inhibition zone, while the positive control group (gentamicin sulfate) exhibited strong antibacterial activity against both tested pathogens. Peptides P1, P2, and P3, under the same experimental concentrations and culture conditions, all demonstrated significant in vitro antibacterial activity against two types of inflammatory pathogens: *Escherichia coli* and *Staphylococcus aureus*, effectively inhibiting their growth and reproduction and possessing excellent adjunctive anti-inflammatory potential. Among them, peptide P1 showed the closest antibacterial effect to the positive control drug, with significantly better antibacterial activity than peptides P2 and P3. Specifically, peptide P1 showed an inhibition zone diameter of 18.62±0.58 mm against *Escherichia coli* and 20.15±0.62 mm against *Staphylococcus aureus*, demonstrating outstanding antibacterial effects close to the positive control group. Peptide P2 exhibited moderate antibacterial activity, while peptide P3 showed the weakest adjunctive antibacterial and anti-inflammatory effect in vitro. Peptide P1 demonstrated the best in vitro antibacterial and anti-inflammatory potential, showing excellent inhibition of inflammatory-related bacteria. Example 3

[0029] (1) Experimental grouping: Eight experimental groups were set up, with three replicates in each group. The final concentrations of samples in each group were as follows: 1. Blank control group: Equal volume of complete cell culture medium, no modeling or drug administration; 2. Model control group: LPS modeling + equal volume of complete cell culture medium, no anti-inflammatory sample intervention; 3. Positive control group: LPS modeling + dexamethasone (final drug administration concentration 10 μg / mL); 4. Low-dose P1 group: LPS modeling + peptide P1 (final drug administration concentration 50 μg / mL); 5. High-dose P1 group: L 6. LPS modeling + peptide P1 (final concentration 200 μg / mL); 7. Single fish oil group: LPS modeling + fish oil (final concentration 100 μg / mL); 8. Low-dose P1 + fish oil combination group: LPS modeling + peptide P1 (final concentration 50 μg / mL) + fish oil (final concentration 100 μg / mL); 9. High-dose P1 + fish oil combination group: LPS modeling + peptide P1 (final concentration 200 μg / mL) + fish oil (final concentration 100 μg / mL). All working solutions were prepared using cell culture medium, sterilized by filtration through a 0.22 μm sterile filter membrane, and used immediately after preparation.

[0030] (2) Cell modeling: RAW264.7 macrophages (CL-0190, Wuhan Pronosei Life Science Technology Co., Ltd.) were thawed and then... Cells were seeded per well in 96-well plates and cultured for 24 hours using standard adherent culture. Except for the blank control group, the other groups were continuously stimulated with 1 μg / mL LPS for 24 hours to construct an inflammation model. After modeling, the corresponding concentrations of sample and control solution were added according to the group for 24 hours of intervention. After the intervention, the cells were placed in a constant temperature incubator for another 24 hours. After the culture was terminated, the cell supernatant and cell samples were collected, and various indicators were detected.

[0031] (3) Indicator detection: The MTT assay was used to detect the cell viability of each group, accurately evaluate the biosafety of each sample to RAW264.7 inflammatory macrophages, and ensure that the test samples had no cytotoxic side effects; the Griess assay was used in strict accordance with the NO detection kit instructions to measure the secretion of NO, a key inflammatory mediator, in the cell supernatant of each group; the double antibody sandwich ELISA enzyme-linked immunosorbent assay was used, strictly following the instructions. The procedure for using an enzyme-linked immunosorbent assay (ELISA) kit is as follows: core pro-inflammatory factors in cell supernatant are measured. The protein secretion expression level was determined. All experimental data were independently repeated three times, and the results are expressed as mean ± standard deviation (x ± s). Statistical analysis of differences between groups was performed using an independent samples t-test.

[0032] Table 2. Experimental verification of the anti-inflammatory effect of the polypeptide composition of the present invention.

[0033] Table 2 shows that the cell viability of each group was not significantly different from that of the blank control group, and all experimental samples showed no cytotoxicity, indicating good biosafety. Compared with the blank control group, the model control group showed a highly significant increase in all inflammatory markers, indicating that the inflammation model was successfully constructed. Compared with the model control group, the positive control, the low- and high-dose P1 group, the fish oil group, and the P1 and fish oil combination group all significantly reduced NO levels. The levels of inflammatory factors were measured (P<0.05 or P<0.01); and the anti-inflammatory effect was dose-dependent, as shown in the low-dose and high-dose P1 groups. The low-dose P1 + fish oil combination group and the high-dose P1 + fish oil combination group showed significant differences in anti-inflammatory effect compared to the low-dose P1 group (P<0.01), indicating that the combination of peptide P1 and fish oil in this invention can synergistically increase the anti-inflammatory effect compared to P1 peptide or fish oil alone. Example 4

[0034] Take 70 parts by weight of fish oil and 15 parts by weight of polypeptide P1, mix them evenly to obtain the core anti-inflammatory composition; add 10 parts by weight of food-grade microcrystalline cellulose and 5 parts by weight of magnesium stearate as conventional excipients, stir and mix thoroughly, and fill into gelatin empty capsules using conventional capsule filling equipment to prepare an anti-inflammatory health food capsule preparation. The net content of a single capsule is 0.5g. Store at room temperature in a sealed container away from light.

[0035] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. A deep-sea fish-derived anti-inflammatory polypeptide, characterized in that, The anti-inflammatory peptides are selected from peptides P1, P2, and P3; the amino acid sequence of peptide P1 is SEQ ID NO.1: QPMPANCNSTG; the amino acid sequence of peptide P2 is SEQ ID NO.2: VQWMMNHF; and the amino acid sequence of peptide P3 is SEQ ID NO.3: TETSQKE.

2. The anti-inflammatory polypeptide according to claim 1, characterized in that, The anti-inflammatory polypeptide is selected from polypeptide P1, and its amino acid sequence is SEQ ID NO.1: QPMPANCNSTG.

3. The anti-inflammatory polypeptide according to any one of claims 1-2, wherein the anti-inflammatory polypeptide is derived from whitebait.

4. An anti-inflammatory composition containing fish oil and polypeptides, characterized in that, The composition comprises the deep-sea fish-derived anti-inflammatory peptides as described in any one of claims 1-3 and fish oil.

5. The anti-inflammatory composition according to claim 4, characterized in that, The fish oil The total purity of unsaturated fatty acids is ≥85%.

6. The anti-inflammatory composition according to any one of claims 4-5, characterized in that, The composition also includes pharmaceutically, food-grade, or cosmetically acceptable conventional excipients.

7. Use of the anti-inflammatory polypeptide of any one of claims 1-3 or the anti-inflammatory composition of any one of claims 3-6 in the preparation of anti-inflammatory products.

8. The use according to claim 7, characterized in that, The product can be any one of the following: medicine, health product, food, or cosmetic.

9. A medicament comprising the anti-inflammatory polypeptide of any one of claims 1-3 or the anti-inflammatory composition of any one of claims 3-6.

10. The medicament according to claim 9, characterized in that, The dosage forms of the drug include capsules, tablets, granules, oral liquids, gels, or creams.

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