Polypeptide with liver protection function

Abstract

The invention relates to the technical field of bioactive polypeptides and liver health regulation, in particular to a polypeptide with a liver protection function. The polypeptide achieves the liver protection effect by inhibiting inflammatory response and protecting liver cell activity, the effect of the polypeptide does not depend on a single induction factor, and the polypeptide can play a stable and remarkable protection role in different types of liver injury models, which indicates that the polypeptide has broad-spectrum liver protection potential. Compared with the existing micromolecular anti-inflammatory or anti-oxidation liver protection preparation, the polypeptide disclosed by the invention is safe in source, low in toxic and side effects, clear in activity, suitable for being developed into functional foods, medicines or health-care preparations for preventing and treating liver injury, hepatitis and inflammation-related liver diseases, and good in application prospect and industrialization value.

Description

Technical Field

[0001] This invention relates to the field of bioactive polypeptides and liver health regulation technology, and in particular to a polypeptide with liver-protective function. Background Technology

[0002] The liver is the largest internal organ in the human body, undertaking a variety of functions including synthesis, decomposition, metabolism, biotransformation, and endocrine / exocrine processes. Its basic functions include synthesis, excretion, storage, metabolism, and biotransformation, and its function is closely related to many physiological activities. However, while protecting the body, the liver is also susceptible to various pathogenic factors and stimuli from both inside and outside the body, leading to inflammatory responses and damage. There are many causes of liver damage, including chemical, metabolic, drug-induced, alcoholic, and environmental factors. Long-term liver damage can induce diseases such as hepatitis, cirrhosis, and liver cancer, ultimately leading to liver failure. Liver diseases are common in clinical practice and have always been a key focus of medical and pharmaceutical research. Acute liver injury is the beginning of the development and progression of many liver diseases towards liver failure, and its prevention and treatment have always been a key research focus for scientists. It is well known that animal models are indispensable for research on liver diseases and the development of effective drugs for treating various liver diseases. The establishment of animal models of liver injury is of significant practical importance for studying the pathogenesis of liver injury, treating liver injury, and screening drugs. Developing anti-inflammatory drugs that target novel anti-inflammatory mechanisms and disease-specific targets in the liver is an urgent need, especially the discovery of anti-inflammatory natural products.

[0003] Walnuts, belonging to the genus Juglans of the family Juglandaceae, are one of the world's four major dried fruits, rich in various nutrients and pharmacological components, and possess high nutritional and commercial value. my country has become the world's largest walnut-growing country, with a planting area reaching 100 million mu (approximately 6.67 million hectares), mainly distributed in Yunnan, Xinjiang, Hebei, and Shaanxi provinces. Walnut oil cake, a byproduct of walnut oil extraction, is rich in protein (up to 50%); however, its added value is extremely low, and it is often used as animal feed or discarded. In recent years, with in-depth research on the bioactivity of walnut protein peptides, it has been found that walnut protein peptides possess various activities such as antioxidant, antitumor, blood pressure lowering, brain-boosting, and immune-regulating effects. The physiological activity of walnut protein peptides is related to the molecular weight and amino acid sequence composition of the peptides, and the isolation and purification of bioactive peptides from walnuts has become a current research hotspot. Summary of the Invention

[0004] This invention covers the following technical solutions: One aspect of the present invention relates to a polypeptide having the amino acid sequence shown in SEQ ID NO: 1.

[0005] Another aspect of the present invention relates to a nucleic acid capable of expressing the polypeptide as described above.

[0006] Another aspect of the present invention relates to a carrier containing the nucleic acid described above.

[0007] Another aspect of the present invention relates to a host cell containing the nucleic acid as described above or the vector as described above.

[0008] Another aspect of the present invention relates to a pharmaceutical composition comprising the polypeptide as described above and a pharmaceutically acceptable excipient.

[0009] Another aspect of the present invention relates to a health food product comprising the polypeptides described above.

[0010] Another aspect of the present invention relates to the use of the polypeptides described above in the preparation of hepatoprotective drugs.

[0011] The polypeptides of this invention achieve hepatoprotective effects through "inhibiting inflammatory responses and protecting hepatocyte activity." Their effects are independent of a single inducing factor, and they exhibit stable and significant protective effects in various types of liver injury models, indicating their broad-spectrum hepatoprotective potential. Compared with existing small-molecule anti-inflammatory or antioxidant hepatoprotective agents, the polypeptides of this invention are safer in origin, have lower toxicity and side effects, and exhibit well-defined activity. They are suitable for development into functional foods, drugs, or health supplements for the prevention and treatment of liver injury, hepatitis, and inflammation-related liver diseases, demonstrating promising application prospects and industrialization value. Attached Figure Description

[0012] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0013] Figure 1 APWRGPLF separation and identification LC-MS / MS spectra.

[0014] Figure 2 APWRGPLF structure diagram.

[0015] Figure 3 HPLC chromatogram for the determination of the purity of APWRGPLF synthetic material.

[0016] Figure 4 LC-MS spectrum of APWRGPLF molecular weight detection.

[0017] Figure 5 The effect of APWRGPLF on hepatocyte survival.

[0018] Figure 6Effects of APWRGPLF on liver function enzyme activity levels in an acetaminophen model.

[0019] Figure 7 Effects of APWRGPLF on the levels of inflammatory factors in an acetaminophen model.

[0020] Figure 8 Effects of APWRGPLF on liver functional enzyme activity levels in a carbon tetrachloride model.

[0021] Figure 9 Effects of APWRGPLF on inflammatory factor levels in a carbon tetrachloride model.

[0022] Figure 10 Effects of APWRGPLF on liver functional enzyme activity levels in a thioacetamide model.

[0023] Figure 11 Effects of APWRGPLF on the levels of inflammatory factors in a thioacetamide model.

[0024] Figure 12 Effects of APWRGPLF on liver functional enzyme activity levels in a lipopolysaccharide model.

[0025] Figure 13 Effects of APWRGPLF on the levels of inflammatory factors in a lipopolysaccharide model. Detailed Implementation

[0026] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0027] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, immunology-related terms and laboratory procedures used herein are all widely used terms and routine procedures in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0028] The terms “containing,” “comprising,” and “including” as used in this invention are synonyms and are inclusive or open-ended, and do not exclude additional, uncited members, elements, or method steps.

[0029] In this invention, the numerical range represented by endpoints includes all numerical values ​​and fractions contained within that range, as well as the endpoints mentioned.

[0030] As used in this invention, the term "about" or "approximately" means within 20%, preferably within 10%, and more preferably within 5%, of a given value or range. It also includes specific numbers, such as about 20 including 20.

[0031] Furthermore, in describing representative embodiments of the invention, this specification may present the methods and / or processes of the invention as a specific sequence of steps. However, the method or process should not be limited to the specific order of the steps described herein, to the extent that the method or process does not depend on the specific order of the steps presented herein. As will be understood by those skilled in the art, other sequences of steps are also possible. Therefore, the specific order of steps presented in the specification should not be construed as a limitation of the claims. Additionally, the claims relating to the methods and / or processes of the invention should not be limited to the execution of their steps in the order they are written, and those skilled in the art will readily recognize that the sequence can be changed while still remaining within the spirit and scope of the invention.

[0032] The concentration values ​​involved in this invention include fluctuations within a certain range. For example, they can fluctuate within a corresponding precision range.

[0033] As used in this invention, unless otherwise stated, the singular forms of the articles “a,” “an,” and “the” include plural referents.

[0034] In this invention, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity of 2 or more.

[0035] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0036] In this invention, terms such as "preferred," "better," "more suitable," and "ideal" merely describe implementation methods or embodiments with better effects and should be understood not to limit the scope of protection of this invention. In this invention, terms such as "optionally," "optionally," and "optional" mean that something is optional, that is, selected from either "with" or "without" a parallel solution. If multiple "optional" statements appear in a technical solution, unless otherwise specified and without contradiction or mutual constraint, each "optional" statement is independent.

[0037] In this invention, the terms "treatment," "therapeutic," or "method of treatment" refer to the administration of an active ingredient to produce any beneficial physiological or pathological effect, including but not limited to: preventing the occurrence of a disease or pathological state; reducing, alleviating, or inhibiting the development of an existing disease or the worsening of its symptoms; and improving or eliminating an existing disease, lesion, or its symptoms. Therefore, the "treatment" described in this invention includes both prophylactic and therapeutic administration, which can be used in subjects who have not yet developed the disease to reduce the risk of disease, or in subjects who have developed the disease to alleviate, inhibit, or reverse the disease progression.

[0038] In this invention, the term "polypeptide" or "peptide" refers to a linear or cyclic molecule formed by two or more amino acid residues linked by peptide bonds (amide bonds). The polypeptide can be a peptide composed of natural amino acid residues, or it can contain non-natural amino acids, D-type amino acids, modified amino acids, or derivatives thereof. The polypeptide can be a product obtained from a natural source, through chemical synthesis, recombinant expression, or in vitro translation system. The polypeptide may further include its pharmaceutically acceptable salts, isotope labels, protecting derivatives, prodrug forms, fragments, analogs, or modifications, provided that they maintain a substantially identical structural backbone to the original polypeptide or have similar biological activity. In some embodiments, the polypeptide may be an oligopeptide (typically containing 2–20 amino acid residues) or a high molecular weight polypeptide.

[0039] In this invention, the term "nucleic acid" refers to a polymer molecule formed by nucleotide units linked by phosphodiester bonds, including both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The nucleic acid can be single-stranded or double-stranded, linear or circular, and can be a naturally occurring sequence or a sequence that has been artificially modified, synthesized, mutated, or optimized; its nucleotide units may contain natural or non-natural bases, modified nucleotides, isotopic labels, or protecting groups. The nucleic acid described in this invention can be obtained through chemical synthesis, in vitro transcription, recombinant expression, or other biotechnological means, and includes pharmaceutically acceptable salts, complementary strands, antisense sequences, or mRNA forms encoding a target polypeptide (e.g., SEQ ID NO: 1).

[0040] In this invention, the term "liver protection" refers to the effects of reducing the degree of hepatocyte damage, maintaining or improving hepatocyte survival, alleviating inflammatory responses, and maintaining liver function homeostasis. This effect can manifest as reducing the upward trend of serum enzyme activity indicators related to hepatocyte damage (such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH), inhibiting the expression of cytokines such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β), and improving hepatocyte morphology and metabolic homeostasis, thereby reducing hepatocyte damage caused by exogenous or endogenous stimuli. Those skilled in the art will understand that liver protection does not necessarily require reversing existing structural tissue damage, but rather includes preventative, alleviating, and physiologically regulating effects on hepatocyte damage.

[0041] This invention relates to polypeptides, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0042] This invention provides a polypeptide with hepatoprotective activity. The amino acid sequence of the polypeptide, as shown in SEQ ID NO:1, is Ala-Pro-Trp-Arg-Gly-Pro-Leu-Phe (APWRGPLF), belonging to a low molecular weight octapeptide derived from walnut protein. The polypeptide of this invention can be obtained by protease hydrolysis and gel chromatography purification, or it can be prepared using solid-phase chemical synthesis or a recombinant expression system. The polypeptide has a well-defined structure, controllable purity, and good stability, making it suitable for further preparation of pharmaceutical compositions or health foods.

[0043] The polypeptide may further include its pharmaceutically acceptable salts, derivatives, isotope labels, protecting derivatives, prodrug forms, or analogs having the same amino acid backbone structure, provided that they maintain the same or similar biological activity as the polypeptide defined in SEQ ID NO: 1.

[0044] Experimental results showed that the peptide exhibited significant hepatoprotective and anti-inflammatory capabilities in various chemical and inflammatory liver injury models. The study also revealed that the peptide was non-toxic to hepatocellular cells within a concentration range of 0–300 μg / mL and promoted hepatocellular survival and proliferation, indicating its good biocompatibility and cell repair potential.

[0045] In hepatocyte injury models induced by acetaminophen (APAP), carbon tetrachloride (CCl4), thioacetamide (TAA), and lipopolysaccharide (LPS), compared with the injury model group, the levels of liver function injury marker enzymes ALT, AST, and LDH in the peptide treatment group of this invention were significantly reduced (p<0.01), indicating that the peptide can effectively alleviate hepatocyte membrane rupture and cell necrosis. Simultaneously, the peptide can significantly inhibit the expression of inflammation-related cytokines TNF-α, IL-6, and IL-1β (p<0.01), thereby blocking the cascade amplification of inflammatory factors and alleviating inflammatory damage to liver tissue.

[0046] According to one aspect of the invention, a vector containing nucleic acids as described above is also involved.

[0047] To improve expression efficiency in different hosts, the nucleic acid sequence of the present invention can be codon optimized according to the codon usage preferences of the target host (such as Escherichia coli, yeast, insect cells or mammalian cells). The optimized nucleic acid is still within the protection scope of the present invention without changing the encoded amino acid sequence.

[0048] The nucleic acid can be a single-stranded or double-stranded structure, a linear or circular molecule, and may include its complementary strand, antisense sequence, splice variant, fragment, fusion sequence, leader sequence, tag sequence (such as His tag, FLAG tag), or functional elements such as promoter, enhancer, terminator, etc. that can regulate its expression.

[0049] Furthermore, the nucleic acids of this invention also include their chemically modified or non-natural nucleotide forms, such as those containing modified bases (e.g., methylcytosine, pseudouridine), modified ribose (e.g., 2′-O-methylribose), phosphorylation-thioyl bonds, locked nucleic acid (LNA) units, isotopically labeled nucleotides, or other structural modifications to improve stability and translation efficiency. These modified or optimized nucleic acids, as long as they can express the polypeptide defined in SEQ ID NO: 1, are all within the scope of protection of this invention.

[0050] The term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). In some embodiments, the vector of the present invention contains regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES), and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly-U sequences).

[0051] The present invention also relates to host cells containing the nucleic acids described above or the vectors described above.

[0052] The present invention also relates to pharmaceutical compositions comprising the polypeptides described above and pharmaceutically acceptable excipients.

[0053] Specific examples of substances that can be used as pharmaceutically acceptable excipients include phosphoric acid, citric acid, and other organic acids; antioxidants (e.g., ascorbic acid and methionine); antibacterial agents (e.g., octadecyl dimethylbenzene ammonium chloride, hexachlorocyclohexane quaternary ammonium chloride, benzalkonium chloride, phenol, butanol or benzyl alcohol, alkylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, or m-cresol); and low molecular weight substances (less than about 10). (kDa) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids (e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine); monosaccharides, disaccharides, and other carbohydrates (including, for example, glucose, mannose, or dextran); chelating agents (e.g., EDTA); sugars (e.g., sucrose, mannitol, trehalose, or sorbitol); salt-forming counterions; metal complexes; and / or nonionic surfactants (e.g., including TWEENTM, PLURONICS™, or polyethylene glycol). Furthermore, depending on the formulation method, commonly used fillers, diluents, binders, humectants, disintegrants, and / or surfactants can be appropriately selected by those skilled in the art. The pharmaceutical composition may be in solid, semi-solid, or liquid form, preferably in liquid form.

[0054] In some embodiments, the pharmaceutical composition further comprises a peptide stabilizer or a nucleic acid stabilizer.

[0055] In some embodiments, the pharmaceutical composition is an oral formulation, a rectal formulation, or an enteric-coated formulation for targeted release into the colon.

[0056] In some embodiments, the oral formulation is selected from tablets, capsules, granules, oral solutions, or oral emulsions. Appropriate amounts of diluents, disintegrants, lubricants, binders, solubilizers, or preservatives may be added as needed. For applications requiring action in the colon, enteric coating or pH-responsive polymer encapsulation techniques can be used to ensure drug stability in the stomach and small intestine, releasing the peptide only in the colonic environment, thereby achieving targeted delivery and high local efficacy.

[0057] In another embodiment, the pharmaceutical composition may further comprise a delivery carrier to improve the stability and bioavailability of the peptide. Suitable delivery carriers include, but are not limited to, liposomes, solid lipid nanoparticles, polylactic-co-glycolic acid (PLGA) nanoparticles, or chitosan nanogels. These carriers can bind to the peptide through encapsulation, adsorption, or covalent linkage to form a sustained-release or targeted delivery system, thereby avoiding enzymatic degradation of the peptide in the gastrointestinal tract and increasing the effective concentration and duration of action in the intestinal tract. The pharmaceutical composition may also be formulated as a solution, suspension, emulsion, or lyophilized powder as needed, and can be administered orally or rectally to achieve hepatoprotective effects.

[0058] The present invention also provides a health food product containing the polypeptides described above.

[0059] The liver-protecting function of this health food can be summarized as at least one of the following functions: helping to reduce the damage to the liver caused by adverse stimuli, helping to maintain a normal liver health status, helping to enhance the liver's ability to resist harmful factors, or having an auxiliary protective effect against chemical liver damage.

[0060] The polypeptide is a small-molecule walnut protein peptide with a well-known source and good safety profile. It is easily digested and absorbed, and has high oral stability, making it suitable for the development of health foods with the primary function of improving liver health and enhancing the liver cell's resistance to damage. The health food can be prepared by mixing the polypeptide with an edible carrier or excipients using conventional food processing techniques.

[0061] The aforementioned health food products can be prepared in various dosage forms according to different consumer needs and consumption methods, including but not limited to capsules, tablets, granules, powders, beverages, oral liquids, nutrition bars, or solid beverages. During preparation, pharmaceutically or food-industrially acceptable carriers, excipients, sweeteners, flavorings, emulsifiers, antioxidants, or preservatives can be added as needed to improve taste and stability.

[0062] In a preferred embodiment, the health food can be prepared using encapsulation, spray drying, or microencapsulation techniques to improve the stability of the peptides during storage and digestion, and to achieve targeted release in the intestine.

[0063] The health food product of this invention exhibits physiological regulatory effects in improving liver function indicators and reducing inflammation levels in hepatocyte injury models induced by acetaminophen, carbon tetrachloride, thioacetamide, and lipopolysaccharide. It can reduce liver injury markers such as ALT, AST, and LDH, and inhibit the expression of inflammatory factors such as TNF-α, IL-6, and IL-1β, thereby maintaining the healthy state of hepatocytes, improving the liver's resistance to injury, and promoting the balanced regulation of liver function. This health food product is naturally sourced, highly safe, suitable for long-term conditioning, and has good market prospects.

[0064] The present invention also relates to the use of the polypeptides described above in the preparation of hepatoprotective drugs.

[0065] This peptide exhibits significant hepatoprotective and anti-inflammatory effects in various liver injury models by reducing the release of ALT, AST, and LDH and inhibiting the expression of inflammatory factors such as TNF-α, IL-6, and IL-1β, thereby achieving a broad-spectrum hepatoprotective effect.

[0066] The present invention also relates to a method for treating liver injury by administering a safe and effective amount of the pharmaceutical composition or peptide described above to a subject with liver injury to alleviate hepatocellular damage and / or inflammatory response.

[0067] The phrase "safe and effective amount" means, as used herein, a compound or composition in a reasonable pharmaceutically modifiable amount sufficient to significantly and effectively relieve the symptoms or condition being treated, but small enough to avoid serious side effects (with a reasonable benefit / risk ratio). The safe and effective amount of the active ingredient in the pharmaceutical composition used in the methods of this invention varies depending on the specific symptoms being treated, the age and physical condition of the treated subject, the severity of the disease, the duration of treatment, concurrent treatments, the specific active ingredient used, the specific pharmaceutically acceptable excipients used, and factors including the knowledge and skills of the physicians involved in the treatment.

[0068] The pharmaceutical compositions of the present invention can be administered via any route, as will be known to those skilled in the art. In some embodiments, the delivery system / pharmaceutical compositions of the present invention are administered orally (PO), intravenously (IV), intramuscularly (IM), intra-arterially, intramedullaryly, intrathecally, subcutaneously (SQ), intravenously, percutaneously, intradermally, intradermally, transrectally (PR), transvaginally, intraperitoneally (IP), intragastrically (IG), topically (e.g., using powders, ointments, creams, gels, lotions and / or drops), mucous membranes, intranasally, intrabuccally, transintestinally, vitreously, sublingually; via tracheal instillation, bronchial instillation and / or inhalation; as an oral spray, nasal spray and / or aerosol and / or via a portal vein catheter.

[0069] The term "subject" as used in this invention can refer to a patient or other animal receiving the drug described in this invention to treat, prevent, alleviate, and / or relieve the disease, condition, or symptom described in this invention. Subjects include warm-blooded animals, such as mammals like pandas, elephants, primates (chimpanzees, orangutans, gibbons, macaques, marmosets), and preferably humans. Non-human primates are also considered individuals. The term "individual" includes domesticated animals such as cats and dogs, livestock (e.g., cattle, horses, pigs, sheep, goats), and laboratory animals (e.g., mice, rabbits, rats, gerbils, guinea pigs).

[0070] The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this invention, or follow experimental manuals or conventional conditions in the art, or other experimental methods known in the art, or follow the conditions recommended by the manufacturer.

[0071] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0072] Example 1: Walnut polypeptide information 1. The isolated walnut polypeptides were detected by liquid chromatography-tandem mass spectrometry (LC-MS / MS). Figure 1 A walnut octapeptide was obtained with the amino acid sequence Ala-Pro-Trp-Arg-Gly-Pro-Leu-Phe, Chinese name: alanine-proline-tryptophan-arginine-glycine-proline-leucine-phenylalanine, abbreviation: APWRGPLF.

[0073] PepDraw is used to predict the APWRGPLF structure ( Figure 2The net charge is 1, and the hydrophobicity is 6.59 kcal / mol. Using the Peptide Ranker database, the APWRGPLF score is 0.975484; ToxinPred predicts that APWRGPLF is non-toxic.

[0074] The walnut APWRGPLF peptide was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the purity of the walnut APWRGPLF peptide was determined by high performance liquid chromatography (HPLC). Figure 3 The accuracy rate was 99.499%, which meets the requirements for subsequent functional experiments.

[0075] The molecular weight of peptides was determined by liquid chromatography-mass spectrometry (LC-MS). Figure 4 The molecular weight of the walnut APWRGPLF polypeptide is 943.30 Da.

[0076] Example 2: Preparation of walnut polypeptides 1. Preparation of walnut protein powder: Walnut kernels were pressed for oil (20±5℃, 45 MPa), and the walnut meal was pulverized and sieved (120 mesh) to obtain defatted walnut meal powder (protein content 46.48%, oil content 12.73%). The defatted walnut meal powder was then subjected to alkali dissolution (pH 10.0, 30 min), acid precipitation (pH 4.8, 30 min), water washing (twice), horizontal screw centrifugation, and spray drying to obtain walnut protein powder (protein content 88.37%).

[0077] 2. Preparation of walnut enzymatic hydrolysate: Take 10 g of walnut protein powder, add 50 mL of water at a material-to-liquid ratio of 1:5, add neutral protease for enzymatic hydrolysis (temperature 50℃, time 3 h, enzyme dosage 1.5%), and then inactivate the enzyme in a water bath (temperature 95-100℃, 10 min). After centrifugation (5000 rpm, 20 min), collect the supernatant to obtain the walnut enzymatic hydrolysate.

[0078] 3. Walnut peptide isolation and purification: The walnut enzymatic hydrolysate was ultrafiltered to obtain a walnut peptide solution with a molecular weight <1000 Da. The walnut peptide solution was separated and purified using a Sephadex G-15 gel column, and the purified walnut peptide was obtained after freeze-drying. The purified walnut peptide was analyzed by LC-MS / MS to obtain a walnut octapeptide Ala-Pro-Trp-Arg-Gly-Pro-Leu-Phe (APWRGPLF).

[0079] Example 3: Application of walnut APWRGPLF polypeptide in the preparation of hepatoprotective drugs Artificial synthesis of walnut APWRGPLF polypeptide.

[0080] Hepatocyte culture: SD rat hepatocytes were seeded in DMEM culture medium containing 10% fetal bovine serum and cultured in a 37°C, 5% CO2 incubator.

[0081] The effects of walnut APWRGPLF peptide on hepatocyte viability and safety were investigated. Hepatocytes were seeded in 96-well plates and cultured for 48 h with APWRGPLF at concentrations of 0, 25, 50, 100, 150, 200, and 300 μg / mL, respectively. Hepatocyte viability was then assessed. The walnut APWRGPLF peptide showed no significant effect on hepatocyte viability within the concentration range of 0–300 μg / mL. Figure 5 It has the effect of promoting hepatocyte growth and is non-toxic to hepatocytes.

[0082] Four hepatocyte injury models were established by inducing hepatocytes with four compounds: acetaminophen, carbon tetrachloride, thioacetamide, and lipopolysaccharide. The hepatoprotective efficacy of walnut APWRGPLF peptide was then investigated. In the hepatoprotective experiment, the walnut APWRGPLF peptide was divided into four treatment groups, seeded into 24-well plates, with six replicates per well. The four treatment groups are as follows: Normal group: Normal culture for 2 h + 12~48 h; Model groups: normal culture for 2 h + acetaminophen (2 μg / mL) for 12 h; normal culture for 2 h + carbon tetrachloride (8 mmol / L) for 12 h; normal culture for 2 h + thioacetamide (0.18 mol / L) for 24 h; normal culture for 2 h + lipopolysaccharide (10 μg / mL) for 12 h, establishing four hepatocyte injury models.

[0083] Drug group 1: Added walnut APWRGPLF peptide for 2 h (100 μg / mL) + 4 types of liver injury models for 12~48 h.

[0084] Drug group 2: Walnut APWRGPLF peptide added for 2 h (200 μg / mL) + 4 types of liver injury models for 12~48 h.

[0085] After culture, the supernatant was collected by centrifugation at 3000 r / min for 15 min. The activity of liver functional enzymes (ALT, AST, LDH) and the content of inflammatory factors (TNF-α, IL-6, IL-1β) in the culture medium of each treatment group were measured using a kit.

[0086] (1) Anti-inflammatory effect of walnut APWRGPLF peptide in acetaminophen-induced liver injury model Walnut APWRGPLF peptide significantly increased acetaminophen-induced hepatocyte enzyme activities (ALT, AST, LDH) (p<0.01). Figure 6It significantly inhibited the expression levels of acetaminophen-induced hepatocellular inflammatory factors (TNF-α, IL-6, IL-1β) (p<0.01). Figure 7 The results indicate that the walnut APWRGPLF polypeptide has the effect of improving acetaminophen-induced hepatocellular damage and has anti-inflammatory activity.

[0087] (2) Anti-inflammatory effect of walnut APWRGPLF peptide in carbon tetrachloride-induced liver injury model Walnut APWRGPLF peptide significantly increased carbon tetrachloride-induced hepatocyte enzyme activities (ALT, AST, LDH) (p<0.01). Figure 8 It significantly inhibited the expression levels of hepatocyte inflammatory factors (TNF-α, IL-6, IL-1β) induced by carbon tetrachloride (p<0.01). Figure 9 The results indicate that the walnut APWRGPLF polypeptide has the effect of improving carbon tetrachloride-induced hepatocyte damage and has anti-inflammatory activity.

[0088] (3) Anti-inflammatory effect of walnut APWRGPLF peptide in thioacetamide-induced liver injury model Walnut APWRGPLF peptide significantly increased the activity of hepatocyte enzymes (ALT, AST, LDH) induced by thioacetamide (p<0.01). Figure 10 It significantly inhibited the expression levels of thioacetamide-induced hepatocellular inflammatory factors (TNF-α, IL-6, IL-1β) (p<0.01). Figure 11 The results indicate that the walnut APWRGPLF polypeptide has the effect of improving thioacetamide-induced hepatocyte damage and has anti-inflammatory activity.

[0089] (4) Anti-inflammatory effect of walnut APWRGPLF peptide in lipopolysaccharide-induced liver injury model Walnut APWRGPLF polypeptide significantly increased lipopolysaccharide-induced hepatocyte enzyme activities (ALT, AST, LDH) (p<0.01). Figure 12 It significantly inhibited the expression levels of lipopolysaccharide-induced hepatocyte inflammatory factors (TNF-α, IL-6, IL-1β) (p<0.01). Figure 13 The results indicate that the walnut APWRGPLF polypeptide has the effect of improving lipopolysaccharide-induced hepatocyte damage and has anti-inflammatory activity.

[0090] The above results indicate that the walnut APWRGPLF polypeptide can improve the activity of three hepatocyte enzymes (ALT, AST, LDH) and inhibit the expression of three inflammatory factors (TNF-α, IL-6, IL-1β) in four chemically induced hepatocyte injury models, thereby improving hepatocyte injury and protecting the liver.

[0091] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A polypeptide, characterized in that, The amino acid sequence is shown in SEQ ID NO:

1.

2. Nucleic acid, characterized in that, It can express the polypeptide described in claim 1.

3. A carrier, characterized in that, It contains the nucleic acid as described in claim 2.

4. A host cell, characterized in that, It contains the nucleic acid as described in claim 2 or the vector as described in claim 3.

5. A pharmaceutical composition, characterized in that, It comprises the polypeptide of claim 1 and a pharmaceutically acceptable excipient.

6. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition is an oral formulation, a rectal formulation, or an enteric-coated formulation for targeted release into the colon.

7. The pharmaceutical composition according to claim 6, characterized in that, The oral preparation is selected from tablets, capsules, granules, oral solutions, or oral emulsions.

8. The pharmaceutical composition according to any one of claims 5-7, characterized in that, The pharmaceutical composition further comprises a delivery carrier selected from liposomes, solid lipid nanoparticles, PLGA nanoparticles, or chitosan nanogels.

9. Health food products, characterized in that, It contains the polypeptide as described in claim 1.

10. The use of the polypeptide according to claim 1 in the preparation of hepatoprotective drugs.

Images