IY series modified peptides with antioxidant activity and their applications

Abstract

This invention provides a series of modified IY peptides with antioxidant activity and their applications. The disclosed IY series modified peptides are modified versions of the parent peptide FR24. Their antioxidant activity was tested by in vitro free radical scavenging, and the IY series modified peptides showed improved antioxidant activity compared to the parent peptide FR24. Among them, IY-3 exhibited the best in vitro free radical scavenging activity. Furthermore, IY-3 also demonstrated excellent cytotoxicity and hemolytic activity. The IY series modified peptides disclosed in this invention possess antioxidant activity and can be used to prepare antioxidants and additives for applications in the food, health product, and pharmaceutical industries.

Description

Technical Field

[0001] This invention discloses an antioxidant-active IY series modified peptide and its applications. The IY series modified peptide is a modified form of the parent peptide FR24, which has antioxidant activity and relates to the fields of biotechnology and bioproducts. Background Technology

[0002] Antioxidant peptides have made significant contributions to improving human health by preventing and treating non-communicable chronic degenerative diseases such as diabetes, cardiovascular and cerebrovascular diseases, rheumatoid arthritis, and cancer. Furthermore, peptides with antioxidant properties possess effective metal ion chelating activity and lipid peroxidation inhibition capabilities, making them potential food processing additives. Food spoilage, including oil rancidity and pigment discoloration, leads to unpleasant odors and potential toxic reactions, affecting the nutritional value of food and shortening its shelf life. Therefore, finding effective antioxidants to reduce health and economic losses has become a current research hotspot.

[0003] As a bridge connecting physical principles and biological reactions, quantum chemistry plays a crucial role in drug discovery and activity prediction. In recent years, assessing the relationship between the structure and activity of antioxidants using quantum chemical calculations has become an emerging trend. Quantum chemistry, based on the Schrödinger equation, predicts the activity of substances through certain approximations and assumptions. Among these, semi-empirical algorithms and density functional theory are the most commonly used methods for discovering antioxidant peptides. Based on density functional theory, the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) are the two most fundamental frontier molecular orbitals possessed by a compound. The antioxidant activity of a substance can be reflected by calculating the band gaps of the HOMO and LUMO. Numerous studies have reported on assessing the relationship between the structure and activity of antioxidants using quantum chemical methods, but there are no reports on modifying antioxidant peptides using quantum chemical methods. Summary of the Invention

[0004] This invention provides an IY series modified peptide with strong antioxidant activity, which can be used to prepare antioxidants and additives for application in the food, health products and pharmaceutical industries.

[0005] This invention discloses an IY series modified peptide, wherein the first amino acid at the C-terminus of the IY series modified peptide is Ile, the last amino acid at the N-terminus is Tyr, and the central sequence of the IY series modified peptide is the same: LGWWYYPHYYFY.

[0006] The IY series modified peptide described in this invention includes:

[0007] IY-1; its amino acid sequence is as shown in SEQ ID No. 1 of the sequence listing;

[0008] IY-2; its amino acid sequence is as shown in SEQ ID No. 2 of the sequence listing;

[0009] IY-3; its amino acid sequence is as shown in SEQ ID No. 3 in the sequence listing;

[0010] IY-4, whose amino acid sequence is SEQ ID No. 4 in the sequence listing.

[0011] The IY series modified peptides of this invention are modified based on polypeptide FR24; the IY series modified peptides retain the high E HOMO Amino acids with low E values ​​will have low E values. HOMO One or more amino acids are deleted or replaced.

[0012] The amino acid sequence of FR24 is as follows:

[0013] FCDGISSVLGWCRYPHRRFRCTCR-NH2.

[0014] The sequence characteristics of the IY-1 modified peptide described in this invention are as follows:

[0015] The sequence consists of 20 amino acids. The first four amino acids from the N-terminus of the FR24 sequence are deleted, and Arg is replaced with Tyr and Ser. 6 and Ser 7 Replace with Trp 2 and Pro 3 Cys 12 Replace with Trp 8 Cys 21 Replace with Phe 17 Thr 22 Replace with Phe 18 Cys 23 Replace with Pro 19 The specific amino acid sequence is as follows:

[0016] IWPVLGWWYYPHYYFYFFPY-NH2.

[0017] The sequence characteristics of the IY-2 modified peptide described in this invention are as follows:

[0018] Composed of 20 amino acids, the Trp in the IY-1 sequence 2 Replace with Pro 2 Pro 3 Replace with Trp 3 Phe 18 Replace with Pro 18 The specific amino acid sequence is as follows:

[0019] IPWVLGWWYYPHYYFYFPPY-NH2.

[0020] The sequence characteristics of the IY-3 modified peptide described in this invention are as follows:

[0021] Composed of 20 amino acids, the Trp in the IY-1 sequence 2 Replace with Pro 2 Pro 3 Replace with Trp 3 Phe 17 Replace with Pro 17 The specific amino acid sequence is as follows:

[0022] IPWVLGWWYYPHYYFYPFPY-NH2.

[0023] The sequence characteristics of the IY-4 modified peptide described in this invention are as follows:

[0024] Composed of 20 amino acids, the IY-1 sequence contains Phe 17 Replace with Pro 17 Phe 18 Replace with Pro 18 Pro 19 Replace with Phe 19 The specific amino acid sequence is as follows:

[0025] IWPVLGWWYYPHYYFYPPFY-NH2.

[0026] This invention provides a series of modified peptides with antioxidant functions. The antioxidant activity of the antioxidant peptide FR24 and the IY series of modified peptides was detected according to the national standard "Determination of Antioxidant Activity of Peptides—DPPH and ABTS Methods" (GB / T 39100-2020). The IY series of modified peptides exhibits good scavenging effects against DPPH and ABTS free radicals.

[0027] As a preferred choice, IY-3 among the IY series modified peptides exhibits the strongest antioxidant activity and has a good scavenging effect on DPPH free radicals and ABTS free radicals.

[0028] The IY series antioxidant peptides provided by this invention have low cytotoxicity, exhibiting low cytotoxicity against mouse macrophages RAW264.7 and low hemolytic activity against mouse erythrocytes.

[0029] The beneficial effects of this invention are as follows: The precursor peptide FR24 was modified using quantum chemical calculations to obtain the IY series of modified peptides. Activity verification using the DPPH and ABTS methods showed good antioxidant activity. Toxicity assessment revealed low cytotoxicity and hemolytic activity. The antioxidant peptides obtained by this invention can be applied to food preservation, cosmetics, and health products, and are of great significance for the development of novel antioxidant products. Attached Figure Description

[0030] Figure 1 These are quantum chemical analysis diagrams during the modification process; where A is FR24, B is IY-1, C is IY-2, D is IY-3, and E is IY-4.

[0031] Figure 2 This study analyzes the antioxidant activity of FR24 and IY series modified peptides; where A represents DPPH free radical scavenging ability and B represents ABTS free radical scavenging ability.

[0032] Figure 3 These are transmission electron microscopy images of IY-3 nanoliposomes; where A is an empty nanoliposome and B is an IY-3 nanoliposome encapsulated with chitosan.

[0033] Figure 4 Fourier transform infrared spectrum of nanoliposomes;

[0034] Figure 5 The effect of nanoliposomes on the appearance of strawberries;

[0035] Figure 6 This study analyzes the preservation performance of strawberries coated with nanoliposomes. A represents weight loss assessment, B represents respiration intensity assessment, C represents total phenol content assessment, D represents ascorbic acid content assessment, E represents microbial contamination assessment, and F represents antioxidant activity assessment. Detailed Implementation

[0036] The present invention will now be illustrated with examples, but the invention is not limited to these examples. Unless otherwise specified, the techniques used in the examples are conventional methods well known to those skilled in the art, and the raw materials used are all commercially available products. All raw and auxiliary materials selected in the present invention are well known in the art, and all percentages mentioned in the present invention are by weight and volume percentages.

[0037] Example 1: Preparation of IY series modified peptides

[0038] The antioxidant peptide FR24, derived from *Nonomura jilinensis*, contains 24 amino acids. The electronic contributions of these amino acids were analyzed using quantum chemical calculations. Specifically, the HOMO-LOMO orbitals of the peptide were calculated using the semi-empirical method of PM7 built into the Gaussian program, and the positions of molecules with the strongest electron-contributing capabilities were predicted. The molecular structure of the peptide was plotted using ChemDraw 22.0.0, converted to a 3D structure using the built-in Chem3D tool, and energy minimization of the peptide conformation was achieved using the MM2 force field, with a minimum RMS gradient of 0.01. The results are as follows: Figure 1 As shown, the 20th amino acid Arg in FR24 occupies the highest electron orbital, representing the amino acid most prone to losing electrons. Since short peptides and peptides containing isoleucine (I), leucine (L), and alanine (A) at the N-terminus have stronger antioxidant activity, residues contributing low electron energy were deleted while retaining those contributing high electron energy. Simultaneously, amino acid I was exposed at the N-terminus, and substitutions of low-electron-contributing amino acid residues were attempted, resulting in the IY series of modified peptides IY-1, IY-2, IY-3, and IY-4; their amino acid sequences are SEQ ID Nos. 1-4 in the sequence listing. Information on FR24 and the IY series of modified peptides is shown in Table 1 below.

[0039] Table 1

[0040]

[0041] Based on the above amino acid sequence, its full sequence was synthesized by solid-phase synthesis, desalted by high performance liquid reverse-phase column chromatography, and its molecular weight was determined by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry. The purity of the purified peptide was identified by high performance liquid chromatography.

[0042] The measurement results are as follows:

[0043] The molecular weight of the antioxidant peptide modified IY-1 is 2758.12 Da.

[0044] The molecular weight of the antioxidant peptide modified IY-2 is 2758.12 Da.

[0045] The molecular weight of the antioxidant peptide modified IY-3 is 2758.12 Da.

[0046] The molecular weight of the antioxidant peptide modified IY-4 is 2758.12 Da.

[0047] Example 2: Determination of antioxidant activity of FR24 and IY series modified peptides

[0048] The antioxidant activity of the modified peptides was assessed. The method for detecting the DPPH radical scavenging ability was based on the national standard GB / T 39100-2020, Determination of Antioxidant Activity of Peptides, Method I (DPPH Method); the method for detecting the ABTS radical scavenging ability was based on the national standard GB / T 39100-2020, Determination of Antioxidant Activity of Peptides, Method II (ABTS Method). Results were expressed as the equivalent antioxidant capacity of vitamin E (Trolox) (μmol TE / μmol peptide).

[0049] The results are as follows Figure 2 As shown, all IY series modified peptides exhibited enhanced antioxidant activity compared to the parent peptide FR24. Among the IY series modified peptides, IY-3 showed the most significant improvement in scavenging activity against DPPH and ABTS free radicals, with an approximately 2-fold increase in scavenging activity against both free radicals.

[0050] Example 3: Toxicity assessment of FR24 and IY series modified peptides

[0051] With 2 × 10 per hole 5 Mouse macrophages RAW264.7 were seeded at a density of 10 cells per well in 96-well cell culture plates and incubated for 12 h in a constant temperature incubator with 5% CO2 and 37°C. Different concentrations of peptides were added at half-dilution and incubated for 24 h in a constant temperature incubator with 5% CO2 and 37°C. 10 μL of CCK-8 reagent was added to each well, mixed well, and incubated for 3 h in the dark. The half-maximal toxic concentration (CC50) of the cells was calculated by measuring the absorbance at 450 nm in each well using a microplate reader.

[0052] 2% (v / v) mouse erythrocyte dilution was mixed with different concentrations of antioxidant peptides, incubated at 37°C for 1 hour, centrifuged at 1000 g for 10 minutes, and the supernatant was collected. The absorbance at 540 nm was measured and the half-hemolysis value (HC50) was calculated. 1% Triton X-100 was used as a positive control, and physiological saline was used as a negative control. The specific results are shown in Table 2 below.

[0053] Table 2

[0054]

[0055] As shown in Table 2, the IY series modified peptides provided by this invention exhibit weak cytotoxicity against mouse macrophage RAW264.7 cells, with all half-maximal toxic concentrations (IC50) above 500 μM. FR24, at a concentration of 200 μM, showed a cell viability of less than 50%, indicating that the IY series modified peptides possess lower cytotoxicity than the parent peptide FR24. Furthermore, the half-maximal hemolytic values ​​(IC50) of the IY series modified peptides were also higher than those of the parent peptide FR24.

[0056] Example 4: Characterization of IY-3 nanoliposomes

[0057] As a preferred option, IY-3 among the IY series modified peptides exhibited the strongest antioxidant activity. Therefore, nanoliposomes loaded with IY-3 were prepared and characterized and evaluated for application.

[0058] Specifically, 0.18 g of lecithin, 0.02 g of cholesterol, and 10 μL of Tween-80 were dissolved in 20 mL of anhydrous ethanol. After complete dissolution, the solution was transferred to a round-bottom flask, and the solvent was evaporated using a rotary evaporator. After storing in a desiccator for 16 hours, 20 mL of peptide solutions of different concentrations were added to the membrane. The resulting mixture was vortexed for 2 minutes and stirred at 60 °C for 2 minutes, repeated 3 times. The mixture was then sonicated at 20 kHz for 10 cycles (1 minute on, 1 minute off). Chitosan was then dissolved in 1% (v / v) acetic acid and stirred for 16 hours. Finally, equal volumes of each chitosan solution were added dropwise to the nanoliposome solution. After diluting each sample 20-fold with distilled water, the average particle size, aggregation index, and zeta potential of the nanoliposomes were measured using a dynamic light scattering system at room temperature and a 90-degree angle.

[0059] Further, 0.5 mL of the liposome solution was mixed with 1 mL of acetone and centrifuged at 4 °C and 5,000 g for 30 minutes. The supernatant containing unencapsulated peptides was separated, and the solvent was evaporated at 60 °C. The peptides were then dissolved in distilled water, and the peptide content was determined using the Bradford method. The encapsulation efficiency was finally obtained by calculating the percentage of peptides loaded in the nanoliposomes relative to the total peptide content. The results are shown in Table 3 below:

[0060] Table 3

[0061] .

[0062] Note: Different letters in the same column represent significant differences (p < 0.05).

[0063] The average particle size of the blank-loaded liposomes (Blank-Lip) was 92.63 ± 2.18 nm. The particle size gradually increased after loading with different concentrations (0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, and 0.4 mg / mL) of IY-3. The PDI of different nanoliposome samples ranged from 0.18 to 0.47, indicating a relatively uniform particle size distribution. The encapsulation efficiency of the liposomes gradually increased with increasing IY-3 concentration. The highest encapsulation efficiency was observed when the IY-3 concentration reached 0.2 mg / mL. Therefore, liposomes containing 0.2 mg / mL of IY-3 were selected for chitosan encapsulation. The Zeta potential of Blank-Lip was -51.37 ± 0.95 mV, while liposomes containing peptides showed a gradual increase in charge due to the peptides surrounding and encapsulating within the liposomes.

[0064] Furthermore, the nanoliposome samples were stained with 1% phosphotungstic acid (pH = 6.5-7.0) for 10 s, placed on a copper grid, air-dried, and their morphology was observed by transmission electron microscopy at an accelerating voltage of 100 kV. See the appendix for detailed results. Figure 4 .

[0065] From the appendix Figure 4 The results show that the IY-3 nanoliposomes (IY-3-Lip) provided by this invention have a near-spherical structure, a vesicle structure, and no obvious aggregation or fusion phenomena. After coating with chitosan, the outer membrane thickness and number of layers of the liposomes increased, further demonstrating that chitosan was adsorbed on the outer surface of the liposomes, increasing the thickness and particle size of the liposomes.

[0066] Further, 0.2 g of dried potassium bromide and 0.002 g of the sample were mixed and ground, then pressed into transparent, uniform, crack-free tablets. The FTIR spectra of the samples were recorded using a Fourier transform infrared spectroscopy (FTIR) instrument. The broad spectral range was 400-4000 cm⁻¹. -1 The measurement resolution is 0.1 cm. -1 See the appendix for detailed results. Figure 5 .

[0067] From the appendix Figure 5 The results showed that after loading IY-3 onto nanoliposomes, the stretching vibration peak of CH2 decreased from 2852 cm⁻¹. -1 Moved to 2850 cm -1 This demonstrates that an ionic complexation occurred between the phospholipid and IY-3. 966 cm -1 The peak shifted to 969 cm. -1 This indicates that IY-3 exists in the lipid polar region, i.e., inside the vesicle. 1734 cm -1The peak at 1737 cm⁻¹ reflects the carbonyl (C=O) stretching vibration of the aliphatic ester bond at the junction of the hydrocarbon chain and the head group. -1 The wavenumber shifted to a higher value, confirming an interaction between the C=O group of the phospholipid and IY-3 via hydrogen bonding. After chitosan coating, some peaks in the liposomes were replaced by chitosan. 2850 cm⁻¹ -1 The CH2 stretching vibrations at the site did not shift significantly after chitosan coating, indicating that chitosan modification on the liposome surface has no effect on the bulk liposome structure. Infrared spectroscopy analysis showed that IY-3 is located in the polar region of the liposome, and chitosan can effectively coat the liposome surface.

[0068] Example 5: The effect of IY-3 nanoliposome coating on strawberry preservation

[0069] Strawberries of uniform size, without defects, and with a surface redness of 90% were selected to evaluate the effect of nanoliposome coating on strawberry quality and freshness. Strawberries in each group (n = 5) were immersed in the coating solution for 1 minute, with or without an antioxidant peptide coating, and then air-dried in a fume hood. Untreated strawberries served as a control. All strawberries were stored at 19–25°C and 40–50% humidity. Strawberries were photographed and weighed every 24 hours to calculate weight loss. See Appendix for detailed results. Figure 6 .

[0070] From the appendix Figure 6 The results showed that the quality of strawberries in the control group significantly declined during room temperature storage, exhibiting signs of rotting after 7 days. The strawberries shrank severely, and extensive mold growth was observed. In contrast, only one strawberry in the IY-3-Lip group showed localized mold, while the rest remained relatively fresh. The fruit coated with the chitosan-based IY-3 nanoliposome coating (CS-IY-3-Lip) showed almost no signs of mold growth, maintaining an intact appearance with only slight color darkening. Strawberries in the control group shrank severely after 7 days of storage, with a weight loss of 52.51 ± 4.7%. Under the same storage conditions, the strawberries in the CS-IY-3-Lip group experienced the least weight loss, at 14.97 ± 2.27%.

[0071] Furthermore, strawberries (1 kg) from different treatment groups were left under ambient conditions for 1 hour to allow the CO2 accumulated in the tissues to diffuse into the air. The strawberries were then transferred to a sealed container containing 10 mL of 0.4 mol / L NaOH solution. After 30 minutes, 2 drops of phenolphthalein were added, and the mixture was titrated with 0.2 mol / L oxalic acid. The respiration intensity of the strawberries was expressed as the CO2 production rate. See the appendix for detailed results. Figure 6 .

[0072] From the appendix Figure 6The results showed that the initial respiration rate of strawberries was approximately 56.94 mg CO2 / kg·h. After 3 days of storage, the respiration rate of strawberries coated with IY-3-Lip and CS-IY-3-Lip decreased to about half of its original rate. The respiration rate of the control group strawberries was higher than that of the coated strawberries during storage. After 7 days of storage, the respiration rate of strawberries coated with IY-3-Lip and CS-IY-3-Lip was significantly lower than that of the control group strawberries.

[0073] Furthermore, the total phenolic content of strawberries was determined using the Folin-Ciocalteu method. On days 0, 3, and 7, 0.1 g of strawberries were ground with liquid nitrogen, and 1.5 mL of pre-cooled 95% (v / v) methanol was added, followed by homogenization for 5 minutes. After incubation for 2 hours, the mixture was centrifuged at 10,000 g for 20 minutes. 100 μL of the supernatant was incubated with 200 μL of 10% (v / v) Folin-Ciocalteu phenol reagent for 5 minutes. The reaction was terminated by adding 800 μL of 0.7 mol / L Na₂CO₃, and the mixture was incubated in the dark for 2 hours. The absorbance of each sample at 765 nm was measured using a microplate reader. Gallic acid was used as a standard, and the total phenolic content was expressed as milligrams of gallic acid equivalents (mg GAE / 100 g) in strawberries. See the appendix for detailed results. Figure 6 .

[0074] From the appendix Figure 6 The results showed that the total phenolic content of fresh strawberries was 211.48 mg gallic acid equivalent (GAE) / 100 g strawberries. After 3 days of storage at room temperature, the total phenolic content of the control group strawberries decreased to 151.24 mg GAE / 100 g, while the total phenolic content of strawberries coated with both coatings was significantly higher than that of the control group. After 7 days of storage, the phenolic content of strawberries coated with IY-3-Lip and CS-IY-3-Lip were 189.75 mg GAE / 100 g and 200.12 mg GAE / 100 g, respectively, which were significantly higher than those of the untreated group (30.63 mg GAE / 100 g), indicating that the IY-3-Lip and CS-IY-3-Lip coatings had a good effect on preserving phenolic compounds.

[0075] Further, 100 g of strawberries were crushed and filtered with 100 mL of 2% oxalic acid solution. 20 mL of the liquid was taken and diluted to 100 mL with 2% oxalic acid solution. 5 g of kaolin was added for decolorization, and the mixture was filtered again. 10 mL of the filtrate was then titrated with standardized 2,6-dichlorophenolindophenol solution until the solution turned pink and did not fade within 15 seconds. Simultaneously, 10 mL of 2% oxalic acid solution was used as a blank control. The results are expressed as milligrams of ascorbic acid per 100 mL of sample.

[0076] From the appendix Figure 6The results showed that the trends in ascorbic acid (Vc) content during storage were similar to those in total phenol content across different treatment groups. The Vc content of fresh strawberries was 61.87 mg / 100 g. After 7 days of storage, the Vc content of uncoated strawberries significantly decreased to 8.53 mg / 100 g, but the Vc content of the coated strawberries was significantly higher than that of the control group, indicating that the IY-3-Lip and CS-IY-3-Lip coatings can effectively reduce the loss of Vc in strawberries.

[0077] Further, 5 g of strawberry sample was pulverized and placed in 25 mL of PBS solution containing 0.2% Tween-80. A series of 10-fold dilutions were prepared in PBS after pulverization. 1 mL of each sample was mixed with approximately 15 mL of rose rubes agar. The solidified plates were incubated at 37°C for 48 hours, and the colony count was then performed. See the appendix for detailed results. Figure 6 .

[0078] From the appendix Figure 6 The results showed that the coating treatments inhibited the proliferation of yeast and mold to varying degrees, especially the CS-IY-3-Lip coating treatment group. After 7 days of storage, the number of yeast and mold in the CS-IY-3-Lip coating treatment group was reduced by more than three orders of magnitude compared with the control group (CS-IY-3-Lip coating treatment group: 3.68 lg CFU / g, control group: 6.72 lg CFU / g). The results indicate that the CS-IY-3-Lip coating has the ability to resist the growth or contamination of yeast and mold.

[0079] Further, 1 mL of 0.01% DPPH was mixed with 1 mL of fruit extract. The mixture was stored at room temperature in the dark for 30 minutes, and then the antioxidant activity of strawberries was determined using the DPPH assay. See the appendix for specific results. Figure 6 .

[0080] The fruit extract mixture is made by crushing 25 g of fruit pulp and distilled water.

[0081] From the appendix Figure 6 The results showed that the antioxidant activity of strawberries in the control group gradually decreased throughout the storage process, reaching a DPPH scavenging rate of 59.73% on day 7. The use of IY-3-Lip and CS-IY-3-Lip coatings inhibited the decline in antioxidant activity. The DPPH scavenging rate of the CS-IY-3-Lip coating group was close to the initial DPPH scavenging rate, indicating that the CS-IY-3-Lip coating has an inhibitory effect on strawberry oxidation.

[0082] In summary, the above embodiments are merely descriptions of preferred implementations of this experiment and are not intended to limit the scope of this experiment. Without departing from the spirit of this experiment design, all modifications and improvements made by those skilled in the art to the technical solutions of this experiment should fall within the protection scope determined by this experiment.

Claims

1. An antioxidant peptide IY-3, characterized in that: The amino acid sequence is shown in SEQ ID No.

3.

2. The application of the antioxidant peptide IY-3 as described in claim 1 in the preparation of antioxidant products.

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