A composition for preventing non-viral hepatitis, a method of preparing the same, and use thereof

Abstract

The application discloses a composition for preventing non-viral hepatitis and a preparation method and application thereof, and belongs to the technical field of food processing. The composition comprises a crude peptide, and the amino acid sequence of the crude peptide is Ile-Val-Tyr-Pro-Trp-Thr-Gln-Arg; further comprises lipids and excipients; the crude peptide and the lipids form a water-in-oil emulsion system; and the emulsion system and the excipients are mixed to form the composition. Experiments prove that the composition prepared by the application has the effect of improving non-viral hepatitis, and provides a new way for the prevention and treatment of non-viral hepatitis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of food processing, in particular to a composition for preventing non-viral hepatitis and a preparation method and application thereof. BACKGROUND

[0002] Inflammation accompanying the occurrence of diseases is easy to aggravate the development of diseases, and even lead to malignant tumors. Hepatitis is one of the important pathological characteristics, which can lead to fatty hepatitis, liver fibrosis, and cirrhosis. According to the global burden of disease research, non-viral hepatitis caused by improper dietary habits (including alcohol, high fat, high cholesterol, etc.) is one of the important diseases leading to morbidity and mortality. At present, antibiotics are used to relieve non-viral hepatitis, but excessive use of antibiotics can cause strong side effects, so the treatment of non-viral hepatitis is still a major health problem worldwide. Previous studies have confirmed that improper diet can damage the intestinal barrier function and accelerate the translocation of LPS, thereby activating the downstream pathways and the release of related inflammatory factors to cause the production of hepatitis. Macrophages may produce inflammatory mediators (ROS and NO), a series of cytokines (interleukin 1β, interleukin 6, TNF-α) under the stimulation of LPS, and ROS can induce oxidative stress to mediate apoptosis and inflammation and aggravate the development of diseases. Therefore, inhibiting oxidative stress and inflammatory response is an effective strategy to improve LPS-mediated non-viral hepatitis.

[0003] In recent years, bioactive peptides have been widely used to improve inflammatory response and prevent liver disease from worsening. Although a variety of active polypeptides (such as collagen peptides, Jinhua ham oligopeptides, etc.) have been proved to improve liver damage, it is still a challenge to prevent non-viral hepatitis caused by improper diet. Bioactive peptides are derived from a wide range of sources, including plants, animals, microorganisms, etc., because they contain rich proteins. Poultry blood, as one of the animal by-products, is rich in protein and is one of the high-quality sources. However, the development and utilization of poultry blood is still far from enough, mainly focusing on food, extraction of some medicinal raw materials and animal feed, and a large part of it is directly discarded, resulting in serious waste of resources. Therefore, using poultry blood as the extraction raw material can find a new direction for the high-value processing of poultry blood, provide a new direction for the development of food-grade blood products, and also provide a new way for the prevention of non-viral hepatitis. SUMMARY

[0004] The purpose of the present application is to provide a composition for preventing non-viral hepatitis and a preparation method and application thereof, to solve the problems existing in the prior art, to separate and extract a crude peptide from poultry blood, to prepare a composition with anti-non-viral hepatitis by using the crude peptide, lipids and excipients, to realize the improvement of the utilization value of poultry blood, and to provide a new way for the prevention of non-viral hepatitis.

[0005] To achieve the above object, the present application provides the following scheme:

[0006] The present application provides a composition for preventing non-viral hepatitis, which comprises a crude peptide with an amino acid sequence of Ile-Val-Tyr-Pro-Trp-Thr-Gln-Arg (SEQ ID NO: 1).

[0007] Preferably, the composition further comprises a lipid and an auxiliary material, the crude peptide and the lipid form a water-in-oil emulsion system, and the emulsion system and the auxiliary material are mixed to form the composition.

[0008] Preferably, the crude peptide is derived from poultry blood.

[0009] The present application also provides a preparation method of the composition, comprising the following steps:

[0010] (1) Extraction of crude peptide

[0011] The blood meal derived from poultry blood is enzymatically hydrolyzed, centrifuged and ultrafiltrated to obtain a crude peptide extract;

[0012] The obtained crude peptide extract is sequentially subjected to size exclusion chromatography, ion exchange chromatography and reverse phase high performance liquid chromatography separation to obtain the crude peptide.

[0013] (2) Preparation of lipid

[0014] The poultry fat is collected and refined under high temperature conditions, and after filtering and discarding the oil residue, the lipid is prepared by mixing with palmitate and stearate.

[0015] (3) Emulsification and homogenization

[0016] The crude peptide prepared in step (1), the lipid prepared in step (2), and polyglycerol ricinoleate are mixed and high-speed sheared to form a water-in-oil emulsion system.

[0017] (4) The emulsion system prepared in step (3) and the auxiliary material are mixed and stirred uniformly to obtain the composition for preventing non-viral hepatitis.

[0018] Preferably, in step (1), the enzymatic hydrolysis conditions are that the blood meal and pepsin are mixed at a mass ratio of 400:1 and enzymatically hydrolyzed at 37°C for 4 hours.

[0019] Preferably, in step (2), the refining and extraction conditions are that the refining and extraction are performed at 120-150°C for 20-40 minutes.

[0020] The mass ratio of the poultry fat, palmitate and stearate is (80-82):(11-13):7.

[0021] Preferably, in step (3), the mass ratio of the crude peptide, the lipid and the polyglycerol ricinoleate is (40-60):(40-60):(6-8);

[0022] The high-speed shearing condition is that the emulsion is dispersed into nanoparticles by high-speed shearing at a speed of 25000-30000 rpm / min.

[0023] Preferably, in step (4), the mass ratio of the emulsifying system and the adjuvant is (60-80):(20-40).

[0024] The application further provides use of the composition in preparation of a medicine for preventing non-viral hepatitis.

[0025] The application further provides a medicine comprising the composition.

[0026] The application discloses the following technical effects:

[0027] The crude peptide is obtained from poultry blood, and experiments prove that the crude peptide has strong stability and can resist the digestion of the gastrointestinal tract, thereby playing a role in resisting non-viral hepatitis, so that the application solves the problem of insufficient utilization of poultry blood in the prior art, and provides a new idea for prevention and treatment of non-viral hepatitis.

[0028] The crude peptide prepared by the application is relatively systematic, and is combined with poultry oil to prepare a composition capable of resisting non-viral hepatitis, which can be prepared into a medicine according to needs, and can be prepared into various forms such as granules, powdery tonics and tablets, which is very convenient and provides a new way for healthy and diversified diet. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed in the embodiments will be briefly introduced below, and obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor.

[0030] Figure 1 Toxicity and cell viability of LPS and GBP on RAW264.7 macrophages; A: effect of LPS on RAW264.7 macrophage viability; B: effect of GBP on RAW264.7 macrophage viability; C: effect of LPS+GBP on RAW264.7 macrophage viability; D: effect of LPS+GBP on inflammatory factor IL-1 beta; E: effect of LPS+GBP on inflammatory factor IL-6; F: effect of LPS+GBP on inflammatory factor TNF-alpha;

[0031] Figure 2 Effect of peptides obtained from size exclusion chromatography on LPS-induced RAW264.7 macrophage injury model; A: chromatogram of size exclusion chromatography separation of crude peptide extract; B: evaluate the effect of components A-F on the level of inflammatory factor IL-1 β; C: evaluate the effect of components A-F on the level of inflammatory factor IL-6; D: evaluate the effect of components A-F on the level of inflammatory factor TNF-α;

[0032] Figure 3 Effect of peptides obtained from ion exchange chromatography on LPS-induced RAW264.7 macrophage injury model; A: chromatogram of ion exchange chromatography separation of GBP-E components; B: evaluate the effect of fractions E1 and E2 on the level of inflammatory factor IL-1 β; C: evaluate the effect of fractions E1 and E2 on the level of inflammatory factor IL-6; D: evaluate the effect of fractions E1 and E2 on the level of inflammatory factor TNF-α;

[0033] Figure 4 Effect of polypeptides obtained from reverse phase liquid chromatography on LPS-induced RAW264.7 macrophage injury model; A: chromatogram of reverse phase high performance liquid chromatography separation of E1; B: evaluate the effect of different fractions on the level of inflammatory factor IL-1 β; C: evaluate the effect of different fractions on the level of inflammatory factor IL-6; D: evaluate the effect of different fractions on the level of inflammatory factor TNF-α;

[0034] Figure 5 Analysis and identification of active peptide IIe-Val-Tyr-Pro-Trp-Thr-Gln-Arg (IVYPWTQR); A: RP-HPLC chart; B: primary mass spectrum chart; C: secondary mass spectrum chart;

[0035] Figure 6 Effect of pepsin-trypsin simulated GI (gastrointestinal) digestion on the stability of IVYPWTQR; A: evaluate the effect of different treatment groups on the level of inflammatory factor IL-1 β; B: evaluate the effect of different treatment groups on the level of inflammatory factor IL-6; C: evaluate the effect of different treatment groups on the level of inflammatory factor TNF-α;

[0036] Figure 7 RP-LC chart of IVYPWTQR treatment group after trypsin simulated intestinal digestion;

[0037] Figure 8 Molecular weight and amino acid sequence of 6.2337 min component identified by MS / MS;

[0038] Figure 9 Molecular weight and amino acid sequence of 42.0351 min component identified by MS / MS;

[0039] Figure 10 MS / MS was used to identify the molecular weight and amino acid sequence of the component at 44.7485 min;

[0040] Figure 11 Effects of IVYPWTQR on LPS-induced inflammatory response of RAW264.7 macrophages; A-B: effects on the expression levels of TLR4, CD14, MD-2, NF-KB, p-NF-KB, IKKβ proteins after LPS or LPS and active peptide treatment; C-E: effects on the TLR4 / NF-kB / iNOS pathway after LPS or LPS and active peptide treatment;

[0041] Figure 12 Effects of IVYPWTQR on LPS-induced oxidative stress response of RAW264.7 macrophages; A-C: effects on the expression levels of ROS, SOD and GSH-Px after LPS or LPS and active peptide treatment; D-E: effects on the Keap-1 / NrF2 / HO-1 signaling pathway after LPS or LPS and active peptide treatment. DETAILED DESCRIPTION

[0042] Various exemplary embodiments of the present application will now be described in detail, which should be considered to be illustrative of certain aspects, features and embodiments of the present application and are not intended to limit the scope of the present application.

[0043] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, for any numerical range recited herein, every narrower numerical range that falls within the broader range is also contemplated as being included in this application. Broader and narrower ranges can be combined with other ranges to form still broader ranges. As used herein, the use of the singular includes the plural unless otherwise indicated. It is also to be understood that the use of "or" means "and / or" unless otherwise indicated. It is further to be understood that the use of "about" means "approximately" or "substantially" unless otherwise indicated.

[0044] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict between the present specification and any document incorporated by reference, the present specification will control.

[0045] Many modifications and variations of the specific embodiments of the application can be practiced in accordance with the teachings of the description of the application, which are within the scope of the present application. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples of the application are exemplary only.

[0046] As used herein, the terms "comprise", "comprising", "include", "including", "have" and "having" and the like are open-ended, that is, they mean "including but not limited to".

[0047] Example 1 A method for preparing a composition for preventing non-viral hepatitis, comprising the steps of:

[0048] I. Extraction and separation of active peptides

[0049] 1. Extraction of crude peptides

[0050] Poultry blood (chicken blood, duck blood, goose blood, one or more of which has the same effect, and goose blood is taken as an example, the same below) is collected and freeze-dried into powder. 100 g of blood powder is dissolved in 2000 mL of PBS (pH 7.2), and pepsin is used for enzymolysis at 37°C for 4 hours (blood powder: pepsin = 400: 1 W / W), and then homogenized at 20000 rpm for 5 times, 40 s each time. Centrifugation at 12000 x g for 30 min, take the supernatant, ultrafiltration through a filter membrane with a molecular weight of 3 kDa, take the filtrate, freeze-dry, and it is ready for use as a crude peptide extract.

[0051] 2. Separation and identification

[0052] (1) Size exclusion chromatography is used to separate the crude peptide extract obtained from poultry blood as described above, and the elution conditions are as follows: the mobile phase is 0.1 mol / L HCl, the flow rate is 1 mL / min, each fraction is detected by a 280 nm ultraviolet detector, and each fraction is collected by an automatic fraction collector; and the fractions are dried in a vacuum freeze dryer for later use.

[0053] The process conditions for vacuum freeze drying are: -40°C to -50°C, 24 h.

[0054] (2) Ion exchange chromatography is used to further separate the most effective component for preventing non-viral hepatitis obtained in (1). 20 mM Tris-HCl is used as the starting buffer, and 20 mM Tris-HCl / 1 M NaCl is used as the eluent, and the flow rate is set to 5 mL / min. Fractions are collected by a UV detector at 280 nm to obtain the most effective component for preventing non-viral hepatitis; and the component is dried in a vacuum freeze dryer for later use.

[0055] (3) The most effective component for preventing non-viral hepatitis obtained in (2) is separated and purified by reverse phase high performance liquid chromatography. The specific separation process is as follows: the freeze-dried sample is dissolved in 1 mL of distilled water to prepare a solution with a concentration of 10 mg / mL, which is injected into the RP-HPLC system for gradient elution at a flow rate of 0.5 mL / min. Eluent A: 0.065% TFA (dissolved in 2% acetonitrile), eluent B: 0.050% TFA (dissolved in 80% acetonitrile), elution program settings are as follows: 0-30 min, 100% A; 30-60 min, 80% B; 60-80 min, 100% A. The components are detected at 280 nm using a UV detector and collected, and six components are obtained. After freeze-drying, the effect of each component on preventing non-viral hepatitis is detected, and the most effective component for preventing non-viral hepatitis is obtained.

[0056] (4) The most effective component for preventing non-viral hepatitis obtained in (3) is analyzed and identified by mass spectrometry, and the crude peptide sequence for preventing non-viral hepatitis is obtained as follows: IIe-Val-Tyr-Pro-Trp-Thr-Gln-Arg (IVYPWTQR). The specific separation process is as follows: the most effective component for preventing non-viral hepatitis obtained in step (3) is identified by LC-MS / MS using a reverse phase BEH C18 chromatographic column to separate the component. The gradient elution program settings are as follows: 0-30 min, 100% A; 30-60 min, 80% B; 60-80 min, 100% A. The flow rate is 0.5 mL / min, the UV detection wavelength is 280 nm, and the column temperature is maintained at 25°C. The flow is directly introduced into the MS / MS system for multiple reaction measurement. The mass range of the precursor ion recorded is m / z = 200-4000. The instrument is operated using Mass Lynx V4.1, and the mass spectrum information is analyzed.

[0057] (5) Results

[0058] When separated by size exclusion chromatography, component A is eluted first due to its largest molecular weight, and component F is eluted last. All components are collected and freeze-dried for later use. The anti-inflammatory activity of A-F( Figure 2 's A) is evaluated, and the levels of inflammatory factors IL-1β, IL-6, and TNF-α are determined using a kit. Component E shows the strongest anti-inflammatory activity, with significantly lower levels of IL-1β (291.17 ± 34.53 pg / mL), IL-6 (228.15 ± 5.38 pg / mL), and TNF-α (179.27 ± 6.26 pg / mL) compared to other components( Figure 2 's B-D). Therefore, GBP-E has the strongest anti-inflammatory activity. For further analysis, GBP-E is dried in a vacuum freeze dryer.

[0059] The E fraction was further separated by ion exchange chromatography, and the E fraction was separated according to different anion exchange ability. Two fractions, E1 and E2, were separated in total, and fraction E1 represented weaker anion exchange ability, and E2 represented stronger anion exchange ability. Figure 3 The separated fractions were freeze-dried, and their anti-inflammatory activities in LPS-induced RAW264.7 macrophages were determined (B-D of FIG. 6). Figure 3 In the E-1 group, IL-1β (275.01 ± 12.37 pg / mL), IL-6 (205.14 ± 13.22 pg / mL), and TNF-α (164.76 ± 6.80 pg / mL) were significantly lower than those in the E2 group. Therefore, E1 had the strongest anti-inflammatory activity. E1 was dried in a vacuum freeze dryer.

[0060] The E1 fraction was further purified by reverse phase high performance liquid chromatography. According to the difference in hydrophobicity, six fractions, E-1-I, E-1-II, E-1-III, E-1-IV, E-1-V, and E-1-VI, were separated and purified (A of FIG. 7). Figure 4 The separated fractions were freeze-dried, and their anti-inflammatory activities in LPS-induced RAW264.7 macrophages were determined (B-D of FIG. 7). Figure 4 In the E-1-V group, IL-1β (245.09 ± 12.65 pg / mL), IL-6 (180.14 ± 8.42 pg / mL), and TNF-α (160.04 ± 11.36 pg / mL) were significantly lower than those in the other groups. Therefore, E-1-V had the strongest anti-inflammatory activity, and E-1-V was finally collected and freeze-dried for further analysis.

[0061] The structure of E-1-V was identified by an Acquity (Waters Inc.) high performance liquid chromatography system using a reverse phase BEH C18 column (i.e., reverse phase high performance liquid chromatography mass spectrometry LC-MS / MS) (B-C of FIG. 8), and one target peptide (A of FIG. 8) was obtained by analysis, and the amino acid sequence thereof was Ile-Val-Tyr-Pro-Trp-Thr-Gln-Arg (IVYPWTQR). Figure 5 Figure 5

[0062] 3. Anti-inflammatory activity identification

[0063] (1) IVYPWTQR was synthesized by Biosynthetic Company using solid phase synthesis, and the anti-inflammatory activity of the peptide in LPS (lipopolysaccharide)-induced RAW264.7 macrophages was verified in vitro.

[0064] ​​In vitro simulation: NaCl:pepsin ratio of 1:1.6 was dissolved in 800 mL of ultrapure water. The pH of the solution was adjusted to 3.0 with 6 mmol / L HCl, and the volume was brought up to 1 L with ultrapure water. This solution simulated gastric juice. 0.68 g of potassium dihydrogen phosphate was dissolved in 70 mL of ultrapure water, and 7.7 mL of 0.2 mol / L NaOH solution was added and mixed thoroughly. 1 g of trypsin and 6 g of bile salts were added, and the pH was adjusted to 7.6 with 0.2 mol / L NaOH solution. The volume was then brought up to 1 L. This solution simulated intestinal juice. Control and experimental groups were set up. The control group consisted of LPS, and the experimental groups consisted of LPS+IVYPWTQR, LPS+IVYPWTQR (pepsin), and LPS+IVYPWTQR (trypsin). The peptide concentration was set at 400 μg / mL, and the pH was adjusted to 3.0. Add 5 mL of simulated gastric juice to the solution, incubate in a water bath at 37°C for 2 hours, and then heat at 100°C for 10 minutes to terminate the simulated gastric juice digestion. Adjust the pH of the mixture to 7.2, add 5 mL of simulated intestinal juice, and incubate at 37°C for 2 hours. Centrifuge the digestive fluid at 4°C and 8500g, and then desalt and freeze-dry.

[0065] This invention investigated the toxicity of LPS and GBP on RAW264.7 macrophages to determine the median lethal concentration (LC50) of LPS and the concentration of GBP. With increasing LPS concentration, the viability of RAW264.7 macrophages was significantly lower than that of the control group (…). Figure 1 The median lethal concentration (LC50) of LPS was determined to be 1.0 μg / mL. The addition of GBP did not significantly reduce the viability of RAW264.7 macrophages, demonstrating that GBP has no toxic effect. Figure 1 (B). In the LPS+GBP group, the activity of RAW264.7 macrophages was significantly increased and the level of inflammatory factors was significantly decreased, confirming that GBP can improve LPS-induced inflammation of RAW264.7 macrophages. Figure 1 (CF).

[0066] The bioactive peptides of this invention for preventing nonviral hepatitis can effectively improve LPS (lipopolysaccharide)-induced inflammation of RAW264.7 macrophages. Furthermore, dietary supplementation with GBP (goose blood peptide) can prevent or alleviate LPS-induced inflammation, providing a dietary intervention strategy for preventing hepatocellular inflammation.

[0067] (2) Effect of pepsin-trypsin mimicking GI (gastrointestinal) digestion on IVYPWTQR stability

[0068] Gastrointestinal digestion is a major factor affecting the structure and function of bioactive peptides. In vitro simulated gastrointestinal digestion model was used to explore the stability of IVYPWTQR during gastrointestinal digestion. Control group and experimental groups (LPS, LPS+IVYPWTQR, LPS+IVYPWTQR(pepsin), LPS+IVYPWTQR(trypsin)) were set up to detect the release levels of IL-Iβ, IL-6 and TNF-α.

[0069] As Figure 6 In the experimental group LPS+IVYPWTQR(pepsin), the levels of IL-Iβ, IL-6 and TNF-α were slightly increased compared to the levels of IL-Iβ, IL-6 and TNF-α in the experimental group LPS+IVYPWTQR, but were still significantly lower than the levels of IL-Iβ, IL-6 and TNF-α in the LPS group. In the experimental group LPS+IVYPWTQR(trypsin), the levels of IL-Iβ (336.33±12.58 pg / mL), IL-6 (232.34±9.56 pg / mL) and TNF-α (197.44±13.01 pg / mL) were not significantly increased compared to the levels of inflammatory factors in the experimental group LPS+IVYPWTQR(pepsin), indicating that IVYPWTQR has strong resistance to trypsin digestion.

[0070] As Figures 7-10 IVYPWTQR was partially decomposed into smaller fragments after pepsin treatment, including IVYP (16.85%) and WTQR (16.74%), indicating that pepsin can specifically cleave the N-terminal peptide bond of the tryptophan residue in the peptide segment. Most of the peptide segments were not hydrolyzed and showed strong anti-inflammatory activity. These results indicate that IVYPWTQR has strong stability and resistance to GI digestion.

[0071] (3) The above results show that IVYPWTQR can improve LPS-induced inflammation in RAW264.7 macrophages. LPS is generally considered to be a key activator that is specifically recognized by receptor proteins (TLR4) and auxiliary receptors (CD14 and MD-2) on the cell membrane, followed by accelerated IKB kinase (IKK) phosphorylation to activate the NF-KB signaling pathway, thereby producing excessive inflammatory factors. The inflammatory factor TNF-α, as a key pro-inflammatory mediator of inflammation, can activate the iNOS signaling pathway to exacerbate inflammation. Western blot was used to detect the expression of related proteins.

[0072] As Figure 11A-B, the expression levels of TLR4, CD14, MD-2, NF-KB, p-NF-KB, IKKβ proteins were significantly increased compared with the control group, while the expression levels of the above proteins were significantly decreased after IVYPWTQR treatment. Compared with the control group, the expression level of iNOS protein was significantly increased after LPS treatment, and the level of NO was increased, while after IVYPWTQR treatment, the level was significantly decreased. These results show that IVYPWTQR can reduce the production of NO, IL-Iβ, IL-6 and TNF-α by down-regulating the TLR4 / NF-kB / iNOS pathway to improve LPS-induced inflammatory response Figure 11

[0073] (4) The above results show that IVYPWTQR improves LPS-induced oxidative stress in RAW264.7 macrophages. LPS-mediated inflammation causes macrophages to produce excess reactive oxygen species (ROS), which accelerates immune dysfunction through oxidative stress. The Keap-1 / NrF2 / HO-1 mediated pathway is considered a key factor in activating and regulating SOD and GSH-Px involved in antioxidant defense. The levels of reactive oxygen species (ROS), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were detected using a kit.

[0074] Compared with the control group, after LPS treatment, the level of ROS (61.96±2.27 U / mg prot) was significantly increased, and the levels of SOD ((24.43±1.41 U / mg prot) and GSH-Px (44.63±2.72 U / mg prot) were decreased. After IVYPWTQR treatment, the level of ROS (36.32±3.69 U / mg prot) was significantly reduced, and the levels of SOD (38.68±1.68 U / mg prot) and GSH-Px (79.14±3.41 U / mg prot) were increased Figure 12

[0075] As shown in Figure 12 D, LPS treatment significantly reduced the expression of Keap-1, NrF2, HO-1, but after IVYPWTQR treatment, the expression levels of Keap-1, NrF2, HO-1 were significantly increased, indicating that IVYPWTQR can enhance the antioxidant defense of cells by up-regulating the Keap-1 / NrF2 / HO-1 signaling pathway, reduce the accumulation of ROS and improve LPS-mediated inflammatory response.

[0076] II. Preparation of lipids

[0077] ​​(1) Collect the poultry fat, refine the fat at 120°C for 20 minutes, filter, discard the oil residue, then add palmitate and stearate (poultry fat: palmitate: stearate = 80: 13: 7) in proportion to prepare the lipid, and reserve it;

[0078] (2) Emulsify and homogenize, take 40% of the lipid prepared in step (1) and 60% of the crude peptide prepared in Example 1, and 6% of polyglycerol ricinoleate, and perform high-speed shearing at 25000 rpm to disperse the emulsion into nanoparticles to form a water-in-oil (W / O) emulsion system, and reserve it;

[0079] (3) Preparation of adjuvants: mix sorbitol, vitamin C, fruit essence, fructose, lactose, and carrageenan. The adjuvants include the following components in the following proportions by weight: fruit essence 0.1 part, fructose 1 part, carrageenan 0.3 part, sorbitol 0.5 part, vitamin C 0.5 part, and lactose 1 part;

[0080] (4) Mix the emulsion in step (2) and the adjuvants in step (3) in a mass ratio of 60:40, stir well, and place in a pharmaceutical dosage form preparation device or mold to prepare granular or powdered granules, tablets, etc.

[0081] Example 2 A method for preparing a composition for preventing non-viral hepatitis, comprising the following steps:

[0082] I. Preparation and isolation of active peptides

[0083] 1. Extraction of crude peptides

[0084] Collect poultry blood and freeze-dry it into powder. Dissolve 100 g of the blood powder in 2000 mL of PBS (pH 7.2), and perform enzymatic hydrolysis with pepsin at 37°C for 4 hours (blood powder: pepsin = 400: 1 W / W), then homogenize it at 21000 rpm / min for 4 times, 50 s each time. Centrifuge at 14000 g for 30 min, take the supernatant, and ultrafilter it through a filter membrane with a molecular weight of 3 kDa. Take the filtrate and freeze-dry it to obtain crude peptides, which are reserved.

[0085] Isolate the active peptides from the crude peptides using the same method as in Example 1. Synthesize IVYPWTQR using solid-phase synthesis, and verify its anti-inflammatory activity in LPS (lipopolysaccharide)-induced RAW264.7 macrophages in vitro using the same method as in Example 1, and obtain the same results as in Example 1.

[0086] II. Preparation of lipids

[0087] (1) Collect the poultry fat, refine it at 140°C for 30 minutes, filter it, discard the oil residue, then add palmitate and stearate in proportion (poultry fat: palmitate: stearate = 81: 12: 7) to prepare the lipid, which is ready for use;

[0088] (2) Emulsification and homogenization: take 50% of the lipid prepared in step (1) and 50% of the crude peptide extracted in Example 1, and 7% of polyglycerol ricinoleate, and perform high-speed shearing at 28000 rpm to disperse the emulsion into nanoparticles to form a water-in-oil (W / O) emulsion system, which is ready for use;

[0089] (3) Preparation of excipients: mix starch, malt dextrin, microcrystalline cellulose, and lactose, which include the following components in the following proportions by weight: starch 0.6 parts, malt dextrin 2 parts, microcrystalline cellulose 0.8 parts, and lactose 1 part;

[0090] (4) Mix the emulsion in (2) and the excipients in (3) in a mass ratio of 70:30, stir them evenly, and place them in a pharmaceutical dosage form preparation device or mold to prepare granular or powdered tonics or tablets (as an example of granular tonics, use 60% ethanol as a wetting agent, mix and stir them evenly, then evaporate the water to a degree where the mixture can be formed into a ball and crumbles when pressed, and then press the soft material through a suitable screen to form granules).

[0091] Example 3 A method for preparing a composition for preventing non-viral hepatitis, comprising the following steps:

[0092] I. Extraction and separation of active peptides

[0093] 1. Extraction of crude peptides

[0094] Collect poultry blood, freeze-dry it into powder. Dissolve 100g of the blood powder in 2000mL of PBS (pH 7.2), and perform enzymatic hydrolysis with pepsin at 37°C for 4 hours (blood powder: pepsin = 400: 1 W / W), then homogenize it at 22000 rpm / min for 3 times, 60s each time. Centrifuge it at 15000g for 30min, take the supernatant, ultrafilter it through a filter membrane with a molecular weight of 3kDa, take the filtrate, and freeze-dry it, which is ready for use as crude peptides.

[0095] Use the crude peptides to obtain active peptides, and the method for separating and identifying the active peptides is the same as in Example 1. Use solid-phase synthesis to synthesize IVYPWTQR, and use the same method as in "3. Anti-inflammatory activity identification" in Example 1 to verify its anti-inflammatory activity in LPS (lipopolysaccharide)-induced RAW264.7 macrophages in vitro, and obtain the same results as in Example 1.

[0096] 2. Preparation of lipids:

[0097] (1) Collect poultry fat, refine at 150℃ for 20 minutes, filter, discard the oil residue, then add palmitate and stearate in proportion (poultry fat: palmitate: stearate = 82: 11: 7) to prepare lipid, ready for use;

[0098] (2) Emulsification homogenization: take 60% of the lipid described in (1), 40% of the crude peptide extracted in Example 1, and 8% of polyglycerol ricinoleate, and perform high-speed shearing at 30000 rpm to disperse the emulsion into nanoparticles to form a water-in-oil (W / O) emulsion system, ready for use;

[0099] (3) Preparation of adjuvants: mix starch, malt dextrin, microcrystalline cellulose, and lactose, the above adjuvants include the following components in weight parts: starch 0.8 parts, malt dextrin 3 parts, microcrystalline cellulose 1 part, lactose 1.2 parts;

[0100] (4) Take the emulsion in (2) and the adjuvants in (3), mix them in a weight ratio of 80:20, stir evenly, and place them in a pharmaceutical dosage form preparation device or mold to prepare granular or powdered tonics, tablets, etc. (take tablets as an example, the method for preparing granular tonics is different only in the size of the screen mesh selected).

[0101] The above-described examples only describe the preferred modes of the present application and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. Use of a polypeptide in the preparation of an anti-inflammatory medicament, characterized in that, The sequence of the polypeptide is: He-Val-Tyr-Pro-Trp-Thr-Gln-Arg. The sequence of the polypeptide is: He-Val-Tyr-Pro-Trp-Thr-Gln-Arg. The sequence of the polype

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