Preparation and application of antioxidative, anti-inflammatory and hypoglycemic lactoferrin peptide

Abstract

The invention discloses preparation and application of antioxidative, anti-inflammatory and hypoglycemic lactoferrin peptide, and belongs to the field of food science and nutriology. According to the invention, lactoferrin is used as a raw material, hydrolysis is carried out by using a composite enzymatic hydrolysis method, and the molecular weight is obtained by ultrafiltration; the preparation method comprises the following steps: preparing a component of antioxidant, anti-inflammatory and hypoglycemic peptide from the active peptide YLSW (Tyr-Leu-Ser-Trp), carrying out freeze drying on the component to obtain peptide powder rich in antioxidant, anti-inflammatory and hypoglycemic peptides, identifying peptide fragments contained in the peptide powder by utilizing an LC-MS / MS technology, and screening out the active peptide YLSW (Tyr-Leu-Ser-Trp) with antioxidant, anti-inflammatory and hypoglycemic triple functions. The active peptide is added into the product to obtain the antioxidant, anti-inflammatory and hypoglycemic functional product.

Description

Technical Field

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

[0002] As people's living standards improve, health and nutrition have gradually become the focus of public attention, especially in the context of the high incidence of chronic diseases, the demand for functional foods and health products is rising. Among them, products with antioxidant, anti-inflammatory and hypoglycemic functions have received much attention due to their importance in preventing and managing chronic diseases. Oxidative stress is considered to be the main cause of many chronic diseases, such as cardiovascular disease, diabetes and certain cancers. This process originates from the excessive generation of free radicals in the body, which exceeds the scavenging capacity of the antioxidant system, resulting in cell damage. Therefore, the research and development of natural ingredients with antioxidant functions has become the focus of scientific researchers to enhance the body's antioxidant capacity and slow down aging. At the same time, anti-inflammatory effects are also crucial in the management of chronic diseases. Inflammation is one of the core mechanisms of many chronic diseases (such as cardiovascular disease, diabetes and arthritis). Continuous low-grade inflammation can lead to tissue damage, abnormal cell function, and even aggravate the occurrence and development of diseases. In addition, diabetes, as a chronic disease that is becoming more and more serious worldwide, is characterized by abnormally high blood sugar levels, so methods to regulate blood sugar are crucial for patients. Although there are many types of existing hypoglycemic drugs, they are often accompanied by side effects and may lead to drug resistance, so it is particularly important to find safe and effective natural hypoglycemic ingredients.

[0003] At present, the research on safe and effective natural functional products mainly focuses on a single function, lacking the optimization design for the triple functions of anti-oxidation, 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 all walks of life and are used in industries such as medicines, 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, 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] In order to solve the problems existing in the prior art, the present invention provides an antioxidant, anti-inflammatory and hypoglycemic peptide segment, using lactoferrin as a raw material, proposing a composite enzymatic hydrolysis method for hydrolysis of the protein, and obtaining a component with a molecular weight of <5kDa by ultrafiltration, and then freeze-drying to obtain a peptide powder rich in antioxidant, anti-inflammatory and hypoglycemic peptides, and then using LC-MS / MS technology to identify the peptide segments contained in the peptide powder, and screening out active peptides with triple functions of antioxidant, anti-inflammatory and hypoglycemic. Then, the above active peptides are added to the product to obtain an antioxidant, anti-inflammatory and hypoglycemic functional product.

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

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

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

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

[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: screening out an antioxidant, anti-inflammatory and hypoglycemic lactoferrin peptide from a series of peptide sequences obtained in step S3, denoted as YLSW, and synthesizing it by solid phase method.

[0012] Furthermore, the specific step of adding the complex enzyme in S1 is: adding trypsin at a dosage of 5000U / g to a 2% lactoferrin solution. Subsequently, a hydrolysis process is performed for 1 hour under an initial pH value of 7.5 and a temperature of 37°C. After completing the above steps, papain is immediately added to the reaction system in an amount of 5000U / g, and the reaction is continued under the hydrolysis conditions for 1.5 hours to prepare 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 hydrolyzate 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 < 5 kDa in S2 were subjected to preliminary antioxidant properties (ABTS, DPPH and ·OH free radical scavenging ability), anti-inflammatory activity and α-glucosidase inhibitory activity determination.

[0015] Further, the detection conditions of LC-MS / MS in step S3 are as follows: the sample is analyzed by LC-MS / MS equipped with an online nanospray ion source. The whole system is an Orbitrap Eclipse mass spectrometer (Thermo Fisher Scientific, MA, USA) connected in series with EASY-nanoLC 1200. A total of 2 μL of sample is loaded (analytical column: Acclaim PepMap C18, 75 μm x 25 cm), and the sample is separated by a gradient of 60 min, the column flow is controlled at 400 nL / min, the column temperature is 40 ° C, the electrospray voltage is 2 kV, the gradient starts from 2.2% of phase B, increases to 44% in 51 minutes with a nonlinear gradient, increases to 90% within 3 minutes, and maintains 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: 60000; AGC target: 4e5; maximum injection time: 50 ms; (2) HCD-MS / MS: resolution: 15000; 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 food, medicine, health care product or daily chemical product.

[0019] In one embodiment, the food comprises soy products, meat products or fruit and vegetable products.

[0020] In one embodiment, the drug further comprises conventional pharmaceutical carriers and / or pharmaceutical excipients.

[0021] In one embodiment, the pharmaceutical carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes; and the pharmaceutical excipient comprises excipients and / or additives.

[0022] The present invention also provides the use of the trifunctional peptide in the preparation of health products or medicines that help to resist oxidation and / or maintain healthy blood sugar functions.

[0023] In one embodiment, the content of the trifunctional peptide in the health product or medicine is at least 0.05 g / g.

[0024] The present invention also provides the use of the trifunctional peptide in preparing daily chemical products with antioxidant and / or anti-inflammatory functions.

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

[0026] In one embodiment, the daily chemical products include but are not limited to skin care products.

[0027] Beneficial effects:

[0028] 1. The present invention uses lactoferrin as a raw material, and obtains antioxidant, anti-inflammatory and hypoglycemic lactoferrin peptide YLSW (Tyr-Leu-Ser-Trp) by 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).

[0029] 2. The lactoferrin peptide YLSW of the present invention has a short peptide chain, which is conducive to reducing the synthesis cost, and is safe, healthy, and has no toxic side effects. It can not only be added to various foods as a functional ingredient, but also has auxiliary antioxidant and hypoglycemic effects on consumers with high blood sugar, and can also regulate the immune system and inhibit the release of inflammatory factors, thereby exerting an anti-inflammatory effect. For consumers with normal blood sugar, the YLSW peptide will not produce side effects, but can effectively reduce the inflammatory response in the body and reduce the risk of chronic inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 ABTS free radical scavenging activity of lactoferrin hydrolysate with molecular weight Mw < 5k Da;

[0031] Figure 2 DPPH free radical scavenging activity of lactoferrin hydrolysate with molecular weight Mw < 5kDa;

[0032] Figure 3 Lactoferrin hydrolysate with molecular weight Mw<5kDa·OH radical scavenging activity;

[0033] Figure 4 Effect of lactoferrin hydrolysate with molecular weight Mw<5kDa on cell activity;

[0034] Figure 5 The results of lactoferrin hydrolysate with molecular weight Mw<5kDa inhibiting the release of NO from cells;

[0035] Figure 6 The results of the relative expression of NF-κB in cells inhibited by lactoferrin hydrolysate with molecular weight Mw < 5kDa;

[0036] Figure 7 The results of the relative expression of intracellular IL-1β inhibited by lactoferrin hydrolysate with molecular weight Mw < 5kDa;

[0037] Figure 8 YLSW peptide ABTS free radical scavenging activity;

[0038] Fig. 9 YLSW peptide DPPH free radical scavenging activity;

[0039] Fig.10 YLSW peptide·OH radical scavenging activity;

[0040] Fig.11 Effects of YLSW peptide on cell activity;

[0041] Fig.12 The results of YLSW peptide inhibiting the release of NO from cells;

[0042] Fig.13 The results of YLSW peptide inhibiting the relative expression of NF-κB in cells;

[0043] Fig.14 The results of YLSW peptide inhibiting the relative expression of intracellular IL-6;

[0044] Fig.15 The results of YLSW peptide inhibiting the relative expression of intracellular IL-1β;

[0045] Fig.16 The results of YLSW peptide inhibiting the relative expression of intracellular TNF-α;

[0046] Fig.17 YLSW peptide α-glucosidase inhibitory activity;

[0047] Fig.18 The type of inhibition of α-glucosidase by YLSW peptide;

[0048] Fig.19 The docking results of YLSW peptide with Keap1 and α-glucosidase molecules;

[0049] Fig. 20 HTGLGR peptide ABTS free radical scavenging activity;

[0050] Fig.21 DPPH radical scavenging activity of HTGLGR peptide;

[0051] Fig. 22 HTGLGR peptide·OH radical scavenging activity;

[0052] Fig.23 HTGLGR peptide has inhibitory activity against α-glucosidase. DETAILED DESCRIPTION

[0053] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0054] The trypsin used in the following examples was purchased from Sigma-Aldrich, and papain was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0055] The statistical analysis involved in the following embodiments:

[0056] All experiments were repeated at least three times. GraphPadPrism 9.0.0 software was used to analyze the data, and the results are expressed as the standard error of the mean (SEM). Discovery studio software was used for molecular docking experiment design and analysis.

[0057] Example 1 Isolation and Functional Verification of Lactoferrin-derived Trifunctional Peptides

[0058] 1. Separation of lactoferrin-derived trifunctional peptides

[0059] 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, make the volume to 100 mL and cover with plastic wrap.

[0060] The above solution is subjected to enzymatic hydrolysis, and the specific method is as follows:

[0061] (1) Trypsin hydrolysis

[0062] The temperature of the constant temperature water bath oscillator is set to the optimum temperature of the protease in advance (the optimum temperature of the protease in this patent is selected as 37°C). After the temperature rises to the set temperature, the dissolved solution is placed in it and kept warm for 10 minutes. After 10 minutes, trypsin (enzyme amount: 5000U / g) is added and the timing is started for 1 hour.

[0063] (2) Papain enzymolysis

[0064] When the 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.

[0065] Inactivation of enzyme and adjustment of pH: After the enzymatic hydrolysis reaction is completed, the proteolysis product is placed in a boiling water bath and heated for 10 minutes to inactivate the protease. After it is cooled to room temperature, the pH of the solution is adjusted to 7.

[0066] The pH-adjusted proteolysis product was divided into 50 mL centrifuge tubes and centrifuged at 8000 rpm for 15 minutes. After centrifugation, the supernatant was mixed. The supernatant was graded using an ultrafiltration flat membrane pilot device and passed through a 5KDa flat membrane. The components with a molecular weight Mw < 5k Da were placed in a refrigerator for pre-freezing. Finally, the pre-frozen samples were freeze-dried using a freeze dryer, and the freeze-dried samples were used for subsequent experiments.

[0067] 2. Functional verification of lactoferrin-derived trifunctional peptides

[0068] 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.

[0069] (1) Antioxidant activity

[0070] ABTS free radical scavenging activity:

[0071] Dilute the ABTS stock solution with PB buffer (50mM, pH 7.4) to an absorbance of 0.7±0.02 at a wavelength of 734nm to obtain the ABTS working solution. Thoroughly mix 10μL of samples of different concentrations (dissolved in pure water) and 990μL of ABTS working solution, incubate at room temperature of 27℃ in the dark for 6min, and measure the absorbance at a wavelength of 734nm. Replace the sample solution with pure water as the blank group.

[0072]

[0073] Among them, A1 is the absorbance value of the experimental group, and A0 is the absorbance value of the blank group.

[0074] DPPH free radical scavenging activity:

[0075] Combine equal volumes of sample solutions (dissolved in pure water) with DPPH (dissolved in anhydrous ethanol) of different concentrations and incubate them in the dark at 37°C for 30 minutes. Then measure the absorbance at 517nm. Replace the sample solution with pure water as the blank group.

[0076]

[0077] Among them, A1 is the absorbance value of the experimental group, and A0 is the absorbance value of the blank group.

[0078] OH radical scavenging activity:

[0079] Take 100 μL of samples of different concentrations and mix them 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. Then, add 100 μL of salicylic acid ethanol (9 mM) and continue incubating at 37°C in the dark for 30 minutes. Finally, measure the absorbance at 510 nm. Replace the sample solution with pure water as the blank group.

[0080]

[0081] Among them, A1 is the absorbance value of the experimental group, and A0 is the absorbance value of the blank group.

[0082] 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 the scavenging rate of ABTS free radicals, which was the highest, indicating that they have strong antioxidant capacity and help to reduce the damage of oxidative stress to cells.

[0083] (2) Verification of anti-inflammatory activity

[0084] 1) Cell viability (MTT) assay:

[0085] RAW264.7 macrophages were cultured at 1×10 5 The cells were inoculated at a density of 100 μg / mL in a 96-well plate for overnight culture, and then the old culture medium was discarded. Sample solutions of 1, 10, 100, 250 and 500 μg / mL with a molecular weight of Mw<5k Da prepared in complete culture medium (containing 90% DMEM, 10% FBS and 1% P / S) were added respectively. The complete culture medium was used as the blank control group, and the cells were cultured in the incubator for 24 hours. After that, 10 μL of MTT (5 mg / mL) was added to each well and incubated in the incubator for 4 hours. The culture medium was then discarded, DMSO was added, and its absorbance was measured at 570 nm.

[0086]

[0087] Among them, A1 is the absorbance value of the experimental group, and A0 is the absorbance value of the control group.

[0088] 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.

[0089] 2) NO determination:

[0090] RAW264.7 macrophages were cultured at 5.5 × 10 5 The cells were inoculated at a density of 100 μL / mL in a 96-well plate, and cultured with complete medium for 24 h. CON group, LPS group and experimental group were set up:

[0091] CON group: discard the culture medium, add DMEM, and continue to treat for 24 h;

[0092] LPS group: discard the culture medium, add DMEM and culture for 12 h, then add LPS (1 μg / mL) and incubate for 12 h;

[0093] Experimental group: The culture medium was discarded, and samples with a molecular weight of Mw < 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 with DMEM). Subsequently, LPS (1 μg / mL) was added and incubated with the samples for 12 hours;

[0094] The CON group was used as a blank control group to provide data of normal cell reactions for comparison and to evaluate the natural state of cells without external intervention. The LPS group was a negative control group, which stimulated cells with LPS to simulate inflammatory reactions and provide a comparative basis for the sample effects of the experimental group. Finally, the cell supernatant was collected and the production of NO was determined using a NO detection kit. According to the manufacturer's instructions, the absorbance was measured at a wavelength of 540 nm and calculated.

[0095] The results are as follows Figure 5 As shown, 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.

[0096] 3) RT-PCR experiment:

[0097] RAW264.7 macrophages were counted at 5.5×10 5 The cells were inoculated into a 24-well plate at a density of 1 mL / mL. The cells were cultured in complete medium for 24 h. CON group, LPS group and experimental group were set up:

[0098] CON group: discard the culture medium, add DMEM, and continue to treat for 24 h;

[0099] LPS group: discard the culture medium, add DMEM and culture for 12 h, then add LPS (1 μg / mL) and incubate for 12 h;

[0100] Experimental group: discard the culture medium, add different concentrations (100, 200, 400 and 500 μg / mL) of samples with molecular weight Mw < 5kDa for 12 hours (samples are dissolved and diluted with DMEM), add LPS (1 μg / mL) and incubate with the samples for 12 hours, with the CON group as the blank control group and the LPS group as the negative control group. Then collect the cells. Perform the following operations:

[0101] The cells were washed twice with PBS, lysed with lysis buffer, and RNA was extracted according to the instructions of the reagent. The total RNA was transcribed into cDNA according to the instructions of the reverse transcription kit. The expression levels of NF-κB, IL-6 and IL-1β mRNA were detected using cDNA as a template. Four parallel tests were set up for each sample test group.

[0102] The results are as follows Figures 6-7 As shown in the figure, NF-κB, as a core transcription factor in the inflammatory response, regulates the expression of a variety of pro-inflammatory cytokines, among which IL-1β is a key pro-inflammatory factor, and overexpression may lead to chronic inflammation and tissue damage. Samples with a molecular weight of Mw < 5kDa can significantly reduce the release of these pro-inflammatory factors, further proving that they have anti-inflammatory effects.

[0103] (3) α-Glucosidase inhibitory activity

[0104] First, add 40 μL of phosphate buffer (0.1 mM, pH 6.8) to a 96-well plate, then add 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 of which are dissolved in phosphate buffer. Then, incubate at 37°C for 15 minutes. Subsequently, add 50 μL of PNPG solution (1 mM, as a substrate) and incubate again at 37°C for 20 minutes for reaction. After incubation, add 100 μL of 1 M sodium carbonate solution to terminate the reaction. Finally, measure the absorbance at a wavelength of 405 nm. Use 20 μL of phosphate buffer instead of the sample solution as a blank group.

[0105]

[0106] Among them, A1 is the absorbance value of the experimental group, and A0 is the absorbance value of the blank group.

[0107] The results showed that samples with molecular weight Mw < 5k Da showed 30.59%, 26.04%, 17.27% and 10.58% inhibitory activity against α-glucosidase at 40mg / mL to 15mg / mL, respectively, with the highest reaching 30.59%. Since α-glucosidase plays a key role in carbohydrate metabolism, its inhibition can slow down the absorption of sugar, thus playing an important role in regulating blood sugar levels.

[0108] Example 2 Preparation of Antioxidant, Anti-inflammatory and Hypoglycemic Lactoferrin Peptide

[0109] 1. LC-MS / MS identification of active components

[0110] The components verified to have antioxidant, anti-inflammatory and hypoglycemic activities in Example 1 were identified by LC-MS / MS to obtain a series of peptide sequences.

[0111] The samples were analyzed by LC-MS / MS equipped with an online nanospray ion source. The entire system was an Orbitrap Eclipse mass spectrometer (Thermo Fisher Scientific, MA, USA) connected in series with an EASY-nanoLC1200. A total of 2 μL of sample was loaded (analytical column: Acclaim PepMap C18, 75 μm x 25 cm), and the sample was separated by a gradient of 60 min. The column flow was controlled at 400 nL / min, the column temperature was 40 °C, the electrospray voltage was 2 kV, and the gradient started from 2.2% of phase B, increased to 44% in 51 minutes with a nonlinear gradient, increased to 90% in 3 minutes, and maintained for 6 minutes.

[0112] 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: 60000; AGC target: 4e5; maximum injection time: 50 ms; (2) HCD-MS / MS: resolution: 15000; AGC target: 5e4; maximum injection time: 45 ms; collision energy: 30%; dynamic exclusion time: 40 s.

[0113] 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.

[0114] 2. Screening of antioxidant, anti-inflammatory and hypoglycemic peptides and solid phase synthesis:

[0115] 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 / AntiD MPpred / cgi-bin / AntiDMPpred.pl) was used to screen out lactoferrin peptides with antioxidant, anti-inflammatory and hypoglycemic properties. In order to further test the activity of the isolated and purified peptides, Shanghai Qiangyao Biotechnology Co., Ltd. was commissioned to synthesize the YLSW peptide using solid phase and confirmed by high performance liquid chromatography that the purity of the synthetic peptide was greater than 95%. The amino acid sequence of the YLSW peptide is Tyr-Leu-Ser-Trp.

[0116] Example 3 Functional verification of the antioxidant, anti-inflammatory and hypoglycemic lactoferrin peptide YLSW

[0117] 1. Antioxidant activity verification

[0118] 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.

[0119] like Figure 8-10 As shown in the results, in the antioxidant experiment, the YLSW peptide showed good 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 indicate that the YLSW peptide may exert its antioxidant properties by directly reacting with free radicals or activating the intracellular antioxidant enzyme system, which is of great significance for preventing diseases related to oxidative stress.

[0120] 2. Verification of anti-inflammatory activity

[0121] (1) Cell viability (MTT) assay:

[0122] 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.

[0123] The results are as follows Fig.11 As shown, the YLSW peptide showed no inhibitory effect on cell activity at high and low concentrations, indicating that the sample had good biocompatibility.

[0124] (2) NO determination:

[0125] The experimental method is the same as in Example 1, and the results are as follows Fig.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 was 12.23% at the lowest experimental concentration of 0.1 μg / mL, showing anti-inflammatory potential.

[0126] (3) RT-PCR experiment:

[0127] 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 mRNA expression levels of NF-κB, IL-6, IL-1β and TNF-α were detected.

[0128] The results are as follows Figure 13-16 As shown in the results, YLSW showed stronger anti-inflammatory effects in inhibiting the release of NF-κB and pro-inflammatory factors (IL-6, IL-1β, TNF-α) compared with samples with molecular weight Mw < 5kDa. Compared with the LPS group, YLSW reduced the expression of NF-κB by 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 biological activity and stronger targeting effects, and are suitable for more precise anti-inflammatory treatment.

[0129] 3. Verification of α-glucosidase inhibitory activity and inhibition type

[0130] (1) α-Glucosidase inhibitory activity

[0131] The experimental method was the same as in 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.

[0132] Activity verification results are as follows Fig.17 As shown in the results, YLSW peptide also showed significant effects in hypoglycemic activity, with an inhibition rate of 71.37% on α-glucosidase, effectively slowing down the digestion and absorption of carbohydrates, thus helping to reduce 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.

[0133] (2) Suppressing type verification

[0134] In the experiment, YLSW peptides 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 it was ensured that no sample peptide was added to the blank control group. Subsequently, the mixture was fully shaken and placed in a constant temperature water bath at 37°C for 15 minutes, and then 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 value at a wavelength of 405 nm was first determined. 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 1M Na2CO3 solution was added to the system to terminate the reaction, and the final absorbance value at a wavelength of 405 nm was determined at this time. Based on these data, a Lineweave-Burk double reciprocal curve of PNPG solution concentration and reaction rate was drawn to determine the inhibition type.

[0135] The results show that ( Fig.18 ), and the Lineweaver-Burk double reciprocal method showed that the inhibitory effect of YLSW peptide on α-glucosidase showed a mixed inhibition mechanism. The peptide not only competes with the active site of the free enzyme for substrate binding, resulting in competitive inhibition, but also binds to the enzyme-substrate complex to further inhibit the catalytic activity of the enzyme. The experimental data showed that the inhibitory effect of YLSW peptide is concentration-dependent and has a strong inhibitory effect, which is suitable for the treatment or prevention of diabetes and related diseases.

[0136] (3) Molecular docking experiment of YLSW peptide with Keap1 and α-glucosidase

[0137] The possible binding sites between the YLSW peptide and Keap1 and α-glucosidase were explored by Discovery studio. 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. The interaction process between the receptor and the ligand was visualized using the Discovery studio visualization tool, and parameters such as binding energy and hydrogen bond distance were obtained.

[0138] The results of the mechanism of action of YLSW peptide with Keap1 and α-glucosidase are as follows Fig.19As shown, the YLSW peptide interacts with Keap1 mainly through traditional hydrogen bonds, carbon-hydrogen bonds, Pi-Cation, and Pi-A1ky1, and α-glucosidase mainly through charge attraction, traditional hydrogen bonds, carbon-hydrogen bonds, π-π stacking, alkyl groups, and Pi-A1ky1. The energies of docking of the YLSW peptide with Keap1 and α-glucosidase are shown in Table 1.

[0139] Table 1 Energy of docking of YLSW peptide with Keap1 and α-glucosidase

[0140]

[0141] Docking studies have shown that when binding to Keap1, the YLSW peptide mainly enhances its binding force 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 play an antioxidant role by binding to it. In the binding with 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, Fig.19 As shown in the figure, YLSW peptide forms hydrogen bonds with SER508, ARG415, GLN530 and TYR334 in Keap1, thus enhancing its binding affinity, which may promote the conduction of antioxidant signals. At the same time, YLSW peptide also forms hydrogen bonds with ASP404 and His674 in α-glucosidase. The former interacts with the amino group or polar group of YLSW peptide through its carboxyl group, which helps to inhibit the activity of α-glucosidase and reduce the digestion and absorption of sugars; the latter may regulate the catalytic efficiency by affecting the conformation of the enzyme.

[0142] In summary, the present invention found that the peptide with the sequence of 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 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.

[0143] Example 4 Preparation of a product containing lactoferrin peptide YLSW

[0144] According to the specific needs of the product, lactoferrin peptide YLSW should be uniformly mixed with excipients, including but not limited to antioxidants, stabilizers, fillers, etc. During the mixing process, appropriate amounts of vitamins, minerals and other nutrients can be added as needed to enhance the effect of the final product. The mixing process should be strictly controlled to ensure that the biological activity of lactoferrin peptide YLSW is not damaged throughout the process.

[0145] In terms of adjusting the ratio, the ratio of lactoferrin peptide YLSW to other excipients should be appropriately adjusted according to the purpose of the product (such as health products or medicines) to ensure that the content of lactoferrin peptide YLSW per gram of the final product meets the predetermined requirements. For health products, the content of lactoferrin peptide YLSW should be controlled between 0.05g / g and 0.2g / g; for medicines, the content of lactoferrin peptide YLSW should be between 0.5g / g and 2g / g.

[0146] Comparative Example 1:

[0147] The specific implementation method is as in Example 2-3, except that the peptide HTGLGR (His-Thr-Gly-Leu-Gly-Arg) is screened from the predicted peptides for functional verification and detection, and the results are as follows:

[0148] 1. Antioxidant activity verification

[0149] 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.

[0150] like Figure 20-22 As shown, 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, showing a weak antioxidant activity.

[0151] 2. Verification of α-glucosidase inhibitory activity

[0152] 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.

[0153] Activity verification results are as follows Fig.23 As shown, the HTGLGR peptide did not show significant effect in terms of glucose-lowering activity.

[0154] Although the present invention has been disclosed as above in the form of a preferred embodiment, 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 food, medicine, health care products or daily chemical products.

4. The product according to claim 3, characterized in that The food includes bean products, meat products or fruit and vegetable products.

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

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

7. Use of the trifunctional peptide according to claim 1 in the preparation of health products or medicines that help to resist oxidation and / or maintain healthy blood sugar function.

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

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

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

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