Euphausia superba peptide and application thereof in preparation of immunomodulatory drugs
By screening and preparing Antarctic krill peptides, the problem of underutilization of Antarctic krill resources has been solved, efficient preparation of immunomodulatory drugs has been achieved, immune function has been enhanced and the development value of Antarctic krill has been increased.
Patent Information
- Application Number
- CN202510699204.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-28
- Publication Date
- 2025-09-05
AI Technical Summary
Antarctic krill resources have not been fully developed, bioactive substances such as proteins have not been effectively utilized, and there is a lack of efficient methods for preparing immunomodulatory drugs.
By preparing Antarctic krill peptides, including defatting, hydrolysis, centrifugation, spray drying and other steps, the bioactive peptides DIFDPL, KPWALT, LPFQRL, FYFF, VDDHFLF, RDWPEGRG, and DDHFLF were screened out for application in immunomodulatory drugs.
The prepared Antarctic krill peptide has no cytotoxicity, promotes cell proliferation, enhances cell phagocytosis ability, improves immune function, has significant reducing and antioxidant capabilities, and enhances immune effects.
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Figure CN120590470A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of functional polypeptide preparation, and particularly relates to an Antarctic krill peptide and its application in the preparation of immunomodulatory drugs. Background Art
[0002] Antarctic krill (Euphausia superba), with an estimated annual production of 1 billion tons, is the largest untapped marine resource. It is a keystone species in the Southern Ocean ecosystem. Its enormous biomass makes it a crucial factor in nutrient cycling, carbon export, and trophic interactions in the South Pacific. It is primarily found in the Antarctic waters of the South Pacific. Antarctic krill is rich in protein, comprising 16% of its fresh weight. The basic amino acid profile of Antarctic krill protein fully meets FAO / WHO / UNU requirements, making it considered a high-quality marine protein and a potential source of protein, lipids, and chitin. However, consumption of Antarctic krill accounts for less than 20% of the total resource, making it a valuable resource for development.
[0003] Bioactive substances, also known as physiologically active substances, are compounds with biological activity. They are trace or small amounts of components that affect life phenomena. Therefore, many components of Antarctic krill are being developed into functional foods. The high-quality protein contained in Antarctic krill is one of the bioactive substances. Antarctic krill protein hydrolysates have attracted widespread attention as a key bioactive substance added to functional foods. Bioactive peptides from different sources may have different functional activities and targeting capabilities due to their natural composition and preparation methods. Summary of the Invention
[0004] The present invention provides an Antarctic krill peptide.
[0005] The present invention further provides the use of the Antarctic krill peptide in the preparation of immunomodulatory drugs.
[0006] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are as follows: The present invention provides an Antarctic krill peptide, which is composed of the following peptide segments: DIFDPL (shown in SEQ ID No. 1), KPWALT (shown in SEQ ID No. 2), LPFQRL (shown in SEQ ID No. 3), FYFF (shown in SEQ ID No. 4), VDDHFLF (shown in SEQ ID No. 5), RDWPEGRG (shown in SEQ ID No. 6), and DDHFLF (shown in SEQ ID No. 7).
[0007] The present invention also provides a method for screening the Antarctic krill peptide, comprising the following steps: (1) defatting the Antarctic krill peptide powder and then air-drying it to obtain an air-dried product; (2) Add the air-dried material to distilled water, add neutral protease at a ratio of 2% of the dry sample weight, and perform hydrolysis reaction. After the reaction is completed, inactivate the enzyme, centrifuge, and take the supernatant; add Ca(OH)2 to the supernatant, mix well, let it stand, centrifuge, take the supernatant, and spray dry to obtain the Antarctic krill mixed peptide sample; (3) Screening of peptides with biological activity.
[0008] Preferably, in step (1), the Antarctic krill peptide powder is defatted by adding 95% ethanol.
[0009] Preferably, in step (2), the mass ratio of the air-dried material to distilled water is 1:10; and the amount of the neutral protease added accounts for 2% of the weight of the air-dried material.
[0010] Preferably, in step (2), the hydrolysis is carried out at 45-50° C. for 6 h.
[0011] Preferably, in step (2), the concentration of Ca(OH)2 is 95%; the amount of Ca(OH)2 added accounts for 0.55-0.65% of the volume of the supernatant; and the standing conditions are: standing at 20-25°C for 30-40 minutes.
[0012] The present invention further provides the use of the Antarctic krill peptide in the preparation of immunomodulatory drugs.
[0013] The beneficial effects of the present invention are: (1) The Antarctic krill mixed peptide sample prepared by the present invention has no cytotoxicity and can promote cell proliferation, enhance cell NO secretion, improve cell phagocytosis, and effectively enhance immunity. In addition, the Antarctic krill peptide prepared by the present invention is more easily absorbed and has significant reducing and antioxidant capabilities.
[0014] (2) The polypeptide provided by the present invention is a functional influencing factor with great potential in enhancing immunity. Different bioactive peptides have different mechanisms of action due to their amino acid composition and structural characteristics. The present invention provides theoretical support for the further development of immune-enhancing food using Antarctic krill peptides, thereby increasing the development value of Antarctic krill. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 The peptide composition of the Antarctic krill mixed peptide sample; Figure 2 This is the full-wavelength UV scan of the Antarctic krill mixed peptide sample; Figure 3 Infrared spectrum of Antarctic krill mixed peptide sample; Figure 4This is the relative molecular mass distribution diagram of the Antarctic krill mixed peptide sample; Figure 5 is the primary mass spectrum of peptide DDHFLF; Figure 6 This is the primary mass spectrum of the peptide DIFDPL; Figure 7 is the primary mass spectrum of peptide FYFF; Figure 8 This is the primary mass spectrum of peptide KPWALT; Figure 9 is the primary mass spectrum of peptide LPFQRL; Figure 10 This is the primary mass spectrum of the peptide RDWPEGRG; Figure 11 is the primary mass spectrum of peptide VDDHFLF; Figure 12 This is a graph showing the effect of peptide DIFDPL on the proliferation activity of RAW cells; Figure 13 This is a graph showing the effect of peptide KPWALT on the proliferation activity of RAW cells; Figure 14 This is a graph showing the effect of peptide LPFQRL on the proliferation activity of RAW cells; Figure 15 This is a graph showing the effect of peptide FYFF on the proliferation activity of RAW cells; Figure 16 This is a graph showing the effect of peptide RDWPEGRG on the proliferation activity of RAW cells; Figure 17 This is a graph showing the effect of peptide DDHFLF on the proliferation activity of RAW cells; Figure 18 This is a graph showing the effect of peptide VDDHFLF on the proliferation activity of RAW cells; Figure 19 The effect of Antarctic krill peptides on NO secretion in RAW cells induced by LPS; wherein a is the peptide DIFDPL, b is the peptide LPFQRL, c is the peptide DDHFLF, d is the peptide VDDHFLF, e is the peptide RDWPEGRG, f is the peptide FYFF, and g is the peptide KPWALT; Figure 20 The effect of peptide KPWALT on LPS-induced cytokine mRNA expression in RAW264.7 cells; a is COX-2, b is TNF-α, c is INOS, and d is IL-6; Figure 21 The effect of mixed peptide samples from Antarctic krill on the cell content of RAW264.7 cells after LPS induction. DETAILED DESCRIPTION
[0016] The technical solution of the present invention is further explained and illustrated by means of specific embodiments below.
[0017] The Antarctic krill peptide powder used in the present invention was purchased from Yuwang Group Marine Biotechnology Company.
[0018] The enzymatic activity of the neutral protease of the present invention is 100 U / mg.
[0019] Example 1 Antarctic krill peptide powder purchased from Weihai Yuwang Group Co., Ltd. was defatted in 95% ethanol and air-dried. Distilled water was added to the air-dried material at a material-to-liquid ratio of 1:10. Neutral protease was added at a ratio of 2% by weight of the dry sample and hydrolyzed at 45°C for 6 hours. The enzyme was inactivated, centrifuged, and the supernatant removed. A 0.6% Ca(OH)2 solution (95% by volume) was added, mixed, and allowed to stand at 20°C for 30 minutes. The supernatant was removed and spray-dried to obtain the Antarctic krill peptide mixture. The BIOPEP database (https: / / biochemia.uwm.edu.pl / biopep-uwm / ) was used to verify the peptide sequences. The bioactivity of the identified peptide sequences was then predicted using PeptideRanker. Peptides with a score greater than 0.5 are generally considered to have potential bioactivity. The physicochemical properties of the screened peptides, such as molecular weight, isoelectric point, hydrophobicity, and charge, were analyzed using PepDraw. The immunomodulatory activity of active peptides is closely related to the amino acid sequence composition, the ratio of hydrophobic amino acids and basic amino acid residues, etc.
[0020] (1) LC-MS / MS peptide identification AKP (a mixed peptide sample from Antarctic krill) was subjected to LC-MS / MS structural identification. The mass spectrometry information obtained from the test was compared with the corresponding protein database. AKP contained 104 peptide segments.
[0021] Figure 1 The peptide distribution diagram is shown in Table 1, where 6 peptides account for the largest proportion, with a total of 25. Table 1 is the peptide sequences of 2 to 10 amino acids and biological activity > 0.5 screened from 104 peptides. Table 1 Seven peptides were selected: DIFDPL, KPWALT, LPFQRL, FYFF, VDDHFLF, RDWPEGRG, and DDHFLF. Comparison with the Biopep database revealed no identical anti-inflammatory peptide sequences. These peptides were uniform in size (4 to 10 amino acids) but varied in amino acid composition and polarity.
[0022] Effect Example 1 Physicochemical Properties (1) UV full wavelength scanning Weigh an appropriate amount of AKP (mixed peptide sample from Antarctic krill) freeze-dried powder and prepare a 5 mg / ml Antarctic krill peptide solution with pure water. After adjusting the concentration to zero with pure water, scan the sample at the full UV wavelength range of 190-400 to observe whether it includes the characteristic absorption peaks of the peptide and the absorption peaks of impurities.
[0023] A 5 mg / ml Antarctic krill peptide solution was prepared with pure water. The results of ultraviolet full wavelength scanning are shown in the figure. The characteristic absorption wavelength range of Antarctic krill peptide is 250 nm ~ 260 nm, and the maximum characteristic absorption wavelength is 257.0 nm.
[0024] (2) Fourier transform infrared spectrometer analysis After AKP freeze-dried powder and potassium bromide were evenly mixed, 2 mg was taken out and ground thoroughly in an agate mortar, placed in a grinding tool, and pressed into tablets using a tablet press. The full wavelength was scanned in a Fourier transform infrared spectrometer, and the scanning results were analyzed using origin software.
[0025] AKP (mixed peptide sample from Antarctic krill) was scanned by infrared spectrum in the range of 4000~500cm-1. The results are as follows: Figure 3 It indicates that the functional groups of AKP can be analyzed. Antarctic krill polypeptide has a characteristic absorption peak of polypeptide. -1 There is a strong absorption peak of NH stretching vibration (amide A band) at 2963.50 cm -1 There is a characteristic absorption peak of CN stretching vibration (amide B band) at 1653.23 cm -1 There is a characteristic absorption peak caused by C=O stretching vibration (amide I band), 1565.09 cm -1 The characteristic absorption peak at 1399.71 cm-1 is attributed to the amide II band. -1 There are absorption peaks (amide III band) caused by C=O stretching vibration and NH bending vibration.
[0026] (3) Determination of relative molecular mass distribution Reverse-phase high-performance liquid chromatography (RP-HPLC) was used to analyze the relative molecular weight distribution of AKP. A 100 mg sample was placed in a 10 mL volumetric flask, diluted to volume with mobile phase, sonicated for 5 minutes, centrifuged, and filtered through a membrane filter before injection. Elution was performed using a mobile phase gradient of acetonitrile / water / trifluoroacetic acid (45 / 55 / 0.1, v / v / v) at a column temperature of 30°C and a wavelength of 220 nm at a flow rate of 0.5 mL / min.
[0027] In the Antarctic krill peptide mixed peptide sample, the molecular weight distribution of the peptides is shown in Table 2. The elution time of AKP is from 11min to 28min. Antarctic krill peptide is composed of a variety of components with different relative molecular weights. The molecular weight of the polypeptide is distributed in 180~10000Da. The polypeptide is mainly distributed below 1000Da, accounting for 65.99% of the Antarctic krill peptide, and the content of peptides greater than 2000Da accounts for only 5.68%. This shows that low molecular weight peptides account for the main components of Nanjing and krill peptides, with a relative molecular mass of less than 180Da accounting for 41.19% of AKP. Those less than 180Da are free amino acids, as shown in the following figure. Figure 4 The molecular weight of a peptide fragment is crucial to its biological activity. Generally, peptides with small molecular weight are more conducive to absorption and have higher biological activity.
[0028] Table 2 Molecular weight determination results of Antarctic krill peptides (4) Amino acid composition analysis The amino acid composition of AKP was analyzed with reference to the method of National Food Safety Standard Determination of Amino Acids in Food (GB 5009.124-2016).
[0029] As shown in Table 3, the amino acid composition of most immunomodulatory peptides contains basic amino acids such as lysine (K) and arginine (R), or branched-chain amino acids such as valine (V), leucine (L), and isoleucine (I). The total branched-chain amino acids (V+L+I) in Antarctic krill peptides is 13.68%, while the total basic amino acids (H+K+R) is 16.67%. This indicates that Antarctic krill peptides contain immunomodulatory peptides.
[0030] Table 3 Amino acid composition of Antarctic krill peptide Effect Example 2 Mechanism of Action of Antarctic Krill Peptide AKP Active Peptide (1) Synthesis of Antarctic krill peptide Six immunologically active peptides identified after mass spectrometry identification were synthesized using solid-phase peptide synthesis (commissioned to Nanjing Peptide Ancient Biotechnology Co., Ltd.). RP-HPLC-MS / MS analysis showed that the purity of the synthesized peptides was higher than 95%. Figure 5-Figure 11 These are the primary mass spectra of DDHFLF, DIFDPL, FYFF, KPWALT, LPFQRL, RDWPEGRG, and VDDHFLF.
[0031] (2) Molecular docking The target receptor protein TLR4 / MD-2 (ID: 5IJD) was obtained from the Protein Data Bank database. PyMOL 2.4.0 software was used to remove water and its own small molecule ligands. The receptor protein was modified by hydrogenation and charge balancing using AutoDock Tools software. Finally, molecular docking between the protein receptor and the peptide ligand was performed using AutoDock Vina 1.1.2 software, and the binding ability of the docking system was scored. The present invention selected Antarctic krill peptides (DIFDPL, KPWALT, LPFQRL, FYFF, VDDHFLF, RDWPEGRG, and DDHFLF) for molecular docking analysis. The results are shown in Table 4.
[0032] Table 4 Molecular docking results of binding energy between peptides and relative targets (3) Cell proliferation activity of synthetic peptides The cells in the logarithmic growth phase were blown off from the culture dish and counted using a cell counting plate. 5 Cells were seeded at 1 μg / ml in a 96-well plate and incubated in a 37°C incubator for 24 hours. The supernatant was discarded and 100 μl of AKP solution (1000, 2000, 4000, 6000, and 8000 μg / ml) was added to each well. Three replicates were set up for each well. A blank group without cells and a control group containing only complete medium were also established. LPS at a concentration of 1 μg / ml was added as a model group and incubated at 37°C for 24 hours. The supernatant was aspirated and 100 μL of 10% CCK-8 solution was added to each well. After incubation at 37°C for 2 hours, the absorbance was measured at 450 nm. Cell viability was calculated according to the following formula.
[0033] A : Absorbance value of the experimental group (absorbance value containing culture medium, cells, fatty acids and CCK-8) B : Absorbance value of the control group (absorbance value containing culture medium, cells and CCK-8) C : Blank group absorbance value (absorbance value containing culture medium and CCK-8) At 31~1000uM, different concentrations of DIF, KPW, LPF, FYFF, ERG, HFLF, and VDD peptides had no cytotoxicity to cells after 24h intervention in raw264.7 cells. The results showed that at 31~1000uM, all peptides had no obvious toxicity to cells. Figure 12-18 As shown, except for the FYFF peptide, the other six peptides all have obvious cell proliferation effects.
[0034] (4) Effects of synthetic peptides on the ability of cells to secrete NO after LPS induction like Figure 19 As shown, intervention with the DIF, LPF, HFLF, VDD, and ERG peptides did not significantly alter macrophage NO secretion. However, intervention with the FYFF peptide gradually decreased NO secretion with increasing concentration. The effect was most pronounced at a concentration of 500 μM, reducing NO secretion by 11.77% compared to the model group. The KPW peptide also showed the most significant effect at a concentration of 500 μM, reducing NO secretion by 26.23% compared to the model group. The KPW peptide was selected for further investigation into its ability to eliminate inflammation and its mechanism of action.
[0035] (5) Effects of synthetic peptides on cytokine mRNA secretion Total RNA Extraction and Reverse Transcription: Total RNA was extracted from cells using the Fastpure Cell / Tissue Total RNA Isolation Kit V2. The extracted RNA concentration was then measured using a nucleic acid detector. Reverse transcription was performed using the HiScript II Q Select RT SuperMix for qPCR Kit, and the resulting product was stored at -80°C. Real-time q-PCR: The above product was amplified using the ChamQ Universal SYBR qPCR Master Mix Kit and a PCR instrument. Amplification conditions included a pre-denaturation at 95°C for 5 minutes, followed by 40 cycles of denaturation at 95°C for 5 seconds and annealing at 60°C for 30 seconds. Gene expression was calculated, and data were normalized using the internal reference gene GAPDH.
[0036] The primer sequences used in this experiment are shown in Table 5.
[0037] Table 5 RT-qPCR primers After KPW peptides were used to intervene in cells, the mRNA expression levels of TNF-α, cox-2, INOS, and IL-6 were reduced to varying degrees. Figure 20 It is shown that KPW peptide can exert its anti-inflammatory effect by inhibiting the secretion of pro-inflammatory factors.
[0038] Effect Example 3 Anti-inflammatory Effect of Antarctic Krill Peptide (I) Effects of Antarctic krill peptide on LPS-induced NO secretion in cells The standard curve was drawn according to the standard operating procedures in the kit. RAW264.7 macrophages in the logarithmic growth phase were seeded in 6-well plates at a density of 2×10 5 / ml, incubated for 24 hours, the supernatant removed, and the experimental groups simultaneously added 1 μg / ml LPS and different concentrations of AKP (the Antarctic krill peptide mixture sample prepared in Example 1) (2000, 4000, and 6000 μg / ml). The positive control group received 1 μg / mL LPS and 50 μM DEX; the model group received 1 μg / mL LPS; and the blank group received only fresh culture medium. The final volume was 100 μL, and three replicates were set for each concentration. The cells were incubated in a 5% CO2, 37°C cell chamber for 24 hours. The supernatant was collected, 50 μL added to each well, and Griess Reagent I and Griess Reagent II were added sequentially. The absorbance was measured at 540 nm, and the standard curve was used to calculate the NO release.
[0039] Under LPS stimulation, cells produce a large amount of NO. After DEX treatment, cell NO secretion is significantly reduced. In the drug-treated group, as the concentration of added krill peptide increases, the amount of cell NO release is significantly reduced, indicating that Antarctic krill peptide can inhibit the release of inflammatory factor NO in a dose-dependent manner.
Claims
1. An Antarctic krill peptide, characterized in that: The Antarctic krill peptide is composed of the following peptide segments: DIFDPL, KPWALT, LPFQRL, FYFF, VDDHFLF, RDWPEGRG, and DDHFLF.
2. A method for screening Antarctic krill peptides according to claim 1, characterized in that: The following steps are involved: (1) defatting the Antarctic krill peptide powder and then air-drying it to obtain an air-dried product; (2) Add the air-dried material to distilled water, add neutral protease at a ratio of 2% of the dry sample weight, and perform hydrolysis reaction. After the reaction is completed, inactivate the enzyme, centrifuge, and take the supernatant; add Ca(OH)2 to the supernatant, mix well, let it stand, centrifuge, take the supernatant, and spray dry to obtain the Antarctic krill mixed peptide sample; (3) Screening of peptides with biological activity.
3. The screening method according to claim 2, characterized in that In step (1), the Antarctic krill peptide powder is defatted by adding 95% ethanol.
4. The screening method according to claim 2 or 3, characterized in that In step (2), the mass ratio of the air-dried material to distilled water is 1:10; and the amount of the neutral protease added accounts for 2% of the weight of the air-dried material.
5. The screening method according to claim 2 or 4, characterized in that In step (2), the hydrolysis is carried out at 45-50° C. for 6 h.
6. The screening method according to claim 2, wherein In step (2), the concentration of Ca(OH)2 is 95%; the amount of Ca(OH)2 added accounts for 0.55-0.65% of the volume of the supernatant; and the standing condition is: standing at 20-25°C for 30-40 minutes.
7. Use of the Antarctic krill peptide according to claim 1 in the preparation of immunomodulatory drugs.