Walnut polypeptide, preparation method and application thereof

Abstract

The present invention discloses a walnut polypeptide, a preparation method and an application thereof, and belongs to the technical field of active peptides. The amino acid sequence of the walnut peptide from the N-terminus to the C-terminus is: Val-Leu-Arg-Trp-Pro-Arg and Leu-Pro-Ser-Tyr, referred to as VLRWPR and LPSY. The present invention prepares an anti-inflammatory polypeptide from walnut by enzymatic hydrolysis, and purifies it by ultrafiltration and reversed-phase high-performance liquid chromatography system (RP-HPLC), and then purifies it by ultra-high performance liquid chromatography-quadrupole-orbitrap mass spectrometry (UPLC-Q-rbitrap-MS 2 ) were used to identify peptide sequences, and peptides with scores greater than 0.5 were selected using the Peptide Ranker database. Antioxidant activity was predicted using AnOxPePred, and anti-inflammatory activity was predicted using AIP Pred. Peptides with higher scores were artificially synthesized and their anti-inflammatory activity was verified. It was found that VLRWPR and LPSY had anti-inflammatory activity, and had different degrees of inhibition rates on the production of four inflammatory mediators (NO, TNF-α, IL-6, and COX-2).

Description

TECHNICAL FIELD

[0001] The present application relates to a walnut polypeptide, a preparation method and application thereof, and belongs to the technical field of active peptides. BACKGROUND

[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 present in immune cells, and then trigger inflammatory responses through several signaling pathways. Inflammatory mediators refer to chemical substances that can cause inflammatory responses released by immune cells or produced by body fluids during the occurrence of inflammation. According to biochemical characteristics, they can be divided into seven categories: vasoactive amines, vasoactive peptides, lipid mediators, complement component fragments, cytokines, chemotactic factors, and proteolytic enzymes. According to the source, 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 pro-inflammatory effects, which are very important in the process of inflammation and development. Common inflammatory mediators mainly include nitric oxide (NO), arachidonic acid, cytokines, and complement.

[0003] NO belongs to cell-derived inflammatory mediators, which acts as a second messenger in signal transduction in the body and participates in the normal physiology and inflammatory response process of the body. 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 combined expression ability and are widely distributed in the body; while inducible NOS (iNOS, also known as NOS2) is only induced to express after cell damage and does not express in resting cells. Under normal conditions, the combined expression of nNOS and eNOS is activated by calcium signals, and in calcium signal dynamics, the activation of the enzyme is rapid and short, so the production of NO is small and short. A small amount of NO is beneficial to the body and can effectively promote the body's immunity and maintain its normal life activities. iNOS is usually not activated by calcium signals and is continuously activated once expression occurs. When the body is stimulated, lipopolysaccharide (LPS) or cytokines induce iNOS to express in large quantities, and then continuously produce a large amount of NO, which damages health. On the one hand, a large amount of NO can cause vasodilation, leading to a decrease in blood pressure and a decrease in blood flow, causing microcirculation disorders in the body; on the other hand, excessive NO can interact with superoxide anions to produce a large amount of highly toxic peroxynitrite anions, leading to oxidative damage to the body.

[0004] Cytokines are a class of small molecular proteins released by cells after stimulation, have a wide range of biological activities, and are involved in various stages of inflammatory response. According to the function, 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, and have the effects of activating endothelial cells and inducing acute inflammatory response in inflammatory response. Anti-inflammatory cytokines include IL-4, IL-10, IL-11, etc., which can inhibit the production of pro-inflammatory cytokines and chemotactic factors in macrophages and dendritic cells, and play an anti-inflammatory role.

[0005] The inflammation model mainly includes in vitro cell model and in vivo animal model. In vitro model is the most common mouse monocyte macrophage RAW264.7 cell line model induced by lipopolysaccharide (LPS). After LPS combines with the receptor on the membrane of RAW264.7 cells, the mouse macrophage model is converted into M1 type, which can induce the activation of corresponding inflammatory signaling pathways (such as NF-κB signaling pathway and MAPK signaling pathway) by secreting various inflammatory mediators, and the activated inflammatory pathways in turn stimulate the production of inflammatory mediators to coordinate the development of inflammation.

[0006] Walnut meal is a by-product after walnut oil is pressed, which is rich in high-quality protein, but the deep processing and utilization are insufficient. The deep development and utilization of walnut meal and the development of new products are the research hotspots at present, among which the most studied is the preparation of active polypeptide by enzymolysis of walnut meal protein. Bioactive polypeptides usually have different biological activities due to different structures, among which anti-inflammatory active polypeptides are a kind 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, so finding safe and non-toxic anti-inflammatory drugs has become the key to research, among which polypeptides are considered as a safe substitute for anti-inflammatory drugs as a natural small molecule active compound due to their safety and non-toxic side effects. SUMMARY

[0007] In order to solve the problems existing in the prior art, one of the purposes of the present application 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)

[0008] The second purpose of the present application is to provide a preparation method of the walnut polypeptide, which specifically comprises the following steps:

[0009] (1) Take walnut kernels to soak, then remove the seed coat of the soaked walnut kernels, dry to constant weight, and repeat pressing to obtain cake meal, which is finely ground and passed through a 120-mesh sieve to obtain defatted walnut powder, which is stored at 4 DEG C for later use.

[0010] (2) The defatted walnut powder is weighed, mixed with ultrapure water, and adjusted to pH 7.0 with HCl and NaOH, and then bromelain is added for enzymatic reaction, and the enzyme activity is inactivated in a boiling water bath, and the supernatant is obtained by centrifugation, and the freeze-dried product is obtained, which is stored at -20 DEG C for later use.

[0011] (3) The enzymatic product obtained in step (2) is prepared into a solution, and separated by ultrafiltration membranes with molecular weights of 10 kda and 3 kda to obtain enzymatic products with molecular weights of <3 kda, between 3-10 kda, and >10 kda,

[0012] (4) The enzymatic product with a molecular weight of less than 3 kDa is purified by RP-HPLC, and the purification conditions are as follows: mobile phase A is water containing 0.1% trifluoroacetic acid, and mobile phase B is acetonitrile containing 0.1% trifluoroacetic acid; the flow rate is 10 mL / min, and the elution gradient is 10-50% B for 30 min; the purified component F4 is obtained at 16-17.5 min of elution, and the walnut peptides are obtained by vacuum freeze-drying, and the anti-inflammatory best amino acid sequences Val-Leu-Arg-Trp-Pro-Arg and Leu-Pro-Ser-Tyr are detected from the walnut peptides.

[0013] Preferably, the soaking temperature in step (1) is 40 DEG C, and the soaking time is 20 min.

[0014] Preferably, the oil content of the cake meal obtained in step (1) is less than 10%.

[0015] Preferably, the amount of defatted walnut powder added to the ultrapure water in step (2) is 50 g / L, and the amount of bromelain added to the ultrapure water is 2 g / L.

[0016] Preferably, the enzymatic reaction conditions in step (2) are as follows: 55 DEG C constant temperature water bath, 100 rpm shaking incubation for 4 h.

[0017] Preferably, the enzyme inactivation time in step (2) is 15 min.

[0018] Preferably, the centrifugation conditions in step (2) are 5000 r / min for 15 min.

[0019] The third object of the present application is to provide an application of walnut peptides, which includes the following two

[0020] (1) The application of the walnut peptides in preparing anti-inflammatory drugs.

[0021] The walnut peptide described in the application can also be obtained by artificial synthesis.

[0022] Advantages of the application

[0023] The application separates and obtains two new walnut polypeptides, and the two polypeptides obtained have good anti-inflammatory effects.

[0024] The two polypeptides obtained have good anti-inflammatory effects and do not produce toxic side effects on the body, and are an excellent choice for preparing anti-inflammatory drugs. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Inhibition activity of walnut meal protein hydrolysate hydrolyzed by different proteases for food industry on NO.

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

[0027] Figure 3 Inhibition activity of different RP-HPLC components on NO.

[0028] Figure 4 Inhibition activity of different peptide segments on NO.

[0029] Figure 5 Effect of different peptide segments on the viability of RAW264.7 cells.

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

[0031] The application will be described in further detail below in conjunction with the accompanying drawings and specific examples, but the scope of protection of the application is not limited to the described content.

[0032] When the anti-inflammatory peptide provided by the application is used by a person skilled in the art, it can be obtained by artificial synthesis or walnut meal enzymatic separation.

[0033] Some experimental methods used in the application are as follows:

[0034] (1) Cell culture

[0035] RAW264.7 mouse macrophages were maintained in DMEM medium containing 10% fetal bovine serum, and then the cells were placed in a 37℃ incubator containing 5% CO2 for culture, and subcultured once every 24h.

[0036] (2) Cell viability determination

[0037] Cell viability was determined by CCK-8 method. Cells in logarithmic phase were seeded in 96-well plates at a density of 5x10 4 cells per well in 100 μL. The culture plates were placed in an incubator overnight, and after the cells adhered (usually 12-24 h), the old liquid in the culture plates was discarded, and then LPS or complete medium containing different concentrations of the sample to be tested was added to each well for incubation for 24 h. Then 10 μL of CCK-8 solution was added to each well. After incubation in the incubator for 1 h, the cell viability was calculated by measuring the absorbance at 450 nm with a microplate reader, and the cell viability was calculated using the following formula 1-1:

[0038] (1-1)

[0039] In the formula, 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 represents the absorbance of the culture medium without cells and the sample to be tested, containing CCK-8.

[0040] (3) Determination of NO production

[0041] RAW264.7 cells (5x10 4 cells per well) were seeded in 96-well plates and cultured for 24 h, and then the old culture medium was discarded, and complete medium containing different concentrations of the sample to be tested was added, and the NO release amount at the corresponding concentration was determined, and a blank group and an induction group induced by LPS (lipopolysaccharide) and a positive control group were set up, as follows:

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

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

[0044] Experimental group: addition of sample, normal culture for 2 h + LPS (1 μg / mL) stimulation for 24 h;

[0045] Six replicates were set up per well, and after the culture was completed, the supernatant was collected; the Griess method was used to determine the NO concentration in the supernatant, and the principle was that NO was oxidized to nitrite in water, and the method determined the content of NO by measuring the content of nitrite. The specific steps were as follows: 50 μL of cell supernatant was mixed with 50 μL of Griess I reagent and 50 μL of Griess II reagent, and after 5 min, the absorbance was measured at 540 nm. The NO content in the sample was calculated according to the standard curve of nitrite, and the inhibition rate of NO was calculated using formula 1-2 (model group, i.e. induction group, sample group, i.e. experimental group):

[0046] (1-2)

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

[0048] RAW264.7 cells (5 x 10 4 cells / well) were inoculated into 96-well plates, with 6 replicates per well. The experimental group, blank group and induction group were set as in the determination of NO production, and the supernatant was collected after culture. The effects of VLRWPR and LPSY on the production of inflammatory cytokines (TNF-α, IL-6, COX-2) of the induced cells were determined by ELISA kit. The inhibition rate of each cytokine was calculated by formula 1-2. Example

[0049] The preparation, structure identification and anti-inflammatory activity screening of walnut polypeptide are as follows:

[0050] (1) Take Yunnan deep-textured walnut kernels and soak them in 40°C warm water for 20 min. Then, remove the seed coat of the soaked walnut kernels in a walnut kernel peeling machine, and dry them in a 40°C drying machine until the weight is constant. Repeat the pressing and squeezing of the dried walnut kernels until the oil content of the cake meal is less than 10%. The cake meal is finely pulverized and sieved through a 120-mesh sieve to obtain defatted walnut powder, which is stored at 4°C.

[0051] (2) Take 5 g of defatted walnut powder and add 100 mL of ultrapure water to mix well. Adjust the pH to the optimum pH of bromelain, alkaline protease, protease, flavor protease, animal protease, neutral protease, papain, trypsin, trypsin and pepsin with 2 mol / L HCl and 1 mol / L NaOH. Then, add 0.2 g of protease and incubate in a constant-temperature water bath at 100 rpm for 4 h. After removal, inactivate the enzyme activity 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 to take the supernatant. Freeze-dry to obtain the hydrolysate, which is stored at -20°C.

[0052] Table 1 Hydrolysis conditions of different proteases

[0053]

[0054] (3) The inhibitory activity of the above 10 protease hydrolysates on NO was determined, and the results are shown in Table 2. According to the determination results, the inhibitory activity of bromelain and alkaline protease hydrolysates was the highest at different protease hydrolysate concentrations of 0.5 mg / mL and 1 mg / mL. The bromelain and alkaline protease hydrolysates were purified for subsequent use. Figure 1 Figure 1

[0055] ​​(4) The hydrolyzate obtained by enzymatic hydrolysis 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 <3 kDa, 3-10 kDa, and >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 test results showed that the NO inhibition rate of the hydrolysates obtained by bromelain and alkaline protease gradually decreased 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 the 29.62±2.04% of alkaline protease. Therefore, the component of the ultrafiltration product of bromelain with a molecular weight of less than 3kDa was selected for RP-HPLC purification to determine the NO inhibition rate.

[0056] (5) The ultrafiltration product with a molecular weight less than 3 kDa, which was hydrolyzed with bromelain, was purified by RP-HPLC under the following purification conditions: 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 against NO was determined, and the results are shown in the figure below. Figure 3 As shown. Figure 3 The test results showed that the F4 component exhibited the best NO inhibitory 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, the targeted screening found that component 4 (F4) had the highest inhibitory activity, and F4 was vacuum freeze-dried to obtain walnut peptide.

[0057] (6) Using UPLC-Q-Orbitrap-MS 2The mass spectrometry information of the peptides in F4 obtained in the high-throughput analysis step (5). Chromatographic conditions: chromatographic column: Infinitylab Poroshell 120EC-C18 (2.1x100mm, 1.9μm, Agilent Technologies), column temperature 30℃; 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: 0-3min 5%A, 3-10min 10%A, 10-15min 20%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℃, dryer temperature 350℃, 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 Peaks Studio 8.0 software, and the F4 components were analyzed by de novo sequencing.

[0058] (7) The results obtained in step (6) were used to select peptides with scores greater than 0.5 using the Peptide Ranker database. The screened peptides were compared with peptides reported in the BIOPEP-UWM and AHTPDB databases, and antioxidant activity was predicted using AnOxPePred (scores > 0.3 were considered to have antioxidant activity), and 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.

[0059] Table 2 Prediction of anti-inflammatory activity of different peptides

[0060]

[0061] (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 synthesized by Shanghai Jier Biochemical Co., Ltd. using solid phase synthesis method, with a purity of >95%, meeting the requirements of activity detection related experiments. By measuring the NO inhibitory activity of VLRWPR, YWNR, NPYYFSH, LPSY, LRHF, and HGFR, as shown in Figure 5, the NO inhibitory activity of VLRWPR, YWNR, NPYYFSH, LPSY, LRHF, and HGFR was 0. Figure 4 As shown, VLRWPR and LPSY were found to have higher NO inhibition rates.

[0062] (9) First, the amino acid sequences of polypeptides VLRWPR and LPSY were converted into simplified molecular input line entry system (SMILES). The ADME properties of the two screened peptide segments were predicted using AdmetSAR (http: / / lmmd.ecust.edu.cn / admetsar1), and peptide segments with good solubility, easy absorption, good metabolic performance, and no toxicity were further analyzed by molecular docking. The screening results are shown in Table 3. According to Table 3, the two screened peptide segments have good solubility, can be absorbed by the human intestinal tract, have good metabolic performance, and are non-toxic.

[0063] Table 3. ADME properties of screened peptide segments using SMILES code

[0064]

[0065] (10) The safety of polypeptides VLRWPR and LPSY was investigated, as shown in Table 4, VLRWPR and LPSY had different degrees of promotion of cell proliferation at a concentration of 0-200 ug / mL, i.e., they were non-toxic. The above data indicate that polypeptides VLRWPR and LPSY are non-toxic at a concentration of 0-200 ug / mL and can be used for further determination of anti-inflammatory activity. Figure 5

[0066] (11) To verify the results of virtual screening, the effects of polypeptides VLRWPR and LPSY on the secretion of TNF-α, IL-6, and COX-2 in LPS-induced RAW264.7 cells were determined. As shown in Table 5, VLRWPR and LPSY had different degrees of inhibition of the production of the three inflammatory mediators (TNF-α, IL-6, and COX-2). Figure 6 ​​

Claims

1. Walnut polypeptide, characterized by: The amino acid sequence of the walnut polypeptide is Val-Leu-Arg-Trp-Pro-Arg.

2. The method for preparing the polypeptide according to claim 1, characterized in that: The polypeptide is prepared from Yunnan deep-grained walnut kernels by enzymatic hydrolysis with bromelain.

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

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