Preparation and application of an antioxidant, anti-inflammatory and hypoglycemic lactoferrin peptide

Abstract

The invention discloses a kind of preparation of antioxidant, anti-inflammatory and hypoglycemic lactoferrin peptide and its application, belong to the field of food science and nutrition. The present invention uses lactoferrin as raw material, utilizes composite enzymolysis method to be hydrolyzed, and obtains the component of molecular weight <5kDa by ultrafiltration, and the component is freeze-dried, obtains the peptide powder rich in antioxidant, anti-inflammatory and hypoglycemic peptide, then utilizes LC-MS / MS technology to identify the peptide section contained in the peptide powder, screens out the active peptide YLSW (Tyr-Leu-Ser-Trp) with triple function of antioxidant, anti-inflammatory and hypoglycemic. The above-mentioned active peptide is added to product, and antioxidant, anti-inflammatory and hypoglycemic functional product can be obtained.

Description

Technical Field

[0001] The present invention relates to preparation and application of an antioxidant, anti-inflammatory and blood sugar-lowering lactoferrin peptide, and belongs to the field of food science and nutrition. Background Art

[0002] As living standards improve, health and nutrition are becoming increasingly important. Especially with the increasing prevalence of chronic diseases, demand for functional foods and health supplements is growing. Products with antioxidant, anti-inflammatory, and glucose-lowering properties are gaining traction due to their importance in preventing and managing chronic diseases. Oxidative stress is considered a major contributing factor to numerous chronic diseases, such as cardiovascular disease, diabetes, and certain cancers. This process stems from the excessive production of free radicals in the body, which outstrips the antioxidant system's ability to clear them, leading to cellular damage. Therefore, the development of natural ingredients with antioxidant properties has become a key focus for researchers, aiming to enhance the body's antioxidant capacity and slow aging. Anti-inflammatory effects are also crucial in the management of chronic diseases. Inflammation is a core mechanism underlying many chronic diseases, such as cardiovascular disease, diabetes, and arthritis. Persistent low-grade inflammation can lead to tissue damage, cellular dysfunction, and even exacerbate the onset and progression of disease. Furthermore, diabetes, a chronic disease increasingly prevalent worldwide, is characterized by abnormally elevated blood sugar levels, making glucose regulation crucial for patients. While existing glucose-lowering medications are numerous, they often come with side effects and can lead to drug resistance, making the search for safe and effective natural glucose-lowering ingredients particularly important.

[0003] At present, research on safe and effective natural functional products mainly focuses on a single function, lacking optimized design for the triple functions of antioxidant, anti-inflammatory and hypoglycemic. Small molecule peptides are a widely accepted natural functional product with advantages such as low preparation cost, safety, no toxic side effects, and easy absorption by the human body. They have gradually become the focus of research and development in various fields and are used in industries such as medicine, beverages, health foods and cosmetics. However, the existing technology lacks multifunctional peptides with antioxidant, anti-inflammatory and hypoglycemic functions. Therefore, the development of a new type of peptide with triple functions of antioxidant, anti-inflammatory and hypoglycemic functions, which can enhance these three functions at the same time and broaden the application range of small molecule peptides, has become an urgent problem to be solved in the current technical field. Summary of the Invention

[0004] To address the problems in the prior art, the present invention provides an antioxidant, anti-inflammatory, and glucose-lowering peptide segment. Lactoferrin is used as a raw material, and a composite enzymatic hydrolysis method is proposed for hydrolysis of the protein. A fraction with a molecular weight of <5 kDa is obtained by ultrafiltration, followed by freeze-drying to obtain a peptide powder rich in antioxidant, anti-inflammatory, and glucose-lowering peptides. The peptide segments contained in the peptide powder are then identified using LC-MS / MS technology to screen for active peptides with triple antioxidant, anti-inflammatory, and glucose-lowering functions. These active peptides are then added to a product to obtain an antioxidant, anti-inflammatory, and glucose-lowering functional product.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a lactoferrin trifunctional peptide with antioxidant, anti-inflammatory and glucose-lowering properties. The amino acid sequence of the trifunctional peptide is Tyr-Leu-Ser-Trp, which is denoted as YLSW.

[0007] The present invention also provides a method for preparing a lactoferrin trifunctional peptide having antioxidant, anti-inflammatory and hypoglycemic properties, the specific steps of which are as follows:

[0008] S1 is calculated by mass fraction. 5000 U / g of composite protease is added to a 2% lactoferrin solution. The solution is enzymatically hydrolyzed at a pH of 7.5 and 37°C for 2.5 hours. The enzyme is inactivated, the pH is adjusted, and the supernatant is obtained by centrifugation to obtain the enzymatic hydrolyzate.

[0009] S2 ultrafilters the enzymatic hydrolysate, and freeze-dries the enzymatic hydrolysate with a molecular weight Mw < 5k Da to obtain peptide powder with antioxidant, anti-inflammatory and hypoglycemic effects.

[0010] S3 identified the components with antioxidant, anti-inflammatory and hypoglycemic activities by LC-MS / MS and obtained a series of peptide sequences.

[0011] S4: A series of peptide sequences obtained in step S3 are screened to identify an antioxidant, anti-inflammatory and hypoglycemic lactoferrin peptide, designated as YLSW, and synthesized using a solid phase method.

[0012] Furthermore, the specific steps for adding the complex enzyme in S1 are as follows: trypsin is added at a dosage of 5000 U / g to a 2% lactoferrin solution. Subsequently, a hydrolysis process is performed for 1 hour at an initial pH of 7.5 and a temperature of 37°C. After completing the above steps, papain is immediately added to the reaction system at an amount of 5000 U / g, and the reaction is continued under the hydrolysis conditions for 1.5 hours to produce the desired in vitro enzymatic hydrolysis product.

[0013] After the enzymatic hydrolysis is completed, the enzyme is inactivated in a boiling water bath for 10 to 15 minutes. After the enzymatic hydrolysis solution reaches room temperature, the pH is adjusted to 7.0. The obtained hydrolyzate is centrifuged and the supernatant is collected. The centrifugation conditions are: 8000 rpm, 15 to 20 minutes, and 4°C.

[0014] Furthermore, the components with molecular weight Mw < 5k Da were subjected to preliminary antioxidant (ABTS, DPPH and ·OH free radical scavenging ability), anti-inflammatory activity and α-glucosidase inhibitory activity determination in S2.

[0015] Furthermore, the LC-MS / MS detection conditions in step S3 were as follows: the sample was analyzed by LC-MS / MS equipped with an online nanospray ion source. The entire system consisted of an Orbitrap Eclipse mass spectrometer (Thermo Fisher Scientific, MA, USA) connected in series with an EASY-nanoLC 1200. A total of 2 μL of sample was loaded onto an Acclaim PepMap C18 analytical column, 75 μm x 25 cm. The sample was separated using a 60-min gradient with a column flow rate of 400 nL / min, a column temperature of 40°C, an electrospray voltage of 2 kV, and an initial gradient of 2.2% phase B. The gradient was increased nonlinearly to 44% at 51 minutes, then to 90% within 3 minutes, and then maintained for 6 minutes.

[0016] The mass spectrometer was operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition. The mass spectrometry parameters were set as follows: (1) MS: scan range (m / z): 375–1200; resolution: 60,000; AGC target: 4e5; maximum injection time: 50 ms; (2) HCD-MS / MS: resolution: 15,000; AGC target: 5e4; maximum injection time: 45 ms; collision energy: 30%; dynamic exclusion time: 40 s.

[0017] The present invention also provides a product containing the trifunctional peptide.

[0018] In one embodiment, the product includes medicines or daily chemical products.

[0019] In one embodiment, the drug further comprises conventional pharmaceutical carriers and / or pharmaceutical excipients.

[0020] In one embodiment, the pharmaceutical carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes; and the pharmaceutical excipient comprises excipients and / or additives.

[0021] The present invention also provides the use of the trifunctional peptide in the preparation of medicines that help to resist oxidation and / or maintain healthy blood sugar function.

[0022] In one embodiment, the content of the trifunctional peptide in the pharmaceutical product is at least 0.05 g / g.

[0023] The present invention also provides the use of the trifunctional peptide in preparing daily chemical products with antioxidant and / or anti-inflammatory functions.

[0024] In one embodiment, the daily chemical products include but are not limited to cosmetics.

[0025] In one embodiment, the daily chemical products include but are not limited to skin care products.

[0026] Beneficial effects:

[0027] 1. The present invention uses lactoferrin as raw material and obtains antioxidant, anti-inflammatory and hypoglycemic lactoferrin peptide YLSW (Tyr-Leu-Ser-Trp) through composite protease hydrolysis. The lactoferrin peptide YLSW has antioxidant activity (ABTS 90.98%, DPPH 40.38%, OH 21.78%), anti-inflammatory activity (inhibiting NF-κB and pro-inflammatory factors) and α-glucosidase inhibitory activity (inhibition rate 71.37%, half inhibition concentration IC 50 =0.46 mg / mL).

[0028] 2. The lactoferrin peptide YLSW described in the present invention has a short peptide chain, which helps reduce synthesis costs. It is safe and healthy, has no toxic side effects, and has auxiliary antioxidant and hypoglycemic effects for consumers with hyperglycemia. It also exerts anti-inflammatory effects by regulating the immune system and inhibiting the release of inflammatory factors. For consumers with normal blood sugar, YLSW peptide has no side effects and can effectively alleviate inflammatory responses in the body, reducing the risk of chronic inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 ABTS free radical scavenging activity of lactoferrin hydrolysate with molecular weight Mw < 5k Da;

[0030] Figure 2 DPPH free radical scavenging activity of lactoferrin hydrolysate with molecular weight Mw < 5k Da;

[0031] Figure 3 Lactoferrin hydrolyzate with molecular weight Mw < 5k Da·OH radical scavenging activity;

[0032] Figure 4 Effect of lactoferrin hydrolysate with molecular weight Mw < 5k Da on cell activity;

[0033] Figure 5 The results of the inhibition of NO release by cells by lactoferrin hydrolyzate with molecular weight Mw < 5k Da;

[0034] Figure 6 The results of the inhibition of relative expression of NF-κB in cells by lactoferrin hydrolyzate with molecular weight < 5kDa;

[0035] Figure 7 The results of the inhibition of relative expression of intracellular IL-1β by lactoferrin hydrolyzate with molecular weight < 5kDa;

[0036] Figure 8 YLSW peptide ABTS free radical scavenging activity;

[0037] Figure 9 YLSW peptide DPPH free radical scavenging activity;

[0038] Figure 10 YLSW peptide·OH radical scavenging activity;

[0039] Figure 11 Effects of YLSW peptide on cell activity;

[0040] Figure 12 The results of YLSW peptide inhibiting the release of NO from cells;

[0041] Figure 13 The results of YLSW peptide inhibiting the relative expression of NF-κB in cells;

[0042] Figure 14 The results of YLSW peptide inhibiting the relative expression of intracellular IL-6;

[0043] Figure 15 The results of YLSW peptide inhibiting the relative expression of intracellular IL-1β;

[0044] Figure 16 The results of YLSW peptide inhibiting the relative expression of TNF-α in cells;

[0045] Figure 17 YLSW peptide α-glucosidase inhibitory activity;

[0046] Figure 18 YLSW peptide inhibition type of α-glucosidase;

[0047] Figure 19 Docking results of YLSW peptide with Keap1 and α-glucosidase molecules;

[0048] Figure 20 HTGLGR peptide ABTS free radical scavenging activity;

[0049] Figure 21 DPPH radical scavenging activity of HTGLGR peptide;

[0050] Figure 22 HTGLGR peptide·OH radical scavenging activity;

[0051] Figure 23 HTGLGR peptide inhibits α-glucosidase activity. DETAILED DESCRIPTION

[0052] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0053] Trypsin used in the following examples was purchased from Sigma-Aldrich, and papain was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0054] The statistical analysis involved in the following examples:

[0055] All experiments were repeated at least three times. Data were analyzed using GraphPad Prism 9.0.0 software, and results are presented as standard error of the mean (SEM). Molecular docking experiments were designed and analyzed using Discovery Studio software.

[0056] Example 1 Isolation and Functional Verification of Lactoferrin-derived Trifunctional Peptides

[0057] 1. Isolation of lactoferrin-derived trifunctional peptides

[0058] Accurately weigh 2 g of lactoferrin, add 80 mL of deionized water and a rotor, and place it on a magnetic stirrer. After it is evenly dispersed, adjust the pH value to the optimal pH for trypsin with 0.5 mol / L NaOH or HCl (the optimal pH for trypsin in this experiment is 7.5). Finally, adjust the volume to 100 mL and cover with plastic wrap.

[0059] The above solution is subjected to enzymatic hydrolysis, and the specific method is as follows:

[0060] (1) Trypsin enzymatic hydrolysis

[0061] Set the thermostatic water bath oscillator to the optimal temperature for proteases (37°C is the optimal temperature for proteases in this patent) in advance. Once the temperature reaches the set temperature, place the dissolved solution in the oscillator and incubate for 10 minutes. After 10 minutes, add trypsin (enzyme dosage: 5000 U / g) and start a 1-hour timer.

[0062] (2) Papain enzymatic hydrolysis

[0063] When trypsin hydrolysis is almost finished (take 1 mL to measure the pH, observe whether the pH value is in the optimal pH range of papain, and then adjust it), add papain (enzyme amount: 5000 U / g) and start timing for 1.5 hours.

[0064] Inactivation of enzyme and adjustment of pH: After the enzymatic hydrolysis reaction is completed, the proteolytic product is placed in a boiling water bath and heated for 10 minutes to inactivate the protease. After cooling to room temperature, the pH of the solution is adjusted to 7.

[0065] After pH adjustment, the proteolysis product was aliquoted into 50 mL centrifuge tubes and centrifuged at 8000 rpm for 15 minutes. The supernatants were collected and mixed. The supernatant was fractionated using a pilot ultrafiltration flat-plate membrane system and passed through a 5 kDa flat-plate membrane. Fractions with a molecular weight (Mw) <5 kDa were pre-frozen in a refrigerator. Finally, the pre-frozen samples were freeze-dried using a lyophilizer. The resulting freeze-dried samples were suitable for subsequent experiments.

[0066] 2. Functional verification of lactoferrin-derived trifunctional peptides

[0067] The sample with a molecular weight Mw < 5 kDa obtained in step 1 was dissolved in water to prepare solutions with concentrations of 1 mg / mL, 2.5 mg / mL, 5 mg / mL, and 10 mg / mL, respectively, for activity verification.

[0068] (1) Antioxidant activity

[0069] ABTS free radical scavenging activity:

[0070] Dilute the ABTS stock solution with PB buffer (50 mM, pH 7.4) to an absorbance of 0.7 ± 0.02 at 734 nm to prepare the ABTS working solution. Thoroughly mix 10 μL of sample of varying concentrations (dissolved in pure water) with 990 μL of the ABTS working solution. Incubate at 27°C in the dark for 6 minutes, and measure absorbance at 734 nm. Replace the sample solution with pure water to serve as a blank control.

[0071]

[0072] Among them, A1 is the absorbance value of the experimental group, and A0 is the absorbance value of the blank group.

[0073] DPPH free radical scavenging activity:

[0074] Combine equal volumes of sample solutions (dissolved in pure water) of varying concentrations with DPPH (dissolved in anhydrous ethanol) and incubate in the dark at 37°C for 30 minutes. Measure absorbance at 517 nm. Replace the sample solution with pure water to serve as a blank.

[0075]

[0076] Among them, A1 is the absorbance value of the experimental group, and A0 is the absorbance value of the blank group.

[0077] OH radical scavenging activity:

[0078] Take 100 μL of sample of varying concentrations and mix with equal amounts of FeSO₄ (9 mM) and H₂O₂ (8 mM). After mixing, incubate the solution at 37°C in the dark for 10 minutes. Subsequently, add 100 μL of ethanolic salicylate (9 mM) and incubate at 37°C in the dark for another 30 minutes. Finally, measure the absorbance at 510 nm. Replace the sample solution with pure water to serve as a blank control.

[0079]

[0080] Among them, A1 is the absorbance value of the experimental group, and A0 is the absorbance value of the blank group.

[0081] The results are as follows Figure 1-3 The results showed that samples with a molecular weight of Mw < 5k Da showed significant activity in scavenging a variety of free radicals (including ABTS, DPPH and ·OH radicals), especially in the scavenging rate of ABTS free radicals, which reached the highest level, indicating that they have strong antioxidant capacity and help to reduce the damage of oxidative stress to cells.

[0082] (2) Verification of anti-inflammatory activity

[0083] 1) Cell viability (MTT) assay:

[0084] RAW264.7 macrophages were cultured at a rate of 1 × 10 5 Cells were seeded at a density of 1 μg / mL in a 96-well plate and cultured overnight. The old medium was then discarded and sample solutions with a molecular weight <5 kDa (Mw <5 kDa) prepared in complete medium (90% DMEM, 10% FBS, and 1% P / S) were added at 1, 10, 100, 250, and 500 μg / mL, respectively. Complete medium served as a blank control, and the cells were cultured in an incubator for another 24 hours. Then, 10 μL of MTT (5 mg / mL) was added to each well and incubated in an incubator for 4 hours. The culture medium was then discarded, DMSO was added, and absorbance was measured at 570 nm.

[0085]

[0086] Among them, A1 is the absorbance value of the experimental group, and A0 is the absorbance value of the control group.

[0087] The results are as follows Figure 4 As shown, within the concentration range of 1 to 500 μg / mL, samples with a molecular weight Mw < 5 kDa showed no inhibitory effect on cell viability.

[0088] 2) NO determination:

[0089] RAW264.7 macrophages were cultured at a density of 5.5 × 10 5The cells were seeded at a density of 100 μL / mL in a 96-well plate and cultured with complete medium for 24 hours. CON group, LPS group and experimental group were set up:

[0090] CON group: discard the culture medium, add DMEM, and continue treatment for 24 h;

[0091] LPS group: the culture medium was discarded, DMEM was added and cultured for 12 h, and then LPS (1 μg / mL) was added and incubated for 12 h;

[0092] Experimental group: The culture medium was discarded and samples with a molecular weight <5 kDa were added at different concentrations (0.1, 1, 10, 50, 100, 200, 400, and 500 μg / mL) for 12 hours (the samples were dissolved and diluted in DMEM). Subsequently, LPS (1 μg / mL) was added and incubated with the samples for 12 hours.

[0093] The CON group served as a blank control, providing data on normal cell responses for comparison and assessing the natural state of cells without external intervention. The LPS group served as a negative control, stimulating cells with LPS to simulate an inflammatory response and providing a basis for comparison of the effects of the experimental group samples. Finally, cell supernatants were collected and NO production was measured using a NO detection kit. Absorbance was measured at a wavelength of 540 nm and calculated according to the manufacturer's instructions.

[0094] The results are as follows Figure 5 As shown in the results, samples with a molecular weight of Mw < 5 kDa significantly inhibited the release of intracellular NO, and the inhibitory effect was dose-dependent, showing good anti-inflammatory potential.

[0095] 3) RT-PCR experiment:

[0096] RAW264.7 macrophages were collected at a rate of 5.5×10 5 The cells were seeded at a density of 1 mL / mL in a 24-well plate. The cells were cultured in complete medium for 24 hours. The CON, LPS, and experimental groups were set up as follows:

[0097] CON group: discard the culture medium, add DMEM, and continue treatment for 24 h;

[0098] LPS group: the culture medium was discarded, DMEM was added and cultured for 12 h, and then LPS (1 μg / mL) was added and incubated for 12 h;

[0099] Experimental Group: Discard the culture medium and treat with samples (Mw < 5 kDa) at varying concentrations (100, 200, 400, and 500 μg / mL) for 12 hours (dissolve and dilute the samples in DMEM). Add LPS (1 μg / mL) and incubate with the samples for 12 hours. The CON group served as the blank control, and the LPS group served as the negative control. Cells were then collected and the following procedures performed:

[0100] The cells were washed twice with PBS, lysed with lysis buffer, and RNA was extracted according to the reagent instructions. The total RNA was transcribed into cDNA according to the reverse transcription kit instructions. The expression levels of NF-κB, IL-6, and IL-1β mRNA were detected using cDNA as a template. Four parallel tubes were set up for each sample test group.

[0101] The results are as follows Figures 6-7 As shown, NF-κB, a core transcription factor in inflammatory responses, regulates the expression of multiple proinflammatory cytokines, of which IL-1β is a key proinflammatory cytokine. Overexpression can lead to chronic inflammation and tissue damage. Samples with a molecular weight (Mw) < 5kDa significantly reduced the release of these proinflammatory cytokines, further demonstrating their anti-inflammatory effects.

[0102] (3) α-glucosidase inhibitory activity

[0103] First, 40 μL of phosphate buffer (0.1 mM, pH 6.8) was added to a 96-well plate, followed by 20 μL of samples of different concentrations (40 mg / mL, 30 mg / mL, 20 mg / mL, and 15 mg / mL) and 10 μL of α-glucosidase solution (1 U / mL), both dissolved in phosphate buffer. The plates were then incubated at 37°C for 15 minutes. Subsequently, 50 μL of PNPG solution (1 mM, as a substrate) was added, and the reaction was incubated again at 37°C for 20 minutes. After incubation, 100 μL of 1 M sodium carbonate solution was added to terminate the reaction. Finally, the absorbance was measured at a wavelength of 405 nm. 20 μL of phosphate buffer was used instead of the sample solution to serve as a blank control.

[0104]

[0105] Among them, A1 is the absorbance value of the experimental group, and A0 is the absorbance value of the blank group.

[0106] The results showed that samples with a molecular weight (Mw) <5kDa exhibited inhibitory activity against α-glucosidase of 30.59%, 26.04%, 17.27%, and 10.58% at concentrations of 40mg / mL to 15mg / mL, respectively, reaching a maximum of 30.59%. Because α-glucosidase plays a key role in carbohydrate metabolism, its inhibition can slow sugar absorption, thereby playing an important role in regulating blood sugar levels.

[0107] Example 2 Preparation of Antioxidant, Anti-inflammatory and Hypoglycemic Lactoferrin Peptide

[0108] 1. LC-MS / MS identification of active components

[0109] The components verified to have antioxidant, anti-inflammatory and hypoglycemic activities in Example 1 were subjected to LC-MS / MS identification to obtain a series of peptide sequences.

[0110] The samples were analyzed by LC-MS / MS equipped with an online nanospray ion source. The system consisted of an Orbitrap Eclipse mass spectrometer (Thermo Fisher Scientific, MA, USA) connected to an EASY-nanoLC1200. A total of 2 μL of sample was loaded onto an Acclaim PepMap C18 analytical column, 75 μm x 25 cm. The sample was separated using a 60-min gradient with a controlled column flow rate of 400 nL / min, a column temperature of 40°C, and an electrospray voltage of 2 kV. The gradient started at 2.2% phase B and increased nonlinearly to 44% over 51 minutes, then to 90% over 3 minutes, where it was maintained for 6 minutes.

[0111] The mass spectrometer was operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition. The mass spectrometry parameters were set as follows: (1) MS: scan range (m / z): 375–1200; resolution: 60,000; AGC target: 4e5; maximum injection time: 50 ms; (2) HCD-MS / MS: resolution: 15,000; AGC target: 5e4; maximum injection time: 45 ms; collision energy: 30%; dynamic exclusion time: 40 s.

[0112] 821 peptides were identified, ranging in length from 3 to 65 amino acids. This finding suggests that the sample contains a rich peptide composition and may have antioxidant, anti-inflammatory, and hypoglycemic activities.

[0113] 2. Screening of antioxidant, anti-inflammatory and hypoglycemic peptides and solid phase synthesis:

[0114] Lactoferrin peptides with antioxidant, anti-inflammatory, and hypoglycemic properties were screened using the active peptide function prediction website (https: / / services.healthtech.dtu.dk / services / AnOxPePred-1.0 / ; http: / / kurata14.bio.kyutech.ac.jp / PreAIP / auto.php?id=20240327204505 and https: / / i.uestc.edu.cn / AntiDMPpred / cgi-bin / AntiDMPpred.pl). To further test the activity of the isolated and purified peptide, Shanghai Qiangyao Biotechnology Co., Ltd. commissioned the synthesis of the YLSW peptide using solid-phase synthesis. High-performance liquid chromatography confirmed the purity of the synthesized peptide to be greater than 95%. The amino acid sequence of the YLSW peptide is Tyr-Leu-Ser-Trp.

[0115] Example 3 Functional verification of the antioxidant, anti-inflammatory and hypoglycemic lactoferrin peptide YLSW

[0116] 1. Antioxidant activity verification

[0117] The experimental method was similar to that of Example 1, except that the YLSW peptide was diluted to 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL and 0.01 mg / mL for functional verification.

[0118] like Figure 8-10 As shown, in antioxidant experiments, the YLSW peptide exhibited excellent scavenging ability, with an ABTS scavenging rate of 90.98%, indicating that the peptide has a strong scavenging effect on cationic free radicals. The DPPH scavenging rate was 40.38%, and the scavenging rate for ·OH free radicals was 21.78%, further confirming its antioxidant effect. These results suggest that the YLSW peptide may exert its antioxidant properties by directly reacting with free radicals or activating intracellular antioxidant enzyme systems, which is of great significance for preventing diseases related to oxidative stress.

[0119] 2. Verification of anti-inflammatory activity

[0120] (1) Cell viability (MTT) assay:

[0121] The experimental method was similar to that of Example 1, except that the YLSW peptide was diluted to 10, 100, 250, and 500 μg / mL for functional verification.

[0122] The results are as follows Figure 11 As shown, the YLSW peptide showed no inhibitory effect on cell activity at high and low concentrations, indicating that the sample has good biocompatibility.

[0123] (2) NO determination:

[0124] The experimental method is the same as in Example 1, and the results are as follows Figure 12 As shown in the results, YLSW peptide also significantly inhibited the release of intracellular NO. Compared with the LPS group, the inhibition rate reached a maximum of 48.08% at a sample concentration of 400 μg / mL and 12.23% at the lowest experimental concentration of 0.1 μg / mL, demonstrating anti-inflammatory potential.

[0125] (3) RT-PCR experiment:

[0126] The experimental method was similar to that of Example 1, wherein the YLSW peptide was diluted to 10, 100, 250 and 500 μg / mL for functional verification, and the expression levels of NF-κB, IL-6, IL-1β and TNF-α mRNA were detected.

[0127] The results are as follows Figure 13-16 As shown, YLSW exhibited a stronger overall anti-inflammatory effect than samples with a molecular weight <5kDa in inhibiting the release of NF-κB and pro-inflammatory factors (IL-6, IL-1β, and TNF-α). Compared with the LPS group, YLSW reduced the expression of NF-κB by up to 39.49%, IL-6 by 39.56%, IL-1β by 28.87%, and TNF-α by 65.60%. This indicates that pure peptide samples have higher bioactivity and stronger targeting effects, making them suitable for more precise anti-inflammatory treatments.

[0128] 3. Verification of α-glucosidase inhibitory activity and inhibition type

[0129] (1) α-glucosidase inhibitory activity

[0130] The experimental method was the same as that of Example 1, wherein the YLSW peptide was diluted to 1.25 mg / mL, 1 mg / mL, 0.75 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.1 mg / mL, 0.075 mg / mL, 0.05 mg / mL and 0.025 mg / mL, respectively.

[0131] Activity verification results are as follows Figure 17 As shown in the results, YLSW peptide also showed significant effect in hypoglycemic activity, with an inhibition rate of 71.37% on α-glucosidase, effectively slowing down the digestion and absorption of carbohydrates, thereby helping to lower postprandial blood sugar levels. Its half inhibitory concentration (IC 50 ) was 0.46 mg / mL, indicating that it can be effective at lower concentrations, which has positive significance for potential product development.

[0132] (2) Suppress type verification

[0133] In the experiment, YLSW peptide at concentrations of 0.1 and 0.175 mg / ml (20 μL each) and α-glucosidase at a concentration of 1 U / ml (10 μL) were mixed, and no sample peptide was added to the blank control group. Subsequently, the mixture was thoroughly shaken and placed in a constant temperature water bath at 37°C for 15 minutes. After that, PNPG solutions (50 μL each) of different concentrations (0.5, 0.75, 0.9, 1.0, 1.5, 2.0, 3.0 mmol / L) were added to the reaction system, and the initial absorbance at a wavelength of 405 nm was first measured. Subsequently, the reaction system was placed in a constant temperature water bath at 37°C for 20 minutes. After the reaction was completed, 100 μL of 1 M Na2CO3 solution was added to the system to terminate the reaction, and the final absorbance at a wavelength of 405 nm was measured. Based on these data, a Lineweave-Burk double reciprocal curve of PNPG solution concentration and reaction rate was drawn to determine the inhibition type.

[0134] The results showed that ( Figure 18 ). Analysis by the Lineweaver-Burk double reciprocal method revealed that the YLSW peptide exhibits a mixed inhibition mechanism against α-glucosidase. The peptide not only competes with the active site of the free enzyme for substrate binding, leading to competitive inhibition, but also binds to the enzyme-substrate complex, further inhibiting the enzyme's catalytic activity. Experimental data demonstrate that the inhibitory effect of the YLSW peptide is concentration-dependent and potent, making it suitable for the treatment or prevention of diabetes and related diseases.

[0135] (3) Docking experiments of YLSW peptide with Keap1 and α-glucosidase

[0136] Discovery Studio was used to explore the potential binding sites between the YLSW peptide and Keap1 and α-glucosidase. The X-ray crystal structures of Keap1 (PDB code: 2FLU) and α-glucosidase (PDB code: 5NN8) were downloaded from the Protein Structure Database (https: / / rcsb.org / ). Water and ligands were removed to obtain a stable YLSW peptide receptor. Using the Discovery Studio visualization tool, the interaction between the receptor and ligand was visualized, and parameters such as binding energy and hydrogen bond distances were obtained.

[0137] The results of the mechanism of action of YLSW peptide on Keap1 and α-glucosidase are as follows Figure 19As shown, the YLSW peptide interacts with Keap1 mainly through traditional hydrogen bonds, carbon-hydrogen bonds, Pi-Cation, and Pi-A1ky1, and with α-glucosidase mainly through charge attraction, traditional hydrogen bonds, carbon-hydrogen bonds, π-π stacking, alkyl groups, and Pi-A1ky1. The docking energies of the YLSW peptide with Keap1 and α-glucosidase are shown in Table 1.

[0138] Table 1. Energy of docking of YLSW peptide with Keap1 and α-glucosidase

[0139]

[0140] Docking studies have shown that when binding to Keap1, the YLSW peptide enhances its binding force mainly through various forms of interactions such as hydrogen bonds, carbon-hydrogen bonds, Pi-Cation and Pi-Alkyl. As an important intracellular signal transduction protein, Keap1 is involved in regulating oxidative stress responses, and the YLSW peptide may exert an antioxidant effect by binding to it. In binding to α-glucosidase, the YLSW peptide mainly shows its potential to inhibit the enzyme through charge attraction, traditional hydrogen bonds, carbon-hydrogen bonds, π-π stacking, alkyl interactions and Pi-Alkyl interactions. This mechanism may help slow down the digestion and absorption of carbohydrates, thereby effectively reducing postprandial blood sugar levels. Judging from the docking energy in Table 1, the binding energy values ​​of the YLSW peptide with these two receptors are within a reasonable range. As shown in Table 1, the binding energy values ​​of the YLSW peptide with these two receptors are within a reasonable range. Figure 19 As shown, the YLSW peptide forms hydrogen bonds with SER508, ARG415, GLN530, and TYR334 in Keap1, enhancing its binding affinity and potentially promoting antioxidant signaling. Furthermore, the YLSW peptide also forms hydrogen bonds with ASP404 and His674 in α-glucosidase. The former, through its carboxyl group, interacts with the amino or polar groups of the YLSW peptide, helping to inhibit α-glucosidase activity and reduce sugar digestion and absorption; the latter may regulate catalytic efficiency by affecting the enzyme's conformation.

[0141] In summary, the present invention discovered that the peptide segment with the sequence YLSW (Tyr-Leu-Ser-Trp) has antioxidant activity (ABTS 90.98%, DPPH 40.38%, ·OH 21.78%), anti-inflammatory activity and α-glucosidase inhibitory activity (inhibition rate 71.37%, half inhibition concentration IC50 = 0.46 mg / mL). It interacts with Keap1 mainly through traditional hydrogen bonds, carbon-hydrogen bonds, Pi-Cation and Pi-A1ky1, and with α-glucosidase mainly through charge attraction, traditional hydrogen bonds, carbon-hydrogen bonds, π-π stacking, alkyl and Pi-A1ky1, playing an important role in antioxidant and hypoglycemic activities.

[0142] Example 4 Preparation of a product containing lactoferrin peptide YLSW

[0143] Depending on the specific product requirements, lactoferrin peptide YLSW should be uniformly mixed with excipients, including but not limited to antioxidants, stabilizers, and fillers. During the mixing process, additional nutrients such as vitamins and minerals may be added as needed to enhance the final product. The mixing process should be strictly controlled to ensure that the biological activity of lactoferrin peptide YLSW is not compromised throughout the entire process.

[0144] In terms of adjusting the ratio, the ratio of lactoferrin peptide YLSW to other excipients should be appropriately adjusted according to the product's intended use (such as a pharmaceutical) to ensure that the lactoferrin peptide YLSW content per gram of the final product meets the predetermined requirements. For pharmaceuticals, the lactoferrin peptide YLSW content should be between 0.5g / g and 2g / g.

[0145] Comparative Example 1:

[0146] The specific implementation method is as in Example 2-3, except that the peptide HTGLGR (His-Thr-Gly-Leu-Gly-Arg) was screened from the predicted peptides for functional verification and detection, and the results are as follows:

[0147] 1. Antioxidant activity verification

[0148] The experimental method was similar to that of Example 1, except that the HTGLGR peptide was diluted to 5 mg / mL, 2.5 mg / mL, 1 mg / mL and 0.5 mg / mL for functional verification.

[0149] like Figure 20-22 As shown in the results, in the antioxidant activity experiment, the HTGLGR peptide failed to significantly scavenge ABTS and DPPH free radicals, and only showed a certain scavenging effect in the ·OH free radical scavenging experiment, indicating weak antioxidant activity.

[0150] 2. Verification of α-glucosidase inhibitory activity

[0151] The experimental method was the same as in Example 1, wherein the HTGLGR peptide was diluted to 1 mg / mL, 2.5 mg / mL and 5 mg / mL, respectively.

[0152] Activity verification results are as follows Figure 23 As shown, HTGLGR peptide did not show significant effect in terms of hypoglycemic activity.

[0153] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A trifunctional peptide, characterized in that The amino acid sequence of the trifunctional peptide is Tyr-Leu-Ser-Trp or YLSW.

2. A product containing the trifunctional peptide according to claim 1.

3. The product according to claim 2, characterized in that The products include medicines or daily chemical products.

4. The product according to claim 3, characterized in that The medicine further comprises conventional pharmaceutical carriers and / or pharmaceutical excipients.

5. The product according to claim 4, characterized in that The pharmaceutical carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes; and the pharmaceutical excipient comprises an excipient.

6. Use of the trifunctional peptide according to claim 1 in the preparation of a medicine that helps to resist oxidation and / or maintain healthy blood sugar function.

7. The use according to claim 6, characterized in that In the medicine, the content of the trifunctional peptide is at least 0.05 g / g.

8. Use of the trifunctional peptide according to claim 1 in the preparation of daily chemical products with antioxidant and / or anti-inflammatory functions.

9. The use according to claim 8, characterized in that The daily chemical products include but are not limited to cosmetics or skin care products.

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