CN115066252A - Peptide-containing composition, method for producing same, and use of peptide - Google Patents

Abstract

The present invention aims to provide a novel composition containing an animal extract and/or a plant extract and a peptide, which has an anti-inflammatory effect. The present invention relates to a composition containing an animal extract and/or a plant extract and a peptide consisting of an amino acid sequence represented by Gly-Pro-Ala-Gly-Pro (SEQ ID NO:1) and/or a salt thereof.

Description

Peptide-containing composition, method for producing same, and use of peptide

Technical Field

The present invention relates to compositions containing animal and/or plant extracts and peptides, and methods for producing the same. The invention also relates to the use of the peptides for the production of anti-inflammatory compositions.

Background

Collagen, known as gelatin, has been widely used in the food field. Since collagen, which is an animal protein, is a main component of dermis and connective tissue, recently, collagen has also received attention in the medical field and the cosmetic field. Generally, oral administration of high molecular weight collagen is not effectively utilized in vivo. Recently, in order to promote the intake of collagen in vivo, low molecular weight collagen peptides obtained by hydrolyzing high molecular weight collagen have been developed, and foods and/or beverages containing such collagen peptides have also been developed.

Inflammation is a phenomenon in which histamine, kinins, etc. are released by damaged cells, causing vasodilation, increase in capillary permeability, and accumulation of macrophages at an inflammation site, which results in increased blood flow at an infection site, edema, immune cell and antibody transfer, pain, fever, etc.

Recently, ingredients capable of inhibiting the expression of proteins associated with inflammation have been studied to provide effective relief of inflammation. Although anti-inflammatory agents having various mechanisms, such as non-steroidal anti-inflammatory drugs (NSAIDs) and steroidal anti-inflammatory drugs (SAIDs), have been developed, these agents may have side effects. Thus, there is still a need for ingredients that are safer and have anti-inflammatory effects.

For example, patent document 1 discloses an anti-inflammatory composition containing, as an active ingredient, a peptide derived from telomerase having an anti-inflammatory activity.

CITATION LIST

Patent document

PTL 1：JP 2015-525768 T

Disclosure of Invention

Technical problem

The present invention aims to provide novel compositions comprising animal and/or plant extracts and peptides. The present invention also aims to provide compositions having an anti-inflammatory effect.

Technical scheme

The present inventors have conducted studies to find peptides having anti-inflammatory activity. As a result, they found that a peptide consisting of the amino acid sequence shown in SEQ ID NO. 1 had anti-inflammatory activity.

The present inventors also used peptides in combination with animal extracts and/or plant extracts. As a result, they found that the anti-inflammatory activity was increased due to a synergistic effect, and thus completed the present invention.

Specifically, the present invention is defined as follows.

(1) A composition comprising an animal extract and/or a plant extract, and a peptide consisting of an amino acid sequence represented by Gly-Pro-Ala-Gly-Pro (SEQ ID NO:1) and/or a salt thereof.

(2) The composition according to the above (1), wherein the peptide and/or salt thereof is a peptide and/or salt of hydrolyzed type II collagen derived from chicken cartilage.

(3) The composition according to the above (1) or (2), wherein the composition comprises hydrolyzed type II collagen of chicken cartilage.

(4) The composition according to any one of the above (1) to (3), wherein the animal extract is a chicken extract.

(5) The composition according to any one of the above (1) to (4), wherein the composition comprises carnosine and/or anserine and/or one or more salts thereof.

(6) The composition as described in (5) above, wherein the weight ratio of the total weight of carnosine, anserine and salts thereof in terms of carnosine and anserine to the weight of the peptide and salts thereof in terms of peptide (total of carnosine and anserine/peptide) is 5000/1 to 50/1.

(7) The composition according to any one of the above (1) to (6), wherein the composition is a food, a drink or a pharmaceutical.

(8) The composition according to any one of the above (1) to (7), wherein the composition is used for reducing inflammation.

(9) The composition according to any one of the above (1) to (8), wherein the composition inhibits production of at least one cytokine selected from the group consisting of: monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1 (MIP-1), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-9 (IL-9), and stimulated regulatory normal T cell expression and secretion factors (RANTES).

(10) The composition according to any one of the above (1) to (9), wherein the composition is used for preventing or alleviating an inflammatory disorder or disease.

(11) An anti-inflammatory composition comprising, as an active ingredient, a peptide consisting of an amino acid sequence represented by Gly-Pro-Ala-Gly-Pro (SEQ ID NO:1) and/or a salt thereof.

(12) The composition of (11) above, wherein the composition inhibits production of at least one cytokine selected from the group consisting of: monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1 (MIP-1), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-9 (IL-9), and interleukin-12 (IL-12).

(13) A method of producing a composition, the method comprising: mixing animal extract and/or plant extract with peptide composed of amino acid sequence shown by Gly-Pro-Ala-Gly-Pro (SEQ ID NO:1) and/or its salt.

(14) The method according to (13) above, wherein the mixing comprises mixing the animal extract and/or the plant extract with hydrolyzed type II collagen of chicken cartilage containing peptides and/or salts thereof.

(15) The method according to (13) or (14) above, wherein the animal extract is a chicken extract.

(16) The method of (15) above, wherein the chicken extract contains carnosine and/or anserine and/or one or more salts thereof.

(17) Use of a peptide consisting of the amino acid sequence shown by Gly-Pro-Ala-Gly-Pro (SEQ ID NO:1) and/or a salt thereof for the production of an anti-inflammatory composition.

Advantageous effects

The present invention provides novel compositions comprising animal and/or plant extracts and peptides. The composition of the present invention can be used as a food or drink composition or a pharmaceutical composition for reducing inflammation and joint pain. An animal extract and/or a plant extract and a plant extract consisting of SEQ ID NO:1 can be consumed as a food or a drink, and is also advantageous in terms of high safety.

Drawings

FIG. 1 is a flow chart schematically illustrating a method for preparing hydrolyzed type II collagen of chicken cartilage.

Fig. 2 is a graph showing the effect of hydrolyzed type II collagen (HCII) and Chicken Extract (CE) added to chicken cartilage of inflammation-inducing cells on inhibiting the production of the inflammation marker MIP-1 β.

Fig. 3 is a graph showing the effect of the combination of fraction P3, fraction P4 and fractions P3-P7 among 7 fractions obtained by fractionation of hydrolyzed type II collagen (HCII) of chicken cartilage on inhibiting the production of the inflammatory marker MIP-1 β.

Fig. 4 is a graph showing the synergistic effect of the combination of hydrolyzed type II collagen (HCII) and Chicken Extract (CE) of chicken cartilage on inhibiting the production of the inflammatory marker MIP-1 β.

FIG. 5 is a graph showing the effect of the peptide GPAGP detected in fractions P3 and P4 of hydrolyzed type II collagen (HCII) of chicken cartilage on the inhibition of the production of the inflammation marker MCP-1.

Fig. 6 is a graph showing the effect of hydrolyzed type II collagen (HCII) added to chicken cartilage of inflammation-inducing cells on the inhibition of the production of inflammatory markers.

Fig. 7 is a graph showing the effect of hydrolyzed type II collagen (HCII) and Chicken Extract (CE) of chicken cartilage, the effect of the combination of HCII and CE, and the synergistic effect of the combination of peptide GPAGP and CE in inhibiting the production of MCP-1, an inflammation marker.

FIG. 8-1(a), FIG. 8-1(b) and FIG. 8-1(c) are graphs showing the effect of the combination of HCII and CE and the combination of peptide GPAGP and CE on the inhibition of the production of the inflammatory markers IL-6 (FIG. 8-1(a)), IL-8 (FIG. 8-1(b)) and IL-9 (FIG. 8-1 (c)).

FIGS. 8-2(d), 8-2(e) and 8-2(f) are graphs showing the effect of the combination of HCII and CE and the combination of the peptides GPAGP and CE on the inhibition of the production of the inflammation markers MCP-1 (FIG. 8-2(d)), MIP-1 β (FIG. 8-2(e)) and RANTES (FIG. 8-2 (f)).

Fig. 9(a) is a graph showing the effect of a 14-day intake of hydrolyzed type II collagen (HCII) of chicken cartilage on VAS pain scores at day 7 and day 14 in each treatment group of a cohort (n ═ 151) that followed the protocol. Fig. 9(b) is a graph showing the effect of hydrolyzed type II collagen (HCII) intake of chicken cartilage on VAS pain scores at day 7 and day 14 in each treatment group of subjects who underwent less than the tenth percentile of the total resistance training period (n-8).

Detailed Description

According to an embodiment, the composition of the invention comprises an animal extract and/or a plant extract and a peptide consisting of the amino acid sequence shown by Gly-Pro-Ala-Gly-Pro (SEQ ID NO:1) and/or a salt thereof.

The composition of the present invention contains an animal extract and/or a plant extract.

Examples of the animal extract include the following extracts: mammals such as cattle, pigs, sheep and goats, birds such as poultry, quail, duck, goose, shellfish, insects, fish, mollusks, and crustaceans. Examples of plant extracts include tea extracts, fruit juices and concentrates thereof, vegetable extracts and concentrates thereof, soy beans and nut extracts.

Among these animal extracts and plant extracts, the extract is preferably an animal extract, more preferably an avian extract, still more preferably a poultry extract, and particularly preferably a chicken extract. The composition of the present invention shows higher anti-inflammatory effect when it contains chicken extract.

Chicken extract

The chicken extract (hereinafter sometimes referred to as "CE") used in the present invention may be an extract obtained by heating chicken used as a raw material in a liquid, or may be a commercially available product. The material may include bone, cartilage, legs, etc., but preferably the material does not contain head or internal organs.

Examples of commercially available products of Chicken Extract (CE) include "Brand's Essence of Chicken (BEC) (by Sun juice)&Produced by Food Asia Pte Ltd) "," Scotch ^TM Expression of Chicken (manufactured by Scotch Industrial Co., Ltd., Ltd.), "Quaker expression of Chicken (manufactured by Standard Foods Corporation Co., Ltd.)," "Chiken stock and cloth of SWANSON manufactured by Campbell Sound Company (NYSE: CPB)") ^TM "," Drip Chicken Essence produced by Eu Yan Sang International Ltd. (Singapore), "Boned Chicken Tonic produced by Eu Yan san Eu Yan Sang International Ltd. (Singapore)," and "bound Essence of Chicken produced by Hao Yi Kang Lao Xie Zhen Co., Ltd. (Taiwan province). Any such commercially available product may be used, but preferably Brand's Essence of Chicken (BEC) is used.

When the chicken extract used in the present invention is produced by hot water extraction, the chicken extract can be produced by a method commonly used in the art. For example, the chicken extract can be produced by performing normal pressure extraction and/or pressure extraction using a liquid having a temperature of 100 ℃ to 125 ℃, and treating or filtering the resulting extract with a membrane. Specifically, the extract was obtained by: a pre-treatment step (1) in which chicken is heated in a liquid; and (2) replacing the liquid with fresh liquid after the pretreatment, and heating the chicken again. The heat treatment in each of step (1) and step (2) is preferably carried out in a solvent. For example, the solvent is preferably water, ethanol or a mixture thereof. The chicken extract comprises liquid extract obtained by the above method; a dilute solution, concentrate or dry powder of the liquid extract; and purified products of these. The purified product can be obtained by, for example, subjecting the chicken extract to ultrafiltration, membrane treatment, liquid separation operation, resin fractionation treatment, or the like to increase the purity. After increasing the purity of the chicken extract, the purified product can be powdered, for example, by freeze drying or spray drying.

The chicken extract used in the present invention preferably contains carnosine and/or anserine and/or one or more salts thereof. Carnosine is beta-alanyl-histidine, which is a dipeptide of beta-alanine and histidine. Anserine is β -alanyl 1-methylhistidine in which histidine is methylated.

Examples of the carnosine salt and anserine salt include the same salts as the salts of GPAGP described later.

When the composition of the present invention contains carnosine and/or a salt thereof, for example, the amount of carnosine and/or a salt thereof in the composition is preferably 0.00001 wt% or more, more preferably 0.0001 wt% or more, and preferably 10 wt% or less, more preferably 1 wt% or less, in terms of carnosine. In an embodiment, for example, the amount of carnosine and/or a salt thereof in the composition is preferably from 0.00001 wt% to 10 wt%, more preferably from 0.0001 wt% to 1 wt%, based on carnosine.

When the composition of the present invention contains anserine and/or a salt thereof, for example, the amount of anserine and/or a salt thereof in the composition is preferably 0.00001 wt% or more, more preferably 0.0001 wt% or more, and preferably 10 wt% or less, more preferably 1 wt% or less, based on the anserine. In an embodiment, for example, the amount of anserine and/or its salts in the composition is from 0.00001 wt% to 10 wt%, more preferably from 0.0001 wt% to 1 wt%, based on anserine.

For example, carnosine, anserine and salts thereof can be quantified by HPLC.

More preferably, the composition of the invention contains both carnosine and anserine.

Peptides

The composition of the present invention contains a peptide consisting of an amino acid sequence shown by Gly-Pro-Ala-Gly-Pro (SEQ ID NO:1) and/or a salt thereof (hereinafter, "peptide consisting of an amino acid sequence shown by Gly-Pro-Ala-Gly-Pro (SEQ ID NO: 1)", which is sometimes referred to as "GPAGP").

GPAGP may be a peptide obtainable by hydrolyzing animal protein, plant protein, or the like, or may be an artificially synthesized peptide. GPAGP is preferably a peptide derived from hydrolyzed type II collagen, more preferably from chicken cartilage.

GPAGP can be purified prior to use. Alternatively, GPAGP in the form of hydrolyzed type II collagen of chicken cartilage containing GPAGP may be included in the composition. In a preferred embodiment, the composition of the invention comprises hydrolyzed type II collagen of chicken cartilage. The composition of the present invention containing hydrolyzed type II collagen of chicken cartilage exhibits higher anti-inflammatory effect.

If desired, GPAGP may be included in the compositions of the present invention as a salt with an inorganic or organic acid or as a salt with an inorganic or organic base. Such acids or bases may be selected based on the application of the salt. The following dietary acceptable salts and pharmaceutically acceptable salts are preferable in view of application to foods, beverages, medicines, and the like. Examples of the inorganic acid salt include hydrochloride, nitrate, sulfate, methanesulfonate and p-toluenesulfonate. Examples of the organic acid salt include salts with dicarboxylic acids such as oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, and salts with monocarboxylic acids such as acetic acid, propionic acid, and butyric acid. Examples of inorganic bases include hydroxides, carbonates and bicarbonates of sodium, lithium, calcium, magnesium and aluminium and of ammonia. Examples of the salt with an organic base include mono-alkylamine salts, di-alkylamine salts or tri-alkylamine salts (for example, salts of methylamine, dimethylamine and triethylamine), mono-hydroxyalkylamine salts, di-hydroxyalkylamine salts or tri-hydroxyalkylamine salts, guanidine salts and N-methylglucamine salts.

In the composition, the weight ratio of the total weight of carnosine, anserine, and salts thereof, calculated as carnosine and anserine, to the weight of GPAGP and salts thereof, calculated as peptide (total of carnosine and anserine/GPAGP) is preferably from 5000/1 to 1/15000. The weight ratio is more preferably 5000/1 to 50/1, and still more preferably 4000/1 to 75/1.

Hydrolyzed type II collagen of chicken cartilage

Hydrolyzed type II collagen of chicken cartilage (hereinafter, "hydrolyzed type II collagen of chicken cartilage" is sometimes referred to as "HCII") can be obtained by hydrolyzing the type II collagen with an enzyme or the like. Type II collagen can be extracted from chicken cartilage by known methods. The hydrolyzed type II collagen used in the chicken cartilage of the present invention can be prepared from cartilage by a method commonly used in the art.

For example, hydrolyzed type II collagen can be obtained by treating chicken cartilage with enzymes. In particular, hydrolyzed type II collagen can be prepared by: a pretreatment step (3) in which chicken cartilage is heated in a liquid, and a step (4) in which the chicken cartilage after the pretreatment step is treated with an enzyme. The enzyme used in step (4) is not limited as long as it is an enzyme commonly used in the art. Examples include collagenase, papain, bromelain, actinidin, ficin, cathepsin, pepsin, chymosin, trypsin, protease, aminopeptidase, subtilisin, endopeptidase and exopeptidase, and enzyme preparations obtained by mixing these enzymes. The method of preparing the hydrolyzed type II collagen is not limited to the enzyme treatment method.

The hydrolyzed type II collagen of chicken cartilage may be a solution obtained by hydrolyzing chicken cartilage, a concentrate of the solution, or a dry powder, or may be a purified product of the concentrate or the dry powder. The purified product of hydrolyzed type II collagen of chicken cartilage can be obtained by increasing the purity by, for example, subjecting a solution obtained by hydrolyzing chicken cartilage to ultrafiltration, membrane treatment, liquid separation operation, resin fractionation treatment, or the like. Hydrolyzed type II collagen of chicken cartilage is a peptide mixture, which typically contains GPAGP, and can be considered a collagen peptide derived from type II collagen.

The hydrolyzed type II collagen of chicken cartilage has a weight average molecular weight of preferably 100-. The molecular weight and weight average molecular weight can be measured by the Eurofins HPAEC-PAD method.

In embodiments, a GPAGP containing fraction, or a combination of a GPAGP containing fraction with one or more other fractions, which can be obtained by fractionation of hydrolyzed type II collagen of chicken cartilage by molecular weight by methods such as gel filtration, can be used in the composition of the present invention.

The fractions obtained by fractionation of the hydrolyzed type II collagen of chicken cartilage for the composition of the present embodiment are preferably a fraction having a molecular weight of less than 2500 and a weight average molecular weight of 900-1100 (fraction 3), and a fraction having a molecular weight of less than 1800 and a weight average molecular weight of 650-850 (fraction 4). In this embodiment, the composition preferably contains a combination of fraction 3 and fraction 4. GPAGP, a hydrolyzed type II collagen derived from chicken cartilage, is usually contained in fraction 3 and fraction 4.

The weight ratio of the hydrolyzed type II collagen of chicken cartilage (as solids) to the sum of carnosine, anserine and salts thereof (as carnosine and anserine) (hydrolyzed type II collagen of chicken cartilage/sum of carnosine and anserine) is preferably 20/1-1/5, more preferably 15/1-1/3.

The amount of each ingredient of the composition of the present invention is not limited and may be set based on, for example, the form of the composition.

In an embodiment, for example, the amount of chicken extract (on a solid basis) in the composition of the invention is preferably 0.1 wt% or more, more preferably 0.5 wt% or more, and also preferably 99 wt% or less, more preferably 90 wt% or less. In an embodiment, for example, the amount of chicken extract (on a solids basis) in the composition is preferably 0.1 wt% to 99 wt%, more preferably 0.5 wt% to 90 wt%.

In an embodiment, for example, the amount of GPAGP and/or a salt thereof in the composition of the present invention is preferably 0.0001 wt% or more, more preferably 0.001 wt% or more, and also preferably 90 wt% or less, more preferably 80 wt% or less. In embodiments, for example, the amount of GPAGP in the composition is preferably from 0.0001 wt% to 90 wt%, more preferably from 0.001 wt% to 80 wt%.

For example, in an embodiment, the amount of hydrolyzed type II collagen (on a solid basis) of chicken cartilage in the composition of the present invention is preferably 0.1 wt% or more, more preferably 0.5 wt% or more, and also preferably 99 wt% or less, more preferably 90 wt% or less. In an embodiment, for example, the amount of hydrolyzed type II collagen (on a solids basis) of chicken cartilage in the composition is preferably 0.1 wt% to 99 wt%, more preferably 0.5 wt% to 90 wt%.

Herein, the amount of hydrolyzed type II collagen of chicken cartilage includes the amount of GPAGP.

The composition of the present invention is preferably used as food, drink or medicine. Examples of the food or drink include functional claims foods (foods with function foods), health-promoting foods, foods for special diet, dietary supplements, health supplements, and general supplements. The form of the food or beverage is not limited. For example, it may be a solid food or a liquid food. Preferably a beverage.

The form of the drug is not limited. Non-limiting examples include oral preparations such as capsules, tablets, powders, granules and dry syrups; external preparations such as ointments, adhesive skin patches, eye drops, suppositories; and an injection. The medicament is preferably an oral preparation (oral medicine).

The compositions of the present invention may contain pharmaceutically or dietetically acceptable additives such as various carriers, excipients, diluents, acidulants, antioxidants, stabilizers, preservatives, flavoring or masking agents, emulsifiers, colors, seasonings, pH adjusting agents and nutritional enhancers.

The compositions of the invention are suitable for therapeutic use (medical use) and non-therapeutic use (non-medical use). "non-treatment" is a concept that does not include medical activities, i.e., a concept that does not include a method of surgery, treatment, or diagnosis in humans.

Anti-inflammatory composition

The compositions of the present invention are useful for reducing inflammation. The composition of the present invention may be an anti-inflammatory composition containing an animal extract and/or a plant extract and GPAGP and/or a salt thereof as active ingredients.

To achieve the anti-inflammatory effect (anti-inflammatory effect) contemplated by the present invention, hydrolyzed type II collagen of chicken cartilage containing GPAGP and/or a salt thereof and an animal extract and/or a plant extract (particularly, a chicken extract) are added to the composition in the same manner as described above for the composition. Hydrolyzed type II collagen of chicken cartilage containing GPAGP and/or a salt thereof and an animal extract and/or a plant extract may be used as they are, or a concentrate, a dry powder or a purified product thereof may be added as described above, as long as the effect of the present invention is not impaired. The same additives as described above may be used.

The composition of the present invention is preferably orally ingested (orally administered). The dosage (also referred to as "intake") of the composition of the present invention is not limited as long as the inflammation-reducing effect is achieved. The dosage of the composition of the present invention can be appropriately determined according to the administration form, the administration method, the body weight of the subject, and the like.

In an embodiment, when the composition of the present invention is fed or orally administered to a human (adult), the intake amount (in terms of solid) of the chicken extract is preferably 0.1mg or more, more preferably 1mg or more, and further preferably 15000mg or less, more preferably 13000mg or less per 60kg body weight per day. In embodiments, the chicken extract is preferably ingested by a human (adult) in an amount of 0.1-15000mg, more preferably 1-13000mg per 60kg body weight per day. The intake of chicken extract includes the amount of carnosine and anserine.

In an embodiment, when the composition of the present invention is orally fed or administered to a human (adult), the total dose of carnosine, anserine, and salts thereof is preferably 0.001mg or more, more preferably 0.01mg or more, and further preferably 500mg or less, more preferably 400mg or less per 60kg body weight per day, in terms of carnosine and anserine. In an embodiment, the total of carnosine, anserine and salts thereof fed to a human (adult) is preferably 0.001-500mg, more preferably 0.01-400mg per 60kg body weight per day in terms of carnosine and anserine.

In an embodiment, when the composition of the present invention is fed or orally administered to a human (adult), the intake amount of GPAGP and/or a salt thereof in terms of GPAGP is preferably 0.001mg or more, more preferably 0.01mg or more, and further preferably 200mg or less, more preferably 100mg or less per 60kg body weight per day. In an embodiment, the amount of GPAGP and/or a salt thereof ingested by a human (adult) is preferably 0.001 to 200mg, more preferably 0.01 to 100mg per 60kg body weight per day, in terms of GPAGP.

In an embodiment, when the composition of the invention is fed or orally administered to a human (adult), the amount of the intake (on a solid basis) of hydrolyzed type II collagen of chicken cartilage is preferably 0.01mg or more, more preferably 0.1mg or more, and also preferably 4000mg or less, more preferably 3000mg or less per 60kg body weight per day. In an embodiment, the amount of hydrolyzed type II collagen ingested by a human (adult) into chicken cartilage is preferably 0.01 to 4000mg, more preferably 0.1 to 3000mg per 60kg body weight per day.

Herein, the intake of hydrolyzed type II collagen of chicken cartilage includes the intake of GPAGP and/or salts thereof.

In the present invention, the above amount of chicken extract and the above amount of hydrolyzed type II collagen of chicken cartilage containing GPAGP and/or a salt thereof are preferably fed or administered at least once a day, for example, once or more times a day (e.g., twice or three times a day). In an embodiment, the aforementioned amount of chicken extract and the aforementioned amount of hydrolyzed type II collagen of chicken cartilage containing GPAGP and/or a salt thereof are preferably fed or orally administered to a human. In an embodiment, the composition of the invention may be used to feed or administer the above amount of chicken extract and the above amount of hydrolyzed type II collagen of chicken cartilage containing GPAGP and/or its salt to a human (per 60kg body weight per day).

The compositions of the present invention inhibit the production of cytokines such as monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1 (MIP-1), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-9 (IL-9), and stimulated regulation of normal T cell expression and secretion factors (RANTES). The compositions of the present invention are particularly effective in inhibiting the production of MCP-1, MIP-1 β, IL-6, IL-8, IL-9 and RANTES.

By inhibiting cytokine production, inflammation in vivo can be reduced.

The compositions of the present invention are useful for preventing or alleviating an inflammatory condition or disease. An inflammatory condition or disease is, for example, a condition or disease caused by inflammation or a condition or disease accompanied by inflammation. Examples of such conditions or diseases include collagen diseases such as arthritis and rheumatoid arthritis, inflammatory bowel disease, osteoarthritis, tendonitis, sciatica, herniated intervertebral disc, stenosis, myelopathy, back pain, facet joint pain, carpal tunnel syndrome, tarsal tunnel syndrome, post-lumbar surgery pain syndrome, AIDS, arteriosclerosis, asthma, diabetes, hepatitis, stroke, dementia, muscle atrophy, viral infection, skin aging including photoaging, cancer, aging, allergic diseases, Parkinson's disease, cerebral infarction, cataracts, epilepsy, spinal cord injury, retinopathy of prematurity, nephropathy, peptic ulcer, pancreatitis, ulcerative colitis, myocardial infarction, adult respiratory distress syndrome, emphysema, vasculitis, edema, diabetic complications, UV injury, altitude sickness, porphyria, burns, frostbite, contact dermatitis, Shock, multiple organ failure, DIC, fatigue, sarcopenia (muscle weakness), mitochondrial dysfunction, Alzheimer's disease, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, and systemic lupus erythematosus (lupus). The composition of the present invention is preferably used for preventing or alleviating these diseases. In particular, the food or beverage composition is preferably used for preventing or alleviating diseases such as osteoarthritis, rheumatoid arthritis and psoriatic arthritis.

As used herein, prevention of a condition or disease includes prevention of onset of disease, delay of onset of disease, reduction in incidence of disease, reduction in risk of onset of disease, and the like. Alleviation of a disorder or disease includes recovery of the subject from the disorder or disease, alleviation of symptoms of the disorder or disease, amelioration of symptoms of the disorder or disease, delay or prevention of progression of the disorder or disease, and the like.

The subject to which the composition of the present invention is fed or administered (which may also be referred to as "subject") is not limited. The subject is preferably a human or non-human mammal, more preferably a human.

In embodiments, the subject may be one who needs or wants to inhibit inflammation. Such a subject may be, for example, one in need or desire to prevent or reduce inflammation, or one in need or desire to prevent or reduce an inflammatory disorder or disease. In embodiments, the subject of the invention may be a middle aged or elderly human. The compositions of the present invention may also be used by healthy people, for example, for the purpose of preventing a condition that can be prevented or alleviated by attenuating inflammation.

The compositions of the present invention may be labeled with functional claims that demonstrate effects through attenuation of inflammation. Such tags are also referred to as tags with functional claims, and the content of the tags is not limited. Examples of such functional claims on the label include "relieving joint pain", "attenuating joint pain", "managing knee condition", "maintaining knee health", "improving joint mobility", and other functional claims equivalent to the above.

In an embodiment of the present invention, the composition of the present invention is preferably a food or beverage, and more preferably a beverage, which is marked with the above-mentioned functional claims. The label may describe the use of the composition of the invention to achieve the above-described functionality. The label may be added to the composition itself or to a container or package for the composition.

The present invention also relates to a method for producing a composition, which comprises the step of mixing an animal extract and/or a plant extract with a peptide consisting of an amino acid sequence shown by Gly-Pro-Ala-Gly-Pro (SEQ ID NO:1) (GPAGP) and/or a salt thereof.

In the above step, GPAGP and/or a salt thereof may be separately mixed with the animal extract and/or the plant extract. However, the composition is preferably produced by mixing hydrolyzed type II collagen of chicken cartilage containing GPAGP and/or a salt thereof with an animal extract and/or a plant extract (preferably a chicken extract). In this case, for example, a hydrolysate obtained by hydrolyzing type II collagen of chicken cartilage with an enzyme or the like can be directly used for mixing with a chicken extract. As described above, a concentrate, a dry powder or a purified product thereof may be added as long as the effect of the present invention is not impaired.

In the production method of the present invention, the order of adding the raw materials is not limited. For example, GPAGP and/or a salt thereof, or hydrolyzed type II collagen of chicken cartilage containing GPAGP and/or a salt thereof, may be first placed in a container and then chicken extract added. Alternatively, the chicken extract may be placed in a container first, and then GPAGP and/or a salt thereof, or hydrolyzed type II collagen of chicken cartilage containing GPAGP and/or a salt thereof, may be added.

Commercially available chicken extracts or chicken extracts produced by hot water extraction can be used directly in admixture with GPAGP and/or salts thereof, or hydrolyzed type II collagen of chicken cartilage containing GPAGP and/or salts thereof. However, when the chicken extract is in the form of a concentrate, a dry powder, or a purified product of the concentrate or dry powder, the chicken extract may be diluted, dissolved, etc. in a liquid (e.g., water, ethanol, or a mixture of water and ethanol) and then mixed with GPAGP, hydrolyzed type II collagen of chicken cartilage, or the like. When the hydrolyzed type II collagen of chicken cartilage containing GPAGP and/or a salt thereof is in the form of a concentrate, a dry powder, or a purified product of the concentrate or dry powder, such product may be similarly diluted, dissolved, etc. in a liquid or the like before mixing. Alternatively, such a product may be added without prior dilution, dissolution, or the like, and then mixed with a liquid to be diluted, dissolved, or the like in the liquid.

In the production method of the present invention, the chicken extract preferably contains carnosine and/or anserine and/or one or more salts thereof.

In the production method of the present invention, the preferred modes of the chicken extract are as described above. Preferred embodiments of hydrolyzed type II collagen of chicken cartilage are also described above.

The composition produced by the production method of the present invention may contain the above-mentioned additives in addition to the chicken extract, GPAGP and/or its salt, and hydrolyzed type II collagen of chicken cartilage.

The present invention also relates to an anti-inflammatory composition comprising, as an active ingredient, a peptide consisting of an amino acid sequence represented by Gly-Pro-Ala-Gly-Pro (SEQ ID NO:1) and/or a salt thereof.

Examples of the anti-inflammatory composition include compositions similar to those described above containing an animal extract and/or a plant extract and GPAGP and/or a salt thereof, however, the animal extract and/or the plant extract are not essential components in the anti-inflammatory composition. The anti-inflammatory composition may comprise the same animal extract, the same plant extract, the same hydrolyzed type II collagen of chicken cartilage containing GPAGP and/or its salt, and the same additives as described above.

The anti-inflammatory composition of the present invention inhibits the production of cytokines such as: monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1 (MIP-1), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-9 (IL-9), and interleukin-12 (IL-12). The compositions of the present invention are particularly effective in inhibiting the production of MCP-1, MIP-1 β, IL-6, IL-7, IL-9 and IL-12.

The invention also relates to the use of peptides consisting of the amino acid sequence shown by Gly-Pro-Ala-Gly-Pro (SEQ ID NO:1) (GPAGP) and/or salts thereof for the production of anti-inflammatory compositions.

Examples of the anti-inflammatory composition include compositions similar to those described above containing an animal extract and/or a plant extract and GPAGP and/or a salt thereof, however, the animal extract and/or the plant extract are not essential components in the anti-inflammatory composition. The anti-inflammatory composition may comprise the same animal extract, the same plant extract, the same hydrolyzed type II collagen of chicken cartilage containing GPAGP and/or its salt, and the same additives as described above.

Examples

The present invention is described more specifically below with reference to examples. The invention is not limited to the embodiments described.

The raw materials, reagents and the like used in the following examples and comparative examples are as follows.

Preparation of hydrolyzed type II collagen from chicken cartilage

FIG. 1 shows a flow chart schematically describing the process for the preparation of HCII. First, frozen chicken cartilage was thawed in water at 40 ℃ and washed in water at 40 ℃ (1 hour). Next, the washing water was poured off, and the clear water was poured into a 1200L pot so that it was 3 times as much as the chicken cartilage. The water was heated to the optimum working temperature for the enzyme treatment and the washed chicken cartilage was immersed therein for the enzyme treatment for several hours. After the enzyme treatment, the pan containing the chicken cartilage was heated to 90 ℃ or higher and incubated at 90 ℃ or higher for 30 minutes, thereby inactivating the enzyme used in the previous enzyme treatment. The resulting mixture (liquid and enzyme treated chicken cartilage) was filtered. The resulting liquid was concentrated. Finally, the resulting concentrate was spray-dried at 200 ℃, thereby preparing HCII powder.

Determination of the molecular weight of HCII powder

The molecular weight distribution of the resulting HCII was determined by the Eurofins HPAEC-PAD method. Table 1 below shows the molecular weight distribution of the resulting HCII. The resulting HCII had a weight average molecular weight of 4582, calculated using methods common in the art.

TABLE 1

Fractionation of HCII fractions and analysis of peptides

(materials and methods for HCII fractionation and biological Activity identification)

(Material)

Formic acid was purchased from Tokyo Chemical Industry co. Ultrapure water was obtained from Merck Milli-Q water purification system. The synthetic peptide GPAGP was obtained from GenScript.

(preparative Gel Permeation Chromatography (GPC))

Fractionation of HCII was performed using a preparative VERITY 271HPLC system (Gilson). HC-II was dissolved in ultrapure water to prepare a solution of 10mg/mL (w/v). After centrifugation at 14000rpm for 5 minutes, a 1000. mu.L aliquot was injected onto a preparative GPC column (BioSep 5. mu.m SEC-s 2000) attached to a guard column (SecurityGuard PREP card C1215X 21.2mm ID (Phenomenex))

LC column 300 × 21.2mm (phenomenex)), and was eluted with eluent a (ultrapure water: formic acid ═ 100:0.1(v/v)) at a rate of 5mL/min for 30 minutes with isocratic elution. Chromatograms were observed at 214 nm.

Fractions were collected every 0.5 min between 8 min to 28 min using a GX series fraction collector (Gilson), and the 40 individual fractions obtained were subsequently combined into 7 fractions, P1-P7 (P1: 8.5-12.0 min, P2: 12.0-13.5 min, P3: 13.5-14.5 min, P4: 14.5-16.0 min, P5: 16.0-17.5 min, P6: 17.5-19.0 min, P7: 19.0-27.5 min). The combined fractions were evaporated to dryness in a lyophilizer (ScanVac) and the dried samples were stored at-20 ℃ until further use.

Table 2 shows the molecular weight of each fraction obtained above. In table 2, "Mw" and "Mp" represent the weight average molecular weight and the peak molecular weight, respectively. As shown in table 2, fraction 3(P3) and fraction 4(P4) consisted of molecules with average molecular weights of 1035(P3) and 784 (P4).

The molecular weights of the fractions of HCII were measured by HPLC gel filtration under the following conditions.

The instrument comprises: agilent 1100 series

And (3) detection: UV 214nm

Flow rate: 1mL/min

Mobile phase: isocratic 0.1mM sodium phosphate buffer pH 6.8

Operating time: 20 minutes

Column: biosep ^TM 5μm SEC-s2000

LC column 300X 7.8mm

TABLE 2

Fraction(s) of	Mw	Mp
			P1	1359	1626
P2	1092	1494
			P3	1035	956
P4	784	783
			P5	422	213
P6	211	225

(computer search for peptides directed to GPAGP)

Fractions P1-P7 were subjected to LC-MS analysis on an Agilent HPLC 1290 series coupled TripleTOF 5600(AB Sciex) with a Duo Spray Turbo V ion source and gas generator (Peak Scientific Ltd.). The main components were separated with UHPLC Guard Zorbax Eclipse Plus C182.1 × 5mm, 1.8 μm (agilent) and eluted with eluent a (ultrapure water: formic acid 100:0.1(v/v)) and eluent B (acetonitrile: formic acid 100:0.1(v/v)) in a linear gradient as follows: 0-0.5 min: 100% eluent A; 0.5-7.5 minutes: 100% -65% of eluent A; 7.5-10.0 min: 65% -0% of eluent A; 11.0-13.0 min: 0% eluent A; 13.0-13.1 min: 0-100% eluent A; 13.1-15.0 min: 100% eluent A. The experiments were performed in position mode using an Independent Data Acquisition (IDA) method, with the Collision Energy (CE) and Declustering Potential (DP) of the MS optimized to 10.0V and 80V, respectively. The analysis was done by PeakView (AB Sciex). The mass spectral data was then analyzed and retrieved using Protein Pilot (AB Sciex) accompanied by a Unit KB fasta file, using as key. The data was further processed by candidate blacklisting and validation was done with peptides purchased from Genscript.

(detection and quantification of peptide markers by LC-MS analysis)

Detection and quantification of GPAGP peptide was performed on LC-MS. LC-MS analysis was performed on an Agilent HPLC 1290 series coupled tripleTOF 5600(AB Sciex) with a Duo Spray Turbo V ion source and gas generator (Peak Scientific Ltd.). The mass spectrometer uses electrospray ionization and Multiple Reaction Monitoring (MRM) at unit mass resolution in positive ion mode, using the following parameters: collision energy diffusion (CES): 10; delayed Ion Release (IRD): 67; ion Release Width (IRW): 25; ion source gas: 40; air curtain gas (CUR): 30, of a nitrogen-containing gas; temperature: 500.0 of the total weight of the mixture; ion spray voltage (isff): 5500. The MS compound-related parameter settings, such as declustering voltage (DP) and Collision Energy (CE), were optimized for different peptides. Analyst1.5.2(AB Sciex) was used for device control, data acquisition and data processing.

GPAGP was reconstituted in ultrapure water and injected onto a Zorbax Eclipse Plus C182.1 × 5mm, 1.8 μm (Agilent) column with UHPLC Guard Zorbax Eclipse Plus C18 RRHD 1.8 μm 2.1 × 50mm (Agilent) and eluted with eluent a (ultrapure water: formic acid 100:0.1(v/v)) and eluent B (acetonitrile: formic acid 100:0.1(v/v)) using the following linear gradient: 0-0.5 min: 100% eluent A; 0.5-10 minutes: 100% -65% of eluent A; 10-11 minutes: 65% -10% of eluent A; 11-12.5 minutes: 10% of eluent A; 12.5-14.1 min: 10% -100% of eluent A. The sample volume was 10. mu.L, the flow rate was 300. mu.3.1 Th, and the retention time was 3.1min as quantitative and qualitative substances. Calibration curves were constructed by injecting standard GPAGP at 31.5ng/mL, 62.5ng/mL, 125ng/mL, 250ng/mL, 500ng/mL into the mass spectrometer. HC-II was redissolved to 125. mu.g/mL as a working solution. Quantification of GPAGP in HC-II and its GPC fractions was done by MultiQuant (AB Sciex).

GPAGP is contained in P3 and P4 fractions of HCII, and the content of GPAGP is 914.5. + -. 121.4. mu.g/g based on 1g of HCII.

Quantification of carnosine and anserine

Quantitation of carnosine and anserine in chicken extracts was performed by HPLC under the following conditions.

A standard stock solution of carnosine was prepared by adding and dissolving carnosine powder in deionized water to a carnosine concentration of 2.50 mg/ml. The anserine standard stock solution was prepared by adding and dissolving L-anserine nitrate powder in deionized water to a L-anserine concentration of 3.96 mg/ml.

The weight of L-anserine was calculated by the following formula.

L-anserine (g) ═ 0.792 XL-anserine nitrate (g)

Analytical conditions of HPLC

The device comprises the following steps: high performance liquid chromatography system with UV detector (Agilent 1100 from Agilent Corp.)

Column: zorbax 300-SCX 4.6mm ID X250 mm (Agilent)

Mobile phase: 50mM potassium dihydrogen phosphate

Flow rate: 1.0mL/min

A flow channel: channel A (50mM potassium dihydrogen phosphate), channel B (acetonitrile), channel D (deionized water)

Wavelength of ultraviolet detector: 210nm

Sample introduction volume: 10 μ L

Method for evaluating anti-inflammatory activity

In examples and comparative examples, the effect of inhibiting the production of inflammatory markers was evaluated by the following method.

(raw materials and reagents)

Chicken Extract (CE): brand's Essence of Chicken (Sun fashion Beverage & Food Asia Pte Ltd, carnosine content in 1 ml: 0.94mg/ml, anserine content in 1 ml: 1.9mg/ml)

Chondrocyte Culture Medium (CM), Fetal Bovine Serum (FBS), Chondrocyte Growth Supplement (CGS), and penicillin/streptomycin (P/S): ScienCell Research Laboratories

IL-1β：R&D Systems

Poly-L-lysine (PLL) -coated 96-well plates (Corning)

Cell culture and pretreatment

Human chondrocytes were isolated from human articular cartilage (HC-a, science cell Research Laboratories) and maintained in CM supplemented with 5% FBS, 1% CGS and 1% P/S. Cells were seeded at a cell density of 4000 cells/well in PLL-coated 96-well plates and at 37 ℃ in the presence of 5% CO ₂ Incubated overnight in the humidified chamber of (1). Chondrocytes were washed once with PBS and pre-treated with different concentrations of HCII, CE, a combination of HCII and CE, and GPAGP alone for 24 hours and further treated by addition of 25ng/mL IL-1 β for 24 hours. Cells were centrifuged at 1100g for 5 minutes and the supernatant was used for cytokine analysis.

Multiplex cytokine assay

Pro-Human Cytokine Multiplex Assays (Bio-Rad, Munich, Germany) were used to analyze cytokines in the culture medium. The following 27 re-analyses were performed: IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8(CXCL8), IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, eotaxin (CCL11), macrophage colony stimulating factor (M-CSF), Interferon (IFN) -gamma, monocyte chemotactic protein 1 (MCP-1; CCL2), macrophage inflammatory protein-1 alpha (MIP-1 alpha; CCL3), MIP-1 beta (CCL4), stimulated regulation of normal T cell expression and secretion factor (TES RANs) (CCL5), TNF-alpha and Vascular Endothelial Growth Factor (VEGF). Multiplex assays were performed according to the manufacturer's instructions and run on the Luminex xPONENT for madix platform. Bio-Plex Manager version 6.0 was used for data processing. Cytokine and chemokine concentrations were calculated by reference to standard curves. The sensitivity of the multiplex kit was <5 pg/mL.

Statistical analysis

Statistical analysis was performed using GraphPad Prism version 5.0 (San Diego, california, usa). All results are expressed as mean ± standard deviation. Statistical analysis was performed by analysis of variance (ANOVA), followed by a post hoc Tukey multiple comparison test. Data were considered significant when P-value P < 0.05.

Comparative example 1, comparative example 2 and example 1

To evaluate the effect of HCII and Chicken Extract (CE) on the inflammatory marker MIP-1 β produced by human chondrocytes, the following four different conditions were compared: cells before treatment (untreated control), cells treated with IL-1 β (comparative example 1), cells treated with IL-1 β + HCII (example 1) and cells treated with IL-1 β + CE (comparative example 2). HCII-treated cells and CE-treated cells were pretreated with HCII or CE. The HCII concentration in the pretreatment was 0.5mg/ml or 2.5mg/ml and the CE concentration was 1.25mg/ml or 6.25 mg/ml.

As shown in fig. 2, 25ng/mL of IL-1 β induced inflammation in human chondrocytes, resulting in a significant increase in the level of MIP-1 β compared to untreated cells (control). Pretreatment of cells with HCII and CE reduced MIP-1 β levels induced by IL-1 β. HCII and CE also have a dose-dependent effect on the inhibition of MIP-1 β production. In fig. 2, the data are expressed as mean ± SD (n ═ 3). Unless otherwise stated, a significant difference of p <0.05, indicates p <0.01, and indicates p <0.001, compared to IL-1 β (IL-1 β treated cells).

Example 2 Effect of the combination of fractions P3, P4 and P3-P7 on inhibiting the production of the inflammatory marker MIP-1 β

Of the seven fractions isolated from HCII, P3-P7 were used. Cells were pretreated with IL-1 β and a combination of HCII, P3, P4, or P3-P7 (each at a concentration of 5mg/mL for the combination of HCII, P3, P4, and P3-P7) as in example 1 to compare the effects.

The results are shown in FIG. 3. Data are presented as mean ± SD (n ═ 3). Unless otherwise stated, a significant difference of p <0.05, p <0.01, compared to IL-1 β (IL-1 β treated cells) indicates p < 0.001.

Example 3 synergistic Effect of combination of HCII and CE

To evaluate the synergistic effect of the combination of HCII and CE in inhibiting the production of the inflammatory marker MIP-1 β, pre-treatment was performed with HCII alone, CE alone or a combination of HCII and CE. In the pretreatment, the concentration of HCII was 0.5mg/ml and the concentration of CE was 1.25 mg/ml.

As shown in FIG. 4, the combination of 0.5mg/mL HCII and 1.25mg/mL CE provided a synergistic effect in reducing inflammation compared to treatment with HCII or CE alone. Data are presented as mean ± SD (n ═ 3). Unless otherwise stated, a significant difference of p <0.05, in comparison to IL-1 β (IL-1 β treated cells), represents p <0.01, and represents p < 0.001.

Example 4 and comparative example 3 effects of GPAGP in inhibiting production of MCP-1, an inflammation marker

To evaluate the effect of HCII and GPAGP on inhibiting the production of the inflammatory marker MCP-1, the following four different conditions were compared: cells before treatment (untreated control), cells treated with IL-1 β (comparative example 3), cells treated with IL-1 β + HCII (example 4) and cells treated with IL-1 β + GPAGP (example 4). Cells were pretreated with HCII or GPAGP (example 4). In the pretreatment, the concentration of HCII was 5mg/mL, and the concentration of GPAGP was 500 mg/mL.

The results are shown in FIG. 5. Data are presented as mean ± SD (n ═ 3). Unless otherwise stated, a significant difference of p <0.05, p <0.01, compared to IL-1 β (IL-1 β treated cells) indicates p < 0.001.

Example 5 and comparative example 4

To evaluate the effect of HCII on the inflammatory markers IL-6, IL-7, IL-9 and IL-12, the following three different conditions were compared: cells before treatment (untreated control), cells treated with IL-1. beta. (comparative example 4) and cells treated with IL-1. beta. + HCII (example 5). IL-1. beta. + HCII treated cells were pretreated with HCII. In the pretreatment, the concentration of HCII was 0.5mg/ml or 2.5 mg/ml.

As shown in FIG. 6, IL-1. beta. at 25ng/mL induced inflammation in human chondrocytes, resulting in a significant increase in the level of IL-6, IL-7, IL-9 or IL-12, as compared to untreated cells (control). Pretreatment of cells with HCII reduced the levels of IL-6, IL-7, IL-9 and IL-12 induced by IL-1 β. HCII inhibits IL-6, IL-7, IL-9 and IL-12 production also has a dose-dependent effect. In fig. 6, the data are represented as mean ± SD (n-3). Unless otherwise stated, a significant difference of p <0.05, in comparison to IL-1 β (IL-1 β treated cells), represents p <0.01, and represents p < 0.001. Example 6 Effect of the combination of HCII and CE and the combination of GPAGP and CE on the inhibition of the production of MCP-1, an inflammation marker

To evaluate the effect of the combination of HCII and CE and the combination of GPAGP and CE on the inhibition of the production of MCP-1, which is an inflammatory marker, a pretreatment was performed with IL-1. beta. and either the combination of HCII and CE or the combination of GPAGP and CE as in example 1, in which the concentration of HCII was 2.5mg/ml or 5mg/ml, the concentration of GPAGP was 2.5mg/ml, and the concentration of CE was 6.25 mg/ml.

Fig. 7 shows the results. Data are presented as mean ± SD (n ═ 3). Unless otherwise stated, a significant difference of p <0.05, in comparison to IL-1 β (IL-1 β treated cells), represents p <0.01, and represents p < 0.001.

Example 7 Effect of the combination of HCII and CE and the combination of GPAGP and CE on inhibiting the production of inflammatory markers

To evaluate the effect of the combination of HCII and CE and the combination of GPAGP and CE on inhibiting the production of the inflammatory markers IL-6, IL-8, IL-9, MCP-1, MIP-1 β and RANTES, a pre-treatment with IL-1 β and the combination of HCII and CE or the combination of GPAGP and CE was performed. In the pretreatment, the concentration of HCII was 2.5mg/ml, the concentration of GPAGP was 25mg/ml, 125mg/ml or 250mg/ml, and the concentration of CE was 6.25 mg/ml.

The results are shown in FIG. 8-1(a), FIG. 8-1(b), FIG. 8-1(c), FIG. 8-2(d), FIG. 8-2(e) and FIG. 8-2 (f). Data are presented as mean ± SD (n ═ 3). Unless otherwise stated, a significant difference of p <0.05, p <0.01, compared to IL-1 β (IL-1 β treated cells) indicates p < 0.001.

Example 8 randomized, double-blind, four-arm trial study to evaluate the effects of HCII and CE on knee pain

This experimental study was conducted as a double-blind, randomized, placebo-controlled study in a single center.

(Subjects grouped and randomized)

A total of 160 subjects 45-75 years of age were selected using inclusion and exclusion criteria summarized in table 3. Subjects were randomly assigned to four groups, namely "placebo", "glucosamine", "HCII" and "HCII and CE". Each group included 40 subjects.

TABLE 3

(test products)

The "placebo" group took a mixture of 6.8g maltodextrin and 7mg xanthan gum once a day after meals in the morning.

The glucosamine group was administered once a day in the morning after meals at 1.5g glucosamine hydrochloride. Glucosamine hydrochloride served as the activity comparator.

The "HCII" group was administered BRAND' S Collagen Hydrolysate (Sun fashion juice & Food Asia Pte Ltd), a hydrolyzed chicken breast cartilage extract consisting of hydrolyzed type II Collagen (HCII) and a naturally occurring matrix of low molecular weight chondroitin sulfate and Hyaluronic Acid (HA). The product comprises HCII containing the peptide GPAGP. Each bottle with a capacity of 68mL contained 2g of HCII, providing a naturally occurring composition of HCII (66.5%), depolymerized chondroitin sulfate (18%) and HA (11%). The uncharacterized thoracic cartilage component accounted for the remaining 4.5%.

The "HCII and CE" group took 68mL of the following mixture: 70g (5-6 g dry weight) of BRAND' S Essence of chicken (BEC) and 2g of HCII.

All test products were made as liquid products in glass bottles, which were isocaloric, identical in appearance, and identical in flavor and texture. Participants had to take the study product once a day in the morning after meals.

Subjects were allowed to continue to take concomitant medications or supplements that were not believed to affect the results of the study. Analgesics or painkillers are allowed as prescribed rescue medication. The proportion of days of analgesic coverage (PDC) was calculated based on the record of the fraction of concomitant medication of the analgesic and was defined as the percentage of days of analgesic coverage to the total days of the interval between visits. Any treatment or dietary supplement that could support joint, bone and muscle health was banned throughout the study including: hormone therapy (growth hormone, progesterone, estrogen or testosterone), calcium and vitamin D, supplements rich in amino acids, peptides, proteins, omega-3, omega-6, glucosamine or chondroitin.

(procedure)

The study included a screening visit (28 days prior to baseline visit), a subsequent baseline visit (baseline visit considered day 0. screening and baseline visits may be on the same day), and 3 follow-up visits (weeks 8, 16, and 24). Subjects were screened from day-28 to day 0 to determine the eligibility of the study. Test product intake started the day after baseline visit for 168 consecutive days (24 weeks). For 168 consecutive days (24 weeks), subjects took a bottle of test product each morning (after meals). Intake compliance was recorded on a diary card.

Visual Analog Scale (VAS) of knee pain was scored at day 0, day 7 and day 14 post-ingestion.

During the study, subjects were advised to perform resistance training (optionally) twice a week at home, 30 minutes each, following the training program. The training is recorded on a diary card. The diet of the previous week of the visit was recorded using a food questionnaire.

(treatment compliance)

Compliance with study product intake was checked by collecting unused product and daily food records kept by subjects. Compliance was defined as the percentage of the indicated dose actually consumed in the days between visits. Consumption of 70% is considered compliant.

(sports plan)

All subjects were encouraged to perform 30 minutes of resistance training at home twice weekly according to the training manual provided at baseline visit. Compliance with the training program was recorded on a log card and calculated using the following formula:

mathematical formula 1

Compliance rates of > 50% are considered compliant.

(visual analog Scale for Knee pain (VAS))

The Visual Analog Scale (VAS) is rated from 0 to 100mm, where 0 means no pain and 100 means the most severe pain. On days 0, 7 and 14 after ingestion, the interviewee was asked to indicate the level of pain felt by drawing a straight line on the scale indicating the position along the solid line.

(tolerance and safety)

Spontaneously reported adverse events were recorded throughout the study. Vital signs were monitored at each visit. To assess the safety of the study products, all participants were assessed for serum and urine at each visit during the study.

(statistical analysis)

Statistical analysis of the compliance program was performed according to a prior statistical analysis plan. Randomized subjects with intake compliance ≧ 70% were included in the protocol-compliant analysis. Safety parameter analysis was performed on subjects who had taken at least 28 bottles of study product. The binary variables were reported using percentages, while the continuous variables were reported as mean and SD. The chi-square test is used for categorical variable comparisons, while the krustal carl-voris test is used to compare between-group differences for continuous variables.

Repeated measures analysis of variance (ANOVA) was performed using a mixed effects model for testing the mean difference in variation between study groups, with the interaction of the interview intake as a fixed effector for consecutive primary and secondary endpoints. All results were considered statistically significant if the corresponding p-value was below 0.05. If the visit intake interaction term is significant, a post-hoc pairwise comparison between treatment groups is performed and an adjusted p-value is reported. Variables expected to affect the endpoint were included as factors in the model. Gender and gender interview interaction terms were included as factors in the joint health analysis.

In addition, a subgroup analysis was performed on subjects who underwent resistance training for less than the tenth percentile of the total training session. Independent statisticians performed analyses using SAS version 9.4 (Cary, USA).

Results

(Baseline characteristics and treatment compliance)

A total of 160 subjects were enrolled and 151 subjects completed the study and were included in the statistical analysis (PP). Nine subjects were withdrawn during the study; there was no significant difference in the number of withdrawals between groups. None of the withdrawers were associated with any side effects caused by the intake of study product or placebo. No adverse events were noted. No clinically significant changes were observed for serum biochemical markers and urinalysis.

Overall, there were no statistical differences in compliance rates between all four groups (table 4). To assess the homogeneity of the data, all parameters between the four arms were compared. There were no statistically significant differences between the study groups at baseline (table 4).

TABLE 4

(VAS Change in Knee pain score)

After 14 days of intake, the pairwise comparison between 2g HCII and placebo was statistically significant, with the 2g HCII group having less pain as measured by the VAS pain score than the placebo (p ═ 0.021). Pain scores for HCII users at day 7 were-11.1% from HCII group baseline and-25% at day 14, compared to pain scores at day 0 (figure 9(a), table 5). In fact, the placebo group showed a tendency to experience increased pain during day 14 with a 33.3% increase in pain score at day 7 and a 13.3% increase at day 14 starting at day 0.

Furthermore, a subgroup analysis of subjects trained on the tenth percentile less than the total resistance training period showed that the pain score of the placebo group experienced a significant increase of 200% from baseline at day 14 compared to the HCII group (p ═ 0.021) increase of 8.7% from day 0 (fig. 9(b), table 6). In tables 5 and 6, "P value ($)" means the P value between groups; "(a)" and "(k)" indicate methods for determining a p value ((a) ═ one-way analysis of variance, (k) ═ kruskarl-wallis test); "+" indicates statistical significance; "95% c.i." means 95% confidence interval.

In fig. 9(a) and 9(b), two values in parentheses of each data are "mean-standard deviation" and "mean + standard deviation".

TABLE 5

TABLE 6

(conclusion)

Overall, HCII is well tolerated and provides rapid and significant symptomatic relief for patients suffering from osteoarthritic pain. Compared to placebo, ingestion of 2g HCII significantly reduced knee pain within only 14 days.

In vitro studies have shown that the mechanism of action may be through modification of the underlying disease process, in particular inhibition of inflammation leading to local pain sensation. In summary, HCII and combinations of HCII and CE can be considered as safe and effective supplements to current medical and dietary options for treating OA symptoms.

INDUSTRIAL APPLICABILITY

The present invention provides novel compositions comprising peptides and animal and/or plant extracts. The composition of the present invention can be used as a food or beverage composition or a pharmaceutical composition for reducing inflammation and joint pain. The animal extract and/or the plant extract and the peptide consisting of the amino acid sequence shown by SEQ ID NO. 1 can be consumed as food or drink, and are also advantageous in terms of safety.

Sequence listing

<110> Sandeli control stock company

<120> peptide-containing composition, method for producing the same, and use of the peptide

<130> SP2020-0086

<150> SG10201913612R

<151> 2019-12-27

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5

<212> PRT

<213> Chicken ()

<400> 1

Gly Pro Ala Gly Pro

1 5

Claims (17)

1. A composition, comprising:

animal extract and/or plant extract; and

a peptide consisting of an amino acid sequence represented by Gly-Pro-Ala-Gly-Pro (SEQ ID NO:1) and/or a salt thereof.

2. The composition of claim 1, wherein the peptides and/or salts thereof are peptides and/or salts of hydrolyzed type II collagen derived from chicken cartilage.

3. The composition of claim 1 or 2, wherein the composition comprises hydrolyzed type II collagen of chicken cartilage.

4. The composition of any one of claims 1-3, wherein the animal extract is a chicken extract.

5. A composition according to any one of claims 1 to 4, wherein the composition comprises carnosine and/or anserine and/or one or more salts thereof.

6. The composition according to claim 5, wherein the weight ratio of the total weight of carnosine, anserine and salts thereof, calculated as carnosine and anserine, to the weight of the peptide and salts thereof, calculated as peptide (sum of carnosine and anserine/peptide) is from 5000/1 to 50/1.

7. The composition of any one of claims 1-6, wherein the composition is a food, beverage, or pharmaceutical.

8. The composition of any one of claims 1-7, wherein the composition is for reducing inflammation.

9. The composition of any one of claims 1-8, wherein the composition inhibits production of at least one cytokine selected from the group consisting of: monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1 (MIP-1), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-9 (IL-9), and stimulated regulatory normal T cell expression and secretion factors (RANTES).

10. The composition of any one of claims 1-9, wherein the composition is used to prevent or alleviate an inflammatory condition or disease.

11. An anti-inflammatory composition comprising, as an active ingredient, a peptide having an amino acid sequence represented by Gly-Pro-Ala-Gly-Pro (SEQ ID NO:1) and/or a salt thereof.

12. The composition of claim 11, wherein the composition inhibits production of at least one cytokine selected from the group consisting of: monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1 (MIP-1), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-9 (IL-9), and interleukin-12 (IL-12).

13. A method of producing a composition, the method comprising: mixing animal extract and/or plant extract with peptide composed of amino acid sequence shown by Gly-Pro-Ala-Gly-Pro (SEQ ID NO:1) and/or its salt.

14. The method of claim 13, wherein the mixing comprises mixing the animal extract and/or the plant extract with hydrolyzed type II collagen of chicken cartilage containing the peptide and/or salt thereof.

15. The method of claim 13 or 14, wherein the animal extract is a chicken extract.

16. The method of claim 15, wherein the chicken extract comprises carnosine and/or anserine and/or one or more salts thereof.

17. Use of a peptide consisting of the amino acid sequence shown by Gly-Pro-Ala-Gly-Pro (SEQ ID NO:1) and/or a salt thereof for the production of an anti-inflammatory composition.

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