Walnut polypeptide as well as preparation method and application thereof

Abstract

The invention discloses walnut polypeptide as well as a preparation method and application thereof, and belongs to the technical field of active peptides. The amino acid sequences of the walnut peptide from the N terminal to the C terminal are Val-Leu-Arg-Trp-Pro-Arg and Leu-Pro-Ser-Tyr, which are called as VLRWPR and LPSY for short. According to the method, the anti-inflammatory polypeptide is prepared through enzymolysis of walnuts, purification is carried out through ultrafiltration and a reversed-phase high performance liquid chromatography (RP-HPLC), the polypeptide sequence is identified through an ultra-high performance liquid chromatography-quadrupole-orbitrap mass spectrometer (UPLC-Q-rbitrap-MS2), a peptide fragment with the score larger than 0.5 is selected through a Peptide Ranker database, anti-oxidation activity prediction is carried out through AnOxPePred, anti-inflammatory activity prediction is carried out through AIP Pred, and the anti-inflammatory activity of the anti-inflammatory polypeptide is determined. Polypeptides with high scores are artificially synthesized, the anti-inflammatory activity of the polypeptides is verified, and it is found that VLRWPR and LPSY have the anti-inflammatory activity and have different degrees of inhibition rates on the generation amounts of four inflammatory media (NO, TNF-alpha, IL-6 and COX-2).

Description

Technical Field

[0001] The invention relates to a walnut polypeptide and a preparation method and application thereof, belonging to the technical field of active peptides. Background Art

[0002] In recent years, various chronic diseases caused by chronic inflammation have seriously threatened human health. Inflammatory inducers trigger the production of various inflammatory mediators through various sensors in immune cells, and then trigger inflammatory responses through several signal pathways. Inflammatory mediators refer to chemical substances that can cause inflammatory responses and are released by immune cells or produced by body fluids during the inflammation process. They can be divided into seven categories according to their biochemical characteristics: vasoactive amines, vasoactive peptides, lipid mediators, complement component fragments, cytokines, chemokines, and proteolytic enzymes. According to their sources, they can be divided into two categories, namely cell-derived inflammatory mediators and plasma-derived inflammatory mediators. Inflammatory mediators not only participate in inflammatory responses, but also have inflammatory effects. They are very important in the generation and development of inflammation. Common inflammatory mediators mainly include nitric oxide (NO), arachidonic acid, cytokines, complement, etc.

[0003] NO is a cell-derived inflammatory mediator that acts as a second messenger in the body's signal transduction and participates in the body's normal physiological and inflammatory response processes. NO is produced by nitric oxide synthase (NOS), which belongs to the nervous system and is divided into three types under normal conditions. Among them, neuronal NOS (nNOS, also known as NOS1) and endothelial NOS (eNOS, also known as NOS3) have the ability to express in combination and are widely distributed in the body; while inducible NOS (iNOS, also known as NOS2) is only induced to express after cell damage and is not expressed in resting cells. Under normal conditions, the combined expression of nNOS and eNOS is activated by calcium signals. In calcium signal dynamics, the activation of the enzyme is rapid and short-lived, so the production of NO is small and short-lived. A small amount of NO is beneficial to the body and can effectively promote the body's immunity and maintain its normal life activities. However, iNOS is usually not activated by calcium signals. Once expression occurs, it will continue to be activated. When the body is stimulated, lipopolysaccharide (LPS) or cytokines induce iNOS to express in large quantities, and then continue to produce a large amount of NO, which damages health. On the one hand, a large amount of NO can cause vasodilation, leading to lower blood pressure, slower blood flow, and microcirculation disorders; on the other hand, excessive NO can interact with superoxide anions to produce a large amount of highly toxic peroxynitrite anions, causing oxidative damage to the body.

[0004] Cytokines are a class of small molecule proteins released by cells after being stimulated. They have a wide range of biological activities and are involved in all stages of the inflammatory response. According to their effects, they can be divided into pro-inflammatory cytokines and anti-inflammatory cytokines. Common pro-inflammatory cytokines include TNF-α, IL-6, COX-2, etc., which are mainly produced by macrophages and mast cells. They have the functions of activating endothelial cells and inducing acute inflammatory responses in inflammatory responses. Anti-inflammatory cytokines include IL-4, IL-10, IL-11, etc., which can exert anti-inflammatory effects by inhibiting the production of pro-cytokines and chemokines in macrophages and dendritic cells.

[0005] Inflammation models mainly include in vitro cell models and in vivo animal models. Among the in vitro models, the mouse mononuclear macrophage RAW264.7 cell line model induced by lipopolysaccharide (LPS) is the most common. After LPS binds to the receptors on the RAW264.7 cell membrane, the mouse macrophage model is converted to the M1 type, which can induce the activation of corresponding inflammatory signaling pathways (such as NF-κB signaling pathway and MAPK signaling pathway, etc.) by secreting a variety of inflammatory mediators. The activated inflammatory pathways in turn stimulate the production of inflammatory mediators and coordinate the control of the development of inflammation.

[0006] Walnut meal is a byproduct of walnut oil extraction. It is rich in high-quality protein, but it is not fully utilized after deep processing. The deep development and utilization of walnut meal and the development of new products are current research hotspots. Among them, the most studied is the preparation of active peptides by enzymatic hydrolysis of walnut meal protein. Bioactive peptides usually have different biological activities due to their different structures. Among them, anti-inflammatory active peptides are a class of active peptides that can regulate the production and development of inflammation. In recent years, various diseases caused by inflammation, especially chronic inflammation, have seriously threatened human health, and traditional anti-inflammatory drugs have various toxic side effects. Therefore, finding safe and non-toxic anti-inflammatory drugs has become the key to research. Among them, peptides, as a small molecule active compound of natural origin, are considered to be a safe alternative to anti-inflammatory drugs because of their safety and lack of toxic side effects. Summary of the invention

[0007] In order to solve the problems existing in the prior art, one of the purposes of the present invention is to provide a walnut polypeptide, wherein the amino acid sequence of the walnut polypeptide is Val-Leu-Arg-Trp-Pro-Arg (VLRWPR) or Leu-Pro-Ser-Tyr (LPSY). The second purpose of the present invention is to provide a method for preparing a walnut polypeptide, and the method specifically comprises the following steps:

[0008] (1) soaking walnut kernels, removing the walnut kernel seed coat from the soaked walnut kernels, drying to constant weight, repeatedly pressing the dried walnut kernels to obtain cake, pulverizing the cake and passing it through a 120-mesh sieve to obtain defatted walnut powder, and refrigerating at 4° C. for later use.

[0009] (2) Weigh defatted walnut powder, add ultrapure water and stir to mix, adjust the pH to 7.0 with HCl and NaOH, then add bromelain to carry out enzymatic hydrolysis, take out and inactivate the enzyme in a boiling water bath, centrifuge to obtain the supernatant, freeze-dry to obtain the enzymatic hydrolyzate, and store at -20°C for later use.

[0010] (3) preparing the enzymatic hydrolysate obtained in step (2) into a solution, and separating it using ultrafiltration membranes with molecular weights of 10 kDa and 3 kDa to obtain enzymatic hydrolysates with molecular weights of less than 3 kDa, between 3 and 10 kDa, and greater than 10 kDa.

[0011] (4) The enzymatic hydrolysis products with a molecular weight less than 3 kDa were purified by RP-HPLC, and the purification conditions were as follows: mobile phase A was water containing 0.1% trifluoroacetic acid, and mobile phase B was acetonitrile containing 0.1% trifluoroacetic acid; the flow rate was 10 mL / min, and the elution gradient was 10-50% B, which lasted for 30 minutes; the purified component F4 was obtained at 16-17.5 minutes of elution, and F4 was vacuum freeze-dried to obtain walnut peptides. The amino acid sequences of Val-Leu-Arg-Trp-Pro-Arg and Leu-Pro-Ser-Tyr were detected to have the best anti-inflammatory activity.

[0012] Preferably, in step (1), the soaking temperature is 40° C. and the soaking time is 20 min.

[0013] Preferably, the oil content of the cake obtained in step (1) is less than 10%.

[0014] Preferably, in step (2), the amount of defatted walnut powder added to the ultrapure water is 50 g / L, and the amount of bromelain added to the ultrapure water is 2 g / L.

[0015] Preferably, the conditions for the enzymatic hydrolysis reaction in step (2) are: incubation in a 55°C constant temperature water bath at 100 rpm for 4 hours.

[0016] Preferably, the time for inactivating the enzyme in step (2) is 15 minutes.

[0017] Preferably, the centrifugation condition in step (2) is 5000 r / min for 15 min.

[0018] The third object of the present invention is to provide an application of walnut peptide, the application includes the following two

[0019] (1) Application of the walnut peptide in the preparation of anti-inflammatory drugs;

[0020] (2) Application of the walnut peptide in the preparation of anti-inflammatory health products.

[0021] The walnut peptide of the present invention can also be obtained by artificial synthesis.

[0022] Beneficial effects of the present invention

[0023] (1) The present invention separates and extracts two new walnut polypeptides. Testing shows that the two polypeptides have good anti-inflammatory effects.

[0024] (2) The two polypeptides obtained by the present invention not only have good anti-inflammatory effects but also have no toxic side effects on the body, and are an excellent choice for preparing anti-inflammatory drugs or anti-inflammatory health products. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The inhibitory activity of walnut meal protein hydrolysates hydrolyzed by proteases used in different food industries on NO.

[0026] Figure 2 The inhibitory activity of ultrafiltration products with different molecular weights obtained by ultrafiltration on NO.

[0027] Figure 3 The inhibitory activity of different RP-HPLC fractions on NO.

[0028] Figure 4 The inhibitory activity of different peptides on NO.

[0029] Figure 5 The effect of different peptides on the viability of RAW264.7 cells.

[0030] Figure 6 The inhibitory activities of VLRWPR and LPSY on TNF-α, IL-6 and COX-2. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited to the described contents.

[0032] When using the anti-inflammatory peptides provided by the present invention, those skilled in the art can obtain them by artificial synthesis or enzymatic separation of walnut meal.

[0033] Some of the experimental methods used in the present invention are as follows:

[0034] (1) Cell culture

[0035] RAW264.7 mouse macrophages were maintained in DMEM medium (containing 10% fetal bovine serum) and then placed in a 5% CO 2 The cells were cultured in a 37°C incubator and passaged every 24 h.

[0036] (2) Determination of cell viability

[0037] Cell viability was determined by CCK-8 assay. Cells in the logarithmic phase were cultured at 5×10 4 The cells were inoculated into a 96-well plate at a density of 100 μL per well. The culture plate was placed in an incubator for overnight culture. After the cells adhered to the wall (usually 12 to 24 hours), the old culture medium was discarded, and LPS or complete culture medium containing different concentrations of the sample to be tested was added to each well and incubated for 24 hours. Then 10 μL of CCK-8 solution was added to each well. After incubation in the incubator for 1 hour, the cell viability was calculated by measuring the absorbance at 450 nm with an enzyme-labeled instrument, and the cell viability was calculated using the following formula 1-1:

[0038]

[0039] Where: As represents the absorbance of the culture medium containing cells, CCK-8, and the sample to be tested; Ac represents the absorbance of the culture medium containing cells and CCK-8 without the sample to be tested; A b It indicates the absorbance of the culture medium without cells and test samples, containing CCK-8.

[0040] (3) Determination of NO generation

[0041] RAW264.7 cells (5x10 4 The cells were inoculated into 96-well plates, and the old culture medium was discarded after culturing for 24 hours. Complete culture medium containing different concentrations of the samples to be tested was added to measure the NO release at the corresponding concentrations. A blank group, an induced group induced by LPS (lipopolysaccharide) and a positive control group were set up as follows:

[0042] Blank group: normal culture for 2h+24h;

[0043] Induction group: normal culture for 2 h + LPS (1 μg / mL) stimulation culture for 24 h;

[0044] Experimental group: normal culture of samples for 2 h + LPS (1 μg / ml) stimulation culture for 24 h;

[0045] Six replicates were set up for each well. After the culture was completed, the supernatant was collected. The NO concentration in the supernatant was determined by the Griess method. The principle is that NO is oxidized to nitrite in water. This method determines the NO content by measuring the nitrite content. The specific steps are: 50 μL of cell supernatant was mixed with 50 μL of Griess I reagent and 50 μl of Griess II reagent, and the absorbance was measured at 540 nm after 5 minutes. The NO content of the cell fluid in each sample was calculated based on the drawn nitrite standard curve. The NO inhibition rate was calculated using formula 1-2 (the model group is the induced group, and the sample group is the experimental group):

[0046]

[0047] (4) Determination of cytokines TNF-α, IL-6, and COX-2:

[0048] RAW264.7 cells (5x10 4 (3) Determination of NO production. After the culture was completed, the supernatant was collected. The effects of VLRWPR and LPSY components on the production of inflammatory factors (TNF-α, IL-6, COX-2) after induction were determined using ELISA kits. The inhibition rate of each cytokine was calculated using formula 1-2.

[0049] Example 1

[0050] The preparation, structural identification and anti-inflammatory activity screening of walnut peptides are as follows:

[0051] (1) Yunnan deep-grained walnut kernels are placed in a soaking tank and soaked in 40°C warm water for 20 min, then the soaked walnut kernels are placed in a walnut kernel peeling machine to remove the walnut kernel inner seed coat, placed in a 40°C drying machine to dry to constant weight, the dried walnut kernels are repeatedly pressed until the oil content of the cake is less than 10%, the cake is finely crushed and then passed through a 120-mesh sieve to obtain defatted walnut powder, and refrigerated at 4°C for use.

[0052] (2) Weigh 5 g of defatted walnut powder, add 100 mL of ultrapure water and stir to mix, adjust the pH to the optimum pH of bromelain, alkaline protease, composite protease, flavor protease, animal protease, neutral protease, papain, pancreatin, trypsin and pepsin with 2 mol / L HCl and 1 mol / L NaOH, then add 0.2 g of protease, incubate in a constant temperature water bath at 100 rpm for 4 h, take out and inactivate the enzyme in a boiling water bath for 15 min (the hydrolysis temperature and optimum pH of different proteases are shown in Table 1), centrifuge at 5000 r / min for 15 min, take the supernatant, freeze-dry to obtain the enzymolysate, and store at -20°C for later use.

[0053] Table 1 Hydrolysis conditions of different proteases

[0054] Temperature / ℃ pH Bromelain 55 7.0 Alkaline protease 55 9.0 Compound protease 55 6.0 Flavor protease 55 7.0 Animal protease 55 7.0 Neutral protease 50 7.0 Papain 55 6.5 Pancreatin 50 7.0 Trypsin 37 7.0 Pepsin 37 1.5

[0055] (3) The inhibitory activity of the above 10 protease hydrolysates on NO was determined. The results are as follows: Figure 1 As shown. Figure 1 The results showed that when the concentrations of different protease hydrolysates were 0.5 mg / mL and 1 mg / mL, bromelain and alkaline protease hydrolysates were screened to have the highest inhibitory activity, and the bromelain and alkaline protease hydrolysates were subsequently purified.

[0056] (4) The hydrolyzate obtained by enzymolysis of bromelain and alkaline protease was prepared into a solution with a concentration of 2.5 mg / mL, and separated using ultrafiltration membranes with molecular weights of 10 kda and 3 kda to obtain ultrafiltration products with molecular weights of less than 3 kda, between 3 and 10 kda, and greater than 10 kda. The components were collected and freeze-dried, and stored at 4° C. for later use. The inhibitory activity of the above components on NO was determined. The determination results are as follows: Figure 2 As shown. Figure 2 The results showed that the NO inhibition rate of the hydrolysates obtained by bromelain and alkaline protease was gradually reduced when the molecular weight was less than 3kDa, the molecular weight was between 3-10kDa and the molecular weight was greater than 10kda. The results of the ultrafiltration products of the two enzymes were similar, but the ultrafiltration product of bromelain had the highest inhibition rate on NO when the molecular weight was less than 3kDa and the concentration was 0.5mg / mL, which was 43.62±1.34%, higher than 29.62±2.04% of alkaline protease. Therefore, the ultrafiltration product of bromelain with a molecular weight less than 3kDa was selected for RP-HPLC purification to determine the NO inhibition rate.

[0057] (5) The ultrafiltration product hydrolyzed with bromelain and having a molecular weight less than 3 kDa was purified by RP-HPLC, and the purification conditions were as follows: mobile phase A was water containing 0.1% trifluoroacetic acid, and mobile phase B was acetonitrile containing 0.1% trifluoroacetic acid. The flow rate was 10 mL / min, and the elution gradient was 10-50% B for 30 minutes. The detection wavelength was 220 nm. Purified component F1 was obtained at 4.0-6.0 minutes of elution, purified component F2 was obtained at 8.5-10 minutes, purified component F3 was obtained at 11-13 minutes, purified component F4 was obtained at 16-17.5 minutes, and purified component F5 was obtained at 19.5-22.5 minutes. The inhibitory activity of the five components on NO was measured, and the measurement results are as follows: Figure 3 As shown. Figure 3 The test results showed that the F4 component exhibited the best NO inhibition activity, and the effective ingredient in the F4 component had the highest NO inhibition rate at a concentration of 1 mg / mL, which was 45.58±1.23%. Therefore, component 4 (F4) was screened to have the highest inhibitory activity, and F4 was vacuum freeze-dried to obtain walnut peptide.

[0058] (6) Using UPLC-Q-Orbitrap-MS 2The mass spectrometry information of the peptide segment in F4 obtained in the high-throughput analysis step (5). Chromatographic conditions: chromatographic column: Infinitylab Poroshell 120EC-C18 (2.1x100mm, 1.9μm, AgilentTechnologies), column temperature 30°C; mobile phase: A: acetonitrile (0.1% formic acid), mobile phase B: ultrapure water (0.1% formic acid); injection volume: 2μL; flow rate: 0.2mL / min; gradient elution: 0-3min5%A, 3-10min 10%A, 10-15min20%A, 15-20min 95%A, 20-22min 5%A, 22-25min 5%A; mass spectrometry conditions: ESI+ ion source, spray voltage 3.2kV, capillary temperature 350°C, dryer temperature 350°C, data acquisition range m / z 200-1000, mass spectrometry data scanning mode Full MS-dd MS 2 The resolution of the primary and secondary mass spectra was 70,000 and 35,000, respectively. The mass spectrometry data were analyzed using PeaksStudio 8.0 software; and the F4 component was analyzed by de novo sequencing.

[0059] (7) The peptides with scores greater than 0.5 were selected from the results obtained in step (6) using the Peptide Ranker database. The screened peptides were compared with the peptides reported in the BIOPEP-UWM and AHTPDB databases, and the antioxidant activity was predicted using AnOxPePred (scores > 0.3 were considered to have antioxidant activity), and the anti-inflammatory activity was predicted using AIP Pred. Finally, six peptides with possible anti-inflammatory activity were identified: VLRWPR, YWNR, NPYYFSH, LPSY, LRHF, and HGFR. The molecular weight, anti-inflammatory activity, and toxicity predictions are shown in Table 2.

[0060] Table 2 Prediction of anti-inflammatory activity of different peptides

[0061]

[0062] (8) In order to further obtain anti-inflammatory active peptides, the top-ranked peptides VLRWPR, YWNR, NPYYFSH, LPSY, LRHF, and HGFR were artificially synthesized to verify their anti-inflammatory activity. The peptides were commissioned to Shanghai Jier Biochemical Co., Ltd. to synthesize by solid phase synthesis, with a purity of >95%, meeting the requirements of activity detection related experiments. The NO inhibitory activity of VLRWPR, YWNR, NPYYFSH, LPSY, LRHF, and HGFR was determined. Figure 4 As shown, VLRWPR and LPSY were found to have higher NO inhibition rates.

[0063] (9) First, the amino acid sequences of the peptides VLRWPR and LPSY were converted into the Simplified Molecular Input Line Entry Standard System (SMILES). AdmetSAR (http: / / lmmd.ecust.edu.cn / admetsar1) was used to predict the ADMET properties of the two peptides screened above, and the peptides with good solubility, easy absorption, good metabolic performance, and non-toxicity were further subjected to molecular docking research and analysis. The screening results are shown in Table 3. According to Table 3, the two peptides screened above have good solubility, can be absorbed by the human intestine, have good metabolic performance, and are non-toxic.

[0064] Table 3 ADMET property prediction of the peptides obtained by screening using SMILES code

[0065]

[0066] (10) The safety of peptides VLRWPR and LPSY was investigated. Figure 5 As shown, it was found that within the concentration range of 0-200ug / mL, VLRWPR and LPSY had different degrees of promoting cell proliferation, that is, they were not toxic. The above data show that the polypeptides VLRWPR and LPSY were not toxic within the concentration range of 0-200ug / mL and can be used to continue to determine the anti-inflammatory activity.

[0067] (11) To verify the results of virtual screening, the effects of peptides VLRWPR and LPSY on the secretion of TNF-α, IL-6, and COX-2 in RAW264.7 cells induced by LPS were measured. Figure 6 As shown, VLRWPR and LPSY have different degrees of inhibition rates on the production of three inflammatory mediators (TNF-α, IL-6, COX-2).

Claims

1. Walnut polypeptide, characterized in that: The amino acid sequence of the walnut polypeptide is Val-Leu-Arg-Trp-Pro-Arg or Leu-Pro-Ser-Tyr.

2. The method for preparing the polypeptide according to claim 1, characterized in that: The polypeptide is prepared from walnut.

3. Use of the polypeptide according to claim 1 in the preparation of anti-inflammatory drugs.

4. Use of the polypeptide according to claim 1 in the preparation of anti-inflammatory health products.

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