CN106699842B

CN106699842B - Novel anti-inflammatory small molecule polypeptide and application thereof

Info

Publication number: CN106699842B
Application number: CN201510791580.5A
Authority: CN
Inventors: 史海明; 马烨; 刘洁
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2015-11-17
Filing date: 2015-11-17
Publication date: 2020-06-12
Anticipated expiration: 2035-11-17
Also published as: CN106699842A

Abstract

The invention relates to a small molecule polypeptide for preventing and treating inflammation. The invention also relates to a preparation method and application of the small molecule polypeptide and a composition containing the polypeptide. The polypeptides of the invention have a number of advantages, such as low molecular weight, permeability through various tissue barriers; good water solubility, and can maintain higher concentration in body fluid. The active polypeptide provided by the invention provides a theoretical basis for revealing the relationship between the sequence structure and the activity of the whey protein anti-inflammatory active peptide, provides a powerful scientific basis for the research, development and utilization of the food-borne anti-inflammatory active peptide, and can be widely applied to various fields.

Description

Novel anti-inflammatory small molecule polypeptide and application thereof

Technical Field

The invention relates to the field of biomedicine, in particular to a novel whey protein-derived small-molecule polypeptide, a composition containing the same and application of the novel whey protein-derived small-molecule polypeptide in medicines, foods and cosmetics.

Background

As a large country in dairy industry, China has abundant dairy resources and urgent needs for research on development of related dairy products. Whey is used as a main byproduct of industrial cheese production, and how to utilize whey is one of the important concerns of food industry in China. Whey protein is the most nutritional product in whey, so the research on the nutritional value of whey protein is very important. Whey protein is a high-quality protein existing in milk whey, and comprises a plurality of bioactive sequences, and the active sequences can be released from the whey protein through hydrolysis of proper protease to form peptide fragments with physiological activity. The research of biologists finds that: the protein shows stronger biological activity in the form of polypeptide. After the protein is hydrolyzed by digestive tract enzyme, the protein is absorbed mainly in the form of small peptide, which is easier and faster to be absorbed and utilized by organism than complete free amino acid, and only trace amount of peptide is needed to exert strong biological activity. Therefore, the active small peptides possibly existing in the whey protein become the research focus of the invention.

With the development of proteomics and the maturation of separation and extraction technologies, more and more new whey protein bioactive peptides are discovered in recent years, milk-derived proteins become the most extensive protein for researching the properties and functions of bioactive peptides, and the development and utilization value of whey proteins is higher and higher. Food taking milk-derived bioactive peptides as functional factors has been developed and sold in the market in foreign countries, but the mechanism of action of bioactive peptides to exert their bioactivity is difficult to be studied, and whether whey protein bioactive peptides can be used as functional factors in the production of food industry is still to be studied further.

Although many scientists in China carry out related research and development on bioactive peptides in laboratories, a series of achievements are obtained. However, due to the shortage of the production technology of separating whey protein from milk in China, a large amount of whey-related products such as concentrated whey protein powder and the like must be imported in China every year, which greatly limits the development of whey protein products in China and makes it more difficult to realize the large-scale production of bioactive peptides of whey protein. However, with the continuous development and maturation of technologies for separating whey protein from whey such as membrane separation, adsorption separation and the like, the development and utilization of whey resources in China must become a new economic growth point in the dairy market, the research and development of bioactive peptides of whey protein must be concerned more and more, and the bioactive peptides of whey protein in the future must be applied and developed more widely in the fields of food and medicine.

At present, much research attention on whey protein active peptides is mainly focused on four types of antihypertensive peptides, antioxidant peptides, antibacterial peptides and immunoregulatory peptides, relatively few research on the anti-inflammatory activity of whey peptides is focused, few types of the discovered anti-inflammatory active peptides are found, the research is mostly focused on optimization of an early enzymolysis process and activity evaluation of enzymolysis crude products, deep exploration is needed, and anti-inflammatory drugs have certain side effects, so that the field needs to actively research on the safe, natural and efficient anti-inflammatory active peptides derived from food.

Disclosure of Invention

The invention aims to provide an anti-inflammatory peptide and a preparation method thereof, which can effectively obtain the anti-inflammatory peptide, provide theoretical basis for the related research, development and utilization of food-borne anti-inflammatory peptide in the future, and enable the anti-inflammatory peptide segments obtained by screening to be more widely applied in food, medicines and other fields.

In a first aspect of the invention, there is provided an isolated polypeptide having the structure of formula I:

A-B-C are of formula I;

wherein, A and C are respectively none, or 1-2 amino acid residues;

b is a core sequence shown by DQWL (SEQ ID No.: 1);

"-" is a peptide bond;

the polypeptide has the activity of preventing or treating inflammatory diseases.

In another preferred embodiment, in said polypeptide, both a and C are absent.

In another preferred embodiment, A is absent or selected from L, I, KL, KI, RL or RI, and/or C is absent or selected from C, S, CE, CD, SE or SD.

In another preferred embodiment, in said polypeptide a is absent, or KL, and/or C is absent or CE.

In a second aspect of the invention, there is provided a collection of polypeptides comprising an isolated polypeptide according to the first aspect of the invention, and one or more short peptides selected from the group consisting of:

DYKKY (SEQ ID NO.:2), LF, AVF, WLA, WYSL (SEQ ID NO.:3), GTWY (SEQ ID NO.:4) and EYGLF (SEQ ID NO.: 5).

In another preferred embodiment, the collection of polypeptides comprises the isolated polypeptides of the first aspect of the invention and the short peptides of SEQ ID No. 2.

In another preferred embodiment, said isolated polypeptide or said collection of polypeptides further comprises a pharmaceutically acceptable salt thereof.

In a third aspect of the invention there is provided an isolated polynucleotide encoding an isolated polypeptide according to the first aspect of the invention or encoding a collection of polypeptides according to claim 2.

In a fourth aspect of the invention, there is provided the use of an isolated polypeptide according to the first aspect of the invention or a collection of polypeptides according to the second aspect of the invention for the preparation of a composition for the prevention and/or treatment of an inflammatory disease, and/or for the inhibition of an inflammatory factor.

In another preferred embodiment, the inflammatory disease comprises an infectious and/or non-infectious inflammatory disease.

In another preferred embodiment, the infectious inflammatory disease comprises an inflammatory disease caused by bacteria and/or fungi.

In another preferred embodiment, the non-infectious inflammatory disease includes red swelling, pain, allergic rhinitis, contact dermatitis, dermatitis medicamentosa, arthritis, eczema, fibrositis, tenosynovitis, ankylosing spondylitis, etc. caused by surgery or trauma, and cardiovascular and cerebrovascular diseases such as cancer, asthma, diabetes, senile dementia, autoimmune disease, osteoporosis, or senilism, which may be induced by some chronic diseases.

In another preferred embodiment, the inflammatory disease comprises ocular inflammatory disease, pancreatitis, inflammatory bowel disease, lung inflammation, skin inflammation, rheumatoid arthritis, ankylosing spondylitis, infectious endocarditis, chronic bronchitis, appendicitis, or rheumatism.

In another preferred embodiment, the inflammatory factor comprises COX-2, TNF- α, IL-1 β, IL-6, IL-8, MCP-1, or TGF- β.

In a sixth aspect of the invention, there is provided a composition comprising as an active ingredient an isolated polypeptide according to the first aspect of the invention, and/or a combination of polypeptides according to the second aspect of the invention, and a pharmaceutically and/or dietetically and/or cosmetically acceptable carrier.

In another preferred embodiment, the composition comprises a pharmaceutical composition, a food composition and/or a cosmetic composition.

In another preferred embodiment, the concentration of the active ingredient is 1-1000. mu.g/ml, preferably 5-500. mu.g/ml, more preferably 10-200. mu.g/ml.

In another preferred embodiment, the dosage form of the composition comprises an oral dosage form, or a parenteral dosage form, such as a topical or topical dosage form.

In another preferred embodiment, the composition is in the form of a tablet, pill, pellet, sustained-release formulation, emulsion, suspension, granule, powder, lyophilized formulation, syrup, ointment, cream, drop, buccal, intravenous injection, suppository, spray, aerosol, lotion, gargle, patch, or eye drop.

In a seventh aspect of the invention, there is provided an in vitro non-therapeutic method of inhibiting inflammatory factors comprising the steps of: adding to a cell culture the isolated polypeptide of the first aspect of the invention, the collection of polypeptides of the second aspect of the invention, and/or the composition of the sixth aspect of the invention, thereby inhibiting an inflammatory factor in said cell.

In an eighth aspect of the present invention, there is provided a method for preventing and/or treating an inflammatory disease and/or inhibiting an inflammatory factor, comprising the steps of: administering to a subject in need thereof a safe and effective amount of an isolated polypeptide according to the first aspect of the invention, a combination of polypeptides according to the second aspect of the invention and/or a composition according to the sixth aspect of the invention.

In another preferred embodiment, the subject is a mammal, including a mouse, rat, or human.

In another preferred embodiment, the subject is a subject suffering from an inflammatory disease, and/or a subject with increased inflammatory factors in vivo.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 is a Sephadex G-25 gel column chromatography chromatogram in which, among 5 collected fractions having anti-inflammatory activity, the F4 fraction showed better anti-inflammatory activity.

Fig. 2 is a semi-preparative HPLC chromatogram in which 9 fractions collected all exhibited a certain degree of anti-inflammatory activity, of which F4e had the most significant anti-inflammatory effect.

Fig. 3 is a secondary mass spectrum of F4 obtained by screening, and it can be seen that the whey protein peptide after primary purification still contains multiple components, and further separation and activity screening experiments are required to determine the peptide fragment with the best anti-inflammatory activity.

FIG. 4 shows DQWL mass spectrum of peptide fragment with best anti-inflammatory activity in F4 e.

FIG. 5 shows the anti-inflammatory results of synthetic peptide RT-PCR.

FIG. 6 shows the synthetic peptide ELISA anti-inflammatory results.

FIG. 7 shows that the peptide DQWL can inhibit the action of inflammatory factors through NF-kappa B, p38MAPK signal pathway, and it can be seen from the figure that the peptide DQWL can significantly inhibit nuclear translocation of NF-kappa B p65 in NF-kappa B pathway, phosphorylation of IKK α/β and degradation of Ikappa B α and effectively reduce the expression of phosphorylated p38 in MAPK pathway.

Detailed Description

The inventor of the invention has conducted extensive and intensive research, and found a novel polypeptide having anti-inflammatory activity from whey protein by subjecting whey protein to enzymolysis to obtain a peptide mixture, subjecting the peptide mixture to ultrafiltration separation to obtain peptide portions having different molecular weights, subjecting the peptide portions to column chromatography and preparative chromatography to obtain a polypeptide sample having a high purity, identifying an amino acid sequence of the polypeptide by mass spectrometry, and subjecting the polypeptide to anti-inflammatory activity test. By analyzing the composition and sequence of the peptide segments of the whey protein after moderate enzymolysis, the invention evaluates the anti-inflammatory activity of different peptide segments and provides good reference for high-value development and utilization of the whey protein. The experiment proves that the whey protein is hydrolyzed by the alkaline protease, and the cytokine release generated by LPS induction in a rodent cell model can be effectively reduced by utilizing the active component obtained by screening, so that the anti-inflammatory effect is achieved. The active polypeptide provided by the invention provides a theoretical basis for revealing the relationship between the sequence structure and the activity of the whey protein anti-inflammatory active peptide, and provides a scientific reference for the research, development and utilization of the food-borne anti-inflammatory active peptide so as to exert the advantages of the anti-inflammatory active peptide to the maximum extent, and the active polypeptide has important theoretical significance and application potential. On the basis of this, the present invention has been completed.

Active polypeptide

In the present invention, the terms "polypeptide of the invention", "polypeptide of formula I", "F4 e small peptide" or "peptide F4 e" are used interchangeably and all refer to an isolated polypeptide of formula I having a DQWL polypeptide (SEQ ID No.:1) as core sequence with inflammation inhibitory activity.

A-B-C are of formula I;

wherein, A and C are respectively none, or 1-2 amino acid residues;

b is a core sequence shown by DQWL (SEQ ID No.: 1);

"-" is a peptide bond;

Wherein A is absent or selected from K, L, R, I, KL, KI, RL or RI, and/or C is absent or selected from C, E, S, D, CE, CD, SE or SD.

In addition, the term also includes a collection of polypeptides having inflammation inhibitory function comprising the polypeptide of formula I, or a variant of the polypeptide sequence of formula I. These variants include, in addition to the core sequence (but are not limited to): deletion, insertion and/or substitution of 1 to 3 (usually 1 to 2, most preferably 1) amino acids, and addition of one or several (usually up to 5, preferably up to 3, more preferably up to 2) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. For another example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the structure and function of the protein.

The invention also includes active fragments, derivatives and analogs of the F4e protein. As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that substantially retains the function or activity of inhibiting inflammation. The polypeptide fragment, derivative or analogue of the present invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which the F4e polypeptide is fused to another compound (such as a compound that increases the half-life of the polypeptide, e.g., polyethylene glycol), or (iv) a polypeptide in which an additional amino acid sequence is fused to the polypeptide sequence (fused to a leader sequence, a secretory sequence or a tag sequence such as 6 His). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein.

Analogs of the F4e protein or polypeptide are also provided, which may differ from the native F4e polypeptide by amino acid sequence differences, by modifications that do not affect the sequence, or by both, and also include analogs having residues other than the native L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., β, gamma-amino acids).

Modified (generally without altering primary structure) forms include: chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modification may be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylase. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to increase their resistance to proteolysis or to optimize solubility.

The polypeptides of the invention can also be used by pharmaceutical or physiologically acceptable acid or base derived salt form. These salts include, but are not limited to, salts formed with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, fumaric acid, maleic acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or isethionic acid. Other salts include: salts with alkali or alkaline earth metals (such as sodium, potassium, calcium or magnesium), and in the form of esters, carbamates or other conventional "prodrugs".

Polypeptide collections

The invention also provides a polypeptide set. The polypeptide set comprises, in addition to the F4e (formula I) polypeptide of the invention, one or more short peptides selected from the group consisting of:

The polypeptides contained in the polypeptide set of the invention are all anti-inflammatory active polypeptides, and also comprise pharmaceutically acceptable salt forms thereof. The polypeptide pool of the invention may also be in the form of a mixture of various polypeptides for the preparation of pharmaceutical, food and/or cosmetic compositions.

The polypeptide set of the invention can be combined or mixed after independently preparing each polypeptide, and can also be used for preparing a mixture of each polypeptide in a recombinant mode. It is understood that the preparation of a polypeptide or collection of polypeptides of the invention is well known to those skilled in the art.

Coding sequence

The invention also relates to polynucleotides encoding a polypeptide or a collection of polypeptides of the invention. Once the amino acid sequences have been obtained, the wild-type coding sequences for these polypeptides or collections of polypeptides can be obtained using conventional methods.

The polynucleotide of the present invention may be in the form of DNA or RNA. The DNA may be the coding strand or the non-coding strand. The sequence of the coding region encoding the mature polypeptide may be identical to the sequence of the coding region shown in formula I or may be a degenerate variant. As used herein, "degenerate variant" means in the present invention a nucleic acid sequence which encodes a polypeptide of the present invention but differs from the sequence of the corresponding coding region in its wild-type protein.

The full-length F4e nucleotide sequence or its fragment of the present invention can be obtained by PCR amplification, recombinant method or artificial synthesis. At present, DNA sequences encoding the polypeptides of the present invention (or fragments or derivatives thereof) have been obtained entirely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art.

The invention also relates to vectors comprising the polynucleotides of the invention, and to host cells genetically engineered with the vectors or H-RN protein coding sequences of the invention.

Enzymolysis reaction

The common tool enzymes for obtaining the bioactive peptide by enzymolysis mainly comprise trypsin, chymotrypsin, alkaline protease, pepsin, papain, compound protease and the like. The protease which is currently applied to the research of the anti-inflammatory peptide mainly comprises trypsin, chymotrypsin, pepsin, plant protease and the like. Among them, alkaline proteases have relatively few studies in this regard, and alkaline proteases have specificity for terminal hydrophobic amino acids, mainly cleaving hydrophobic amino acids such as: leu, Ile, Val, etc. The present inventors have used alkaline protease to optimize the hydrolysis of whey protein, thereby obtaining small fragments of the polypeptide of the present invention. Of course, after obtaining the polypeptide sequence of the present invention, the polypeptide of the present invention can be prepared by a more economical and convenient method.

Preparation method

The polypeptides of the invention may be recombinant polypeptides or synthetic polypeptides. The polypeptides of the invention may be chemically synthesized, or recombinant. Accordingly, the polypeptides of the present invention can be artificially synthesized by a conventional method or can be produced by a recombinant method.

A preferred method is to use liquid phase synthesis techniques or solid phase synthesis techniques, such as Boc solid phase method, Fmoc solid phase method or a combination of both. The solid phase synthesis can quickly obtain samples, and can select proper resin carriers and synthesis systems according to the sequence characteristics of target peptides. For example, the preferred solid support in the Fmoc system is Wang resin with C-terminal amino acid attached to the peptide, Wang resin is polystyrene in structure, and the arm between the Wang resin and the amino acid is 4-alkoxybenzyl alcohol; the Fmoc protecting group was removed by treatment with 25% piperidine/dimethylformamide for 20 minutes at room temperature and extended from the C-terminus to the N-terminus one by one according to the given amino acid sequence. After completion of the synthesis, the synthesized proinsulin-related peptide is cleaved from the resin with trifluoroacetic acid containing 4% p-methylphenol and the protecting groups are removed, optionally by filtration and isolated as a crude peptide by ether precipitation. After lyophilization of the resulting solution of the product, the desired peptide was purified by gel filtration and reverse phase high pressure liquid chromatography. When the solid phase synthesis is performed using the Boc system, it is preferable that the resin is a PAM resin to which a C-terminal amino acid in a peptide is attached, the PAM resin has a structure of polystyrene, and an arm between the PAM resin and the amino acid is 4-hydroxymethylphenylacetamide; in the Boc synthesis system, the protecting group Boc is removed with TFA/Dichloromethane (DCM) and neutralized with Diisopropylethylamine (DIEA)/dichloromethane in cycles of deprotection, neutralization, and coupling. After completion of the peptide chain condensation, the peptide chain was cleaved from the resin by treatment with p-cresol (5-10%) in Hydrogen Fluoride (HF) at 0 ℃ for 1 hour, while removing the protecting group. Extracting peptide with 50-80% acetic acid (containing small amount of mercaptoethanol), lyophilizing, separating and purifying with molecular sieve Sephadex G10 or Tsk-40f, and purifying with high performance liquid chromatography to obtain the desired peptide. The amino acid residues may be coupled using various coupling reagents and coupling methods known in the art of peptide chemistry, for example direct coupling using Dicyclohexylcarbodiimide (DCC), hydroxy benzotriazole (HOBt) or 1,1,3, 3-tetraurea Hexafluorophosphate (HBTU). For the synthesized short peptide, the purity and the structure can be verified by reversed-phase high performance liquid chromatography and mass spectrometry.

In a preferred embodiment, the F4e polypeptide is prepared by a solid phase synthesis method according to the sequence, and is purified by high performance liquid chromatography to obtain high-purity peptide freeze-dried powder which is stored at-20 ℃.

Another method is to produce the polypeptide of the invention by recombinant techniques. The polynucleotides of the present invention may be used to express or produce recombinant polypeptides of the present invention by conventional recombinant DNA techniques. Generally, the following steps are performed:

(1) transforming or transducing a suitable host cell with a polynucleotide (or variant) of the invention encoding a polypeptide of the invention, or with a recombinant expression vector comprising the polynucleotide;

(2) a host cell cultured in a suitable medium;

(3) isolating and purifying the protein from the culture medium or the cells.

The recombinant polypeptide may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

Because the polypeptide of the invention is short, a plurality of polypeptides can be considered to be connected in series, an expression product is obtained after recombinant expression, and then the required small peptide is formed by enzyme digestion and other methods. The polypeptide set of the invention is particularly suitable for the preparation method

Compositions and methods of administration

In another aspect, the present invention provides a composition, including a pharmaceutical, food or cosmetic composition, comprising (a) a safe and effective amount of a polypeptide of the present invention or a pharmaceutically/dietetically/cosmetically acceptable salt thereof; and (b) a pharmaceutically/dietetically/cosmetically acceptable carrier or excipient. The amount of the polypeptide of the present invention is usually 10. mu.g to 100 mg/dose, preferably 100. mu.g to 1000. mu.g/dose.

For the purposes of the present invention, an effective dose is about 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg, of the polypeptide of the invention to a subject. In addition, the polypeptides of the invention may be used alone or in combination with other therapeutic agents (e.g., formulated in pharmaceutical compositions such as glucocorticoids, immunosuppressive agents, or non-steroidal anti-inflammatory drugs).

The compositions of the present invention may also contain a pharmaceutically/dietetically/cosmetically acceptable carrier. The term "pharmaceutically/dietetically/cosmetically acceptable carrier" refers to a carrier for oral and/or parenteral administration. The term refers to such pharmaceutical carriers: they do not themselves induce the production of antibodies harmful to the individual receiving the composition and are not unduly toxic after administration. Such vectors are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's pharmaceutical sciences (Mack pub. co., n.j.1991). Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, adjuvants, and combinations thereof.

The compositions of the present invention can be administered to mammals, including rats, mice, livestock and humans, by conventional routes, such as orally, rectally, intravenously, intramuscularly, subcutaneously, by intrauterine, durally, or by intracerebroventricular (intrabraventicular) injection. Thus, the pharmaceutical composition may be formulated into typical pharmaceutical formulations known in the art. The pharmaceutical composition may be formulated into oral, injectable, suppository, transdermal and nasal preparations, but is not limited thereto. Can be made into any preparation, preferably liquid, suspension, powder, granule, tablet, capsule, pill, emulsion, syrup, aerosol or oral preparation, such as extract.

These compositions can be formulated by mixing, diluting or dissolving according to a conventional method, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic agents (isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizing agents, and the formulation process can be carried out in a conventional manner according to the dosage form.

The pharmaceutically acceptable carriers in the therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol, auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers, examples of which may be any pharmaceutically acceptable examples.

In addition to these ingredients, known additives for improving taste may be included, such as: natural flavouring agents, such as plum, lemon, pineapple or herb flavours, natural fruit juices, natural dyes, such as chlorophyllin or flavonoids, sweetening ingredients, such as fructose, honey, sugar alcohols, or sugar, or acidulants such as citric acid or sodium citrate.

Examples of formulations for parenteral administration include: sterile aqueous solutions, anhydrous solvents, suspensions, emulsions, lyophilizates and suppositories. For the preparation of anhydrous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, or injectable esters such as ethyl oleate may be used. As suppository base, witepsol (a glyceride of a saturated fatty acid mixture), polyethylene glycol, Tween 61, cocoa butter, lauric oil or glycerogelatin may be used.

Generally, the therapeutic compositions can be prepared as injectables, e.g., as liquid solutions or suspensions; solid forms suitable for constitution with a solution or suspension, or liquid carrier, before injection, may also be prepared.

The pharmaceutical compositions of the present invention may also be administered in the form of sustained release formulations. For example, the polypeptide of the invention or a salt thereof may be incorporated into a pellet or microcapsule carried by a sustained release polymer and then surgically implanted into the tissue to be treated. In addition, the polypeptide of the present invention or a salt thereof can be used by inserting an intraocular lens previously coated with a drug. As examples of the sustained-release polymer, ethylene-vinyl acetate copolymer, polyhydroxymethacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymer, lactic acid-glycolic acid copolymer and the like can be exemplified, and biodegradable polymers such as lactic acid polymer and lactic acid-glycolic acid copolymer can be preferably exemplified.

When the pharmaceutical composition of the present invention is used for practical treatment, the dosage of the polypeptide of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient can be determined reasonably according to the body weight, age, sex, and degree of symptoms of each patient to be treated. For example, when administered topically, it is usually administered at a concentration of about 0.1 to 10 wt%, preferably 1 to 5 wt%, 2 to 6 times per day, 1 to 5ml each time.

The food compositions of the present invention may be formulated into typical food products known in the art. The food composition may be prepared in the form of: a powder, granules, tablets, pills, capsules, suspension, emulsion, syrup, liquid, extract, chewing gum, tea, jelly, or beverage. The dietetically acceptable carrier or additive may be any carrier or additive known in the art.

The healthy functional food composition may comprise the polypeptide or the collection of polypeptides of the invention as an active ingredient, which constitutes 0.01 to 100% (wt), preferably 0.2 to 80% (wt), of the composition of the invention, based on the total weight of the food.

In addition to the active ingredients described above, the food compositions of the present invention may also contain other components which may be various flavors or natural carbohydrates used in conventional beverages. Examples of the above natural carbohydrates are conventional sugars such as monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.) and polysaccharides (e.g., dextran, cyclodextrin, etc.), and sugar alcohols such as xylitol, sorbitol and erythritol. Other flavoring agents, natural flavoring agents (e.g., thaumatin (tamarind), stevia extract, etc.), and synthetic flavoring agents (e.g., saccharin, aspartame, etc.) may also be added.

In addition, the composition of the present invention may be a cosmetic composition, and the polypeptide of the present invention may be formulated into an emulsion, cream, ointment, drop, suspension, etc. for external use or topical preparation according to the conventional art.

Indications of

The polypeptide and the derivative polypeptide thereof can be used for preparing a composition for inhibiting inflammation or treating diseases related to inflammation.

As used herein, TNF- α is the earliest and most important inflammatory mediator in the inflammatory response, activates neutrophils and lymphocytes, increases vascular endothelial cell permeability, regulates other tissue metabolic activities, and promotes the synthesis and release of other cytokines.

Inflammation-related diseases

Inflammation-related diseases that may be used in the present invention include ocular inflammatory diseases, pancreatitis, inflammatory bowel disease, lung inflammation, skin inflammation, rheumatoid arthritis, ankylosing spondylitis.

In the present invention, the inflammation-related diseases are commonly associated with increased levels of TNF- α, COX-2, IL-1 β, or IL-6, and therefore, the polypeptide of the present invention has an effect of inhibiting inflammation or treating the inflammation-related diseases, as long as the polypeptide of the present invention can effectively inhibit the proinflammatory cytokines TNF- α, COX-2, IL-1 β, or IL-6.

Industrial applicability

A pharmaceutical composition containing the polypeptide (or polypeptide set) of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient has a significant inhibitory activity against inflammation. The application of the polypeptide mainly focuses on the aspects of polypeptide drugs, tissue engineering materials, diagnostic polypeptides, polypeptide vaccines, polypeptide drug carriers, tissue engineering materials, cosmetic polypeptides, polypeptide nutritious foods and the like.

The main advantages of the invention include:

(a) the polypeptide of the invention has small molecular weight and can permeate various human tissue barriers;

(b) the water solubility is good, and the high concentration can be kept in body fluid;

(c) the safety is high, and the toxic and side effects on biological tissues are small;

(d) can be prepared by a solid-phase synthesis method, and has high purity, large yield and low cost.

Therefore, the polypeptide of the invention is expected to be developed into medicines, foods or cosmetics for treating systemic or local inflammation-related diseases, such as inflammatory bowel diseases, skin inflammation and the like.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the laboratory Manual (New York: Cold Spring Harbor laboratory Press,1989), or according to the manufacturer's recommendations.

General procedure

Amino acid sequence analysis

Liquid chromatography conditions: mobile phase: a-ultrapure water (volume fraction 0.1% formic acid), B-acetonitrile (volume fraction 0.1% formic acid); 0 min: 95% of A; and (5) 55 min: 60% A + 40% B; 80 min: 5% of B.

Mass spectrometer conditions: ion mode: ESI +; capillary voltage: 2.5 kV; taper hole voltage: 30V; ion source temperature: 100 ℃; desolventizing gas temperature: 250 ℃; desolventizing gas flow rate: 600L/h; taper hole gas flow: 50L/h; collision energy: 40V; mass to charge ratio scan range: 500 to 2000 m/z; detecting voltage: 1600V.

Taking a certain amount of RP-HPLC prepared sample, and adding acetonitrile-water solution for dissolving; carrying out mass spectrum analysis on the sample by using UPLC-TOF/MS to obtain an experimental mass spectrum; and performing professional software analysis and database search by using the obtained mass spectrogram, and analyzing and deducing an amino acid sequence.

ELISA assay

Culturing cells in 12-well plate, dissolving anti-inflammatory active peptide in PBS when RAW246.7 cells grow to reach 80% fusion, adding into culture system of RAW246.7 to reach 100 μ g/mL and 10 μ g/mLFinal concentration, biochemical incubator 37 ℃, 5% CO₂The culture was continued for 24 h. Followed by stimulation with Lipopolysaccharide (LPS) at a final concentration of 10ng/mL and the cells were incubated for a further 24 h. Finally, the culture broth was collected for later experiments.

EXAMPLE 1 preparation of whey protein anti-inflammatory active peptides by alkaline enzymatic hydrolysis and purification of the peptides

1.1 preparation of whey protein anti-inflammatory active peptide by alkaline enzyme enzymolysis

Whey protein powder is prepared into solution (6%) with certain substrate concentration, and the solution is stirred and mixed evenly. Pretreating at 80 deg.C for 10min, cooling with 0.1mol/L CH₃COONH₄The buffer was adjusted to pH optimum for the enzyme (pH 8) and alkaline protease ([ E/S) was added]6 percent of whey protein active peptide crude extract is obtained by enzymolysis for 6h at 55 ℃, enzyme deactivation for 10min at 90 ℃ and rapid cooling. And (5) performing refrigerated centrifugation at 8000r/min for 20 min. Collecting supernatant, and vacuum filtering. Collecting the filtrate, adjusting ph to neutral, and vacuum freeze drying.

1.2 Ultrafiltration separation purification

Dissolving the freeze-dried powder obtained by enzymolysis in water, stirring and uniformly mixing by using a glass rod to prepare a to-be-filtered solution, carrying out ultrafiltration (30psi) on the to-be-filtered solution by using a 1kD ultrafiltration membrane, collecting the filtrate with the molecular weight less than 1kD, and carrying out vacuum freeze drying on the filtrate to obtain the whey protein peptide crude product with the anti-inflammatory activity.

1.3 Sephadex column chromatography separation and purification

Taking a sample obtained by ultrafiltration of 50mg, and adding 1mL of ultrapure water for dissolution; separating the whey peptide crude extract by a Sephadex G-25 column, eluting a sample obtained by ultrafiltration by using deionized water as a solvent and a mobile phase (the flow rate is 0.6 mL/min); measuring the absorbance of the collected sample solution at 280nm, collecting the component F4 with good anti-inflammatory activity, and freeze drying.

1.4 semi-preparative RP-HPLC separation purification

Dissolving the freeze-dried powder in ultrapure water, preparing by using reversed phase HPLC, and eluting with an eluent A: water-0.01% trifluoroacetic acid, eluent B: acetonitrile-0.01% trifluoroacetic acid; flow rate: 5 mL/min; detection wavelength: 280 nm.

RP-HPLC gradient elution method

Collecting fractions F4c, F4e and F4i with good antiinflammatory activity, vacuum freeze drying, and obtaining chromatographic chromatogram of the result shown in FIG. 1 and FIG. 2, wherein the peptide DQWL mass spectrum with best antiinflammatory activity is shown in FIG. 4.

The results of mass spectrometric detection are as follows: f4c, mainly comprising Gly-Thr-Trp-Tyr (GTWY), Asp-Tyr-Lys-Lys-Tyr (DYKKY); f4e, mainly comprising Leu-Phe (LF), Ala-Val-Phe (AVF), Trp-Leu-Ala (WLA), Asp-Gln-Trp-Leu (DQWL); f4i mainly comprises Trp-Tyr-Ser-Leu (WYSL), Glu-Tyr-Gly-Leu-Phe (EYGLF). The anti-inflammatory active peptide is subjected to monomer synthesis, and the anti-inflammatory activity on the gene and protein level is verified.

Example 2 solubility testing

The powdery test sample of the peptide DQWL with the best anti-inflammatory activity in example 1 is weighed, placed in a certain volume of pure water at 25 + -2 ℃, shaken vigorously for 30 seconds every 5 minutes, and the dissolution within 30 minutes is observed, if no visible solute particles or liquid drops exist, the test sample is regarded as complete dissolution.

The results show that 25mg of DQWL powder can be dissolved in 1mL of water, indicating a better solubility.

Example 3 anti-inflammatory Activity assay

(1) The experiment adopts abdominal cavity macrophage RAW264.7 of a mouse, and the anti-inflammatory effect of the mouse is detected by detecting the release of inflammatory factors generated by LPS induction through PCR;

(2) anti-inflammatory experiments: culturing cells in 6-well plate, when RAW246.7 cells reach 80% confluency, dissolving the fractions collected from different separation and purification stages in PBS, adding into culture system of RAW246.7 in six-well plate to reach 100 μ g/mL final concentration (peptide monomer concentration of 100 μ g/mL and 10 μ g/mL), biochemical culturing at 37 deg.C with 5% CO₂The culture was continued for 72 h. Followed by stimulation with Lipopolysaccharide (LPS) at a final concentration of 10ng/mL and the cells were incubated for a further 4 h. Finally, the culture medium was discarded and the cells were collected for later experiments.

(3) Extracting total RNA: the medium was aspirated, total RNA extracted by adding Trizol reagent, transferred to a centrifuge tube, 200. mu.L of chloroform was added to each tube, centrifuged at 12000rpm for 20min at 4 ℃, the supernatant was aspirated, and an equal volume of isopropanol was added overnight at-20 ℃.

Taking out the total RNA extract after the overnight, centrifuging at 12000rpm at 4 ℃ for 20 min; removing supernatant by suction, dissolving with 75% ethanol-DEPC water solution, centrifuging at 10000rpm and 4 deg.C for 15 min; removing supernatant by suction, dissolving with 75% ethanol-DEPC water solution, centrifuging at 10000rpm and 4 deg.C for 5 min; the supernatant was aspirated and evaporated to dryness in a fume hood.

Dissolving the volatilized RNA extract with 20 mu L DEPC water, and measuring the total RNA content of each sample under the wavelength of the NanoDrop 280 nm;

(4) reverse transcription PCR: constructing a 20 mu L reaction system, and synthesizing cDNA;

(5) real-time fluorescent quantitative PCR: the cDNA is amplified.

(6) SPSS one-way analysis of variance is adopted to analyze the results of real-time fluorescent quantitative PCR, and the expression level results of different experimental groups TNF- α -1 β -2mRNA can be obtained by comparing the significance of differences among groups.

The results show (fig. 5-6), the 8 peptide fragments show certain anti-inflammatory effects at both gene and protein levels, wherein the anti-inflammatory effect of the DQWL is most remarkable at two levels and two concentrations, and the activity is the best.

Example 4 Western assay

Culturing cells in 6-well plate, dissolving antiinflammatory active peptide in PBS when RAW246.7 cells grow to 80% fusion, adding into culture system of RAW246.7 to reach 10 μ g/mL final concentration, biochemical culturing at 37 deg.C with 5% CO₂The culture was continued for 72 h. Followed by stimulation with Lipopolysaccharide (LPS) at a final concentration of 10ng/mL, and the cells were cultured for an additional 1 h. Finally, the culture broth was discarded, the cells were harvested, the protein was extracted and the total protein content was determined for later experiments, the results are shown in FIG. 7(DQWL 10. mu.g/mL, LPS10 ng/mL).

The results show that DQWL can trigger phosphorylation of IKK α/β and degradation of I kappa B α and transfer of cytoplasmic p65 to nucleus at the same time, the expression of phosphorylated p38 in MAPK pathway can be significantly reduced, therefore the inhibition of inflammatory factors by DQWL is mainly regulated by NF-kappa B and p38MAPK signaling pathways.

Example 5 preparation of DQWL-derived peptides and Activity testing

DQWL-derived polypeptides according to formula I were synthesized according to the following sequence variations:

TABLE 1

	A	B	C	SEQ ID NO.:
					Polypeptide 2	L	DQWL	C	6
Polypeptide 3	I	DQWL	S	7
					Polypeptide 4	KL	DQWL	CE	8
Polypeptide 5	KI	DQWL	CD	9
					Polypeptide 6	RL	DQWL	SE		10
Polypeptide 7	RI	DQWL	SD	11

As a result of anti-inflammatory tests of the sequences synthesized in Table 1 in accordance with the method of example 3, it was found that these polypeptides all had a significant inhibitory effect on inflammatory factors such as TNF- α -1 β -2 at a concentration of 10. mu.g/ml.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. An isolated polypeptide having an amino acid sequence as set forth in SEQ ID NO 1, 6, 7, 9, 10 or 11.

2. The isolated polypeptide of claim 1, wherein the amino acid sequence of said polypeptide is set forth in SEQ ID No. 1.

3. A collection of polypeptides comprising the isolated polypeptides of claim 1, and one or more short peptides selected from the group consisting of:

4. Use of the isolated polypeptide of claim 1 or the collection of polypeptides of claim 3 for the preparation of a composition for the prevention and/or treatment of an inflammatory disease, said composition being capable of inhibiting an inflammatory factor.

5. The use according to claim 4, wherein the inflammatory disease comprises an infectious and/or a non-infectious inflammatory disease.

6. The use of claim 5, wherein said inflammatory agent comprises COX-2, TNF- α, or IL-1 β.

7. A composition comprising as an active ingredient the isolated polypeptide of claim 1, or the combination of polypeptides of claim 3, and a pharmaceutically or dietetically or cosmetically acceptable carrier.

8. The composition of claim 7, wherein the composition comprises a pharmaceutical composition, a food composition, or a cosmetic composition.

9. The composition of claim 8, wherein the composition is in a dosage form comprising an oral dosage form, a parenteral dosage form, or a topical dosage form.

10. An in vitro non-therapeutic method of inhibiting inflammatory factors comprising the steps of: adding the isolated polypeptide of claim 1, the collection of polypeptides of claim 3, or the composition of claim 7 to a cell culture, thereby inhibiting an inflammatory factor in the cell.