WO2015088233A1

WO2015088233A1 - Peptide having antioxidative effect and composition comprising same

Info

Publication number: WO2015088233A1
Application number: PCT/KR2014/012097
Authority: WO
Inventors: 김상재
Original assignee: 주식회사 카엘젬백스; 김상재
Priority date: 2013-12-10
Filing date: 2014-12-10
Publication date: 2015-06-18
Also published as: KR102667428B1; KR20230025935A; KR20160097244A; KR20240073156A

Abstract

Disclosed are an antioxidative peptide having a sequence of any one of SEQ ID NOs: 1 to 340, a peptide which is a fragment of sequences of SEQ ID NOs: 1 to 340, or a peptide having at least 80% sequence identity with the peptide sequence, and an antioxidative composition containing the same as an active ingredient. A peptide having a sequence of any one of SEQ ID NOs: 1 to 340, or a peptide having a sequence having 80% identity with the sequence or a fragment thereof, according to the present invention, has excellent antioxidative activity. Therefore, the composition containing the peptide of the present invention can be used as a pharmaceutical composition or cosmetic composition for an antioxidative effect, and further a food composition, and thus can be widely used to prevent or treat diseases that may be caused by an abnormal operation of an antioxidative system in the human body.

Description

Peptides with Antioxidant Effect and Compositions Comprising the Same

The present invention relates to a peptide having an antioxidant effect and a composition comprising the same.

As the human body ages, aging naturally progresses. However, if the concentration of free radicals in the body increases aging faster and causes other body abnormalities. Free radicals are oxygen, which enters the body during the respiration process, is used in the oxidation process, and is produced by various metabolic processes to attack oxidative tissues and damage cells. Many diseases are related to free radicals so that more than 90% of modern diseases are related to free radicals.

It is recognized that the causes of diseases such as aging, cancer, cerebrovascular disease, cardiovascular diseases, and the like are free radicals or reactive oxygen species (ROS). The reactive oxygen species may refer to oxygen having high reactivity of oxygen, which is essential for biological maintenance, due to stress such as biological stress caused by germs invading living organisms or various environmental stresses caused by deterioration of the global environment. High oxygen can cause serious physiological disorders. For example, there is a superoxide radical, hydroxyl radical, hydrogen peroxide or singlet oxygen.

Specifically, the free radicals or reactive oxygen species destroy cells, cut off the connective tissue of the dermal layer of the skin, or cause cross-linking, so that not only skin-related diseases such as wrinkles, atopic dermatitis, acne or skin cancer, but also cancer, myocardial infarction ( cardiovascular diseases including myocardial infarction, stroke, and atherosclerosis, degenerative neurological diseases such as amyotrophic laternal sclerosis or Parkin's disease (PD) neurodegenerative disease). In addition, free radicals or reactive oxygen species can produce a harmful substance called lipid peroxide by peroxidating lipids in the human body, and the lipid peroxide acts on blood vessels and causes various adult diseases such as arteriosclerosis or blood serum. It is.

As the life expectancy increases and society ages, interest in aging-related diseases, such as aging and degenerative diseases, increases, and interest in metabolic syndrome and adult diseases increases due to economic growth and dietary changes. have. As interest in these diseases increases, interest in free radicals or reactive oxygen species reported as the cause of the diseases increases, and interest in substances having antioxidant effects is increasing.

It is an antioxidant system that removes free radicals or reactive oxygen species in the body, and macromolecular antioxidant enzymes and vitamins such as superoxide dismutase (SOD), peroxidase (POD) or catalase (CAT) Antioxidants such as C, vitamin E, or glutathione. However, due to pollution, ultraviolet rays or various causes including the biological stress and aging, the antioxidant system of the human body gradually becomes unable to operate normally, and in this case, the active oxygen species can be removed to protect by ingesting antioxidants. There is a need for an in vivo supply of a substance that is capable of antioxidants. In accordance with the demand for supplying the antioxidants in the body, efforts to prevent or treat the disease or delay aging by taking antioxidants or foods having an antioxidant effect or the like have been made in various aspects.

In particular, oxidative stress caused by free radicals has been suggested as one of the major mechanisms of cell death in neurological diseases (Curr. Opin. Neurol., 9 (4): 260-264, 1996). It is reported that the major cause of cell death in each tissue in the inside, about 90% of modern diseases are related to free radicals, and other diseases except for the neurological diseases mentioned above are cancer, arteriosclerosis, diabetes, myocardium Infarction, hepatitis, nephritis, atopic dermatitis, autoimmune diseases and the like. Therefore, there is an urgent need for the development of methods or substances that can inhibit the activation of free radicals in the prevention or treatment of related diseases including the neurological diseases.

In addition, conventional synthetic antioxidants having excellent antioxidant power have been reported, such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) or tertiary butylhydropuinone (TBHQ), which are toxic to some of the absorbent substances in the body. Safety factors such as teratogenic factors and carcinogens, can cause hepatomegaly, or increase microsomal enzyme activity in the liver, causing pathological harm to organs such as lungs and kidneys. The problem is being raised.

Therefore, in recent years, many studies have been conducted to develop an antioxidant that is safer to the human body and excellent in antioxidant power. As part of this research, many studies are being conducted on natural antioxidants, that is, antioxidants obtained from natural substances.

[Preceding technical literature]

[Non-Patent Documents]

(Non-Patent Document 1) Curr. Opin. Neurol., 9 (4): 260-264, 1996

(Non-Patent Document 2) Cross, C.E. et al. Ann. Intern. Med. 1987, 107: 526-45; Adelman, R. et al., Proc. Natl. Acad. Sci. USA, 1988, 85 (8): 2706-8,

(Non-Patent Document 3) Moran Benhar et al. EMBO Reports, 2002, 3 (5): 420-425

Accordingly, the present inventors have made a thorough effort to develop an antioxidant that is safer to the human body and has excellent antioxidant power.

We have found that peptides derived from telomerase can have antioxidant activity and have completed the present invention.

It is therefore an object of the present invention to provide novel peptides having antioxidant activity.

Another object of the present invention is to provide a polynucleotide encoding a novel peptide having antioxidant activity.

Another object of the present invention is to provide an antioxidant composition comprising a peptide having antioxidant activity as an active ingredient.

Another object of the present invention to provide a cosmetic composition comprising a peptide having antioxidant activity as an active ingredient.

Another object of the present invention to provide a pharmaceutical composition comprising a peptide having antioxidant activity as an active ingredient.

According to an aspect of the present invention, a peptide having an antioxidant activity, a peptide comprising an amino acid sequence of any one or more of SEQ ID NOS: 1 to 340, a peptide having a sequence homology of 80% or more with the amino acid sequence, or a fragment thereof Is provided.

In one aspect of the invention, the fragment may be a fragment consisting of three or more amino acids.

In one aspect of the invention, the peptide may be composed of up to 30 amino acids.

In one aspect of the invention, the peptide may be composed of any one amino acid sequence of SEQ ID NO: 1 to 340.

In one aspect of the invention, the peptide may be composed of an amino acid sequence comprising SEQ ID NO: 1.

In one aspect of the invention, the peptide is SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 , SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 42, sequence SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 73 SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 85, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 101, SEQ ID NO: SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 186, SEQ ID NO: 189, SEQ ID NO: 192, SEQ ID NO: 197, SEQ ID NO: 200, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 210, SEQ ID NO: 240, SEQ ID NO: 272, SEQ ID NO: 273, SEQ ID NO: 284, SEQ ID NO: 286, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO: 310, SEQ ID NO: 323, SEQ ID NO: 327, SEQ ID NO: 330, SEQ ID NO: 337 It may be selected from the group consisting of.

In one aspect of the invention, the peptide is SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 38 , SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 73, SEQ ID NO: 74, sequence SEQ ID NO: 75, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136 , SEQ ID NO: 137, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 180, SEQ ID NO: 186, SEQ ID NO: 189, SEQ ID NO: 192, SEQ ID NO: SEQ ID NO: 197, SEQ ID NO: 200, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 210, SEQ ID NO: 272, SEQ ID NO: 273 , SEQ ID NO: 284, SEQ ID NO: 286, SEQ ID NO: 295, SEQ ID NO: 310, SEQ ID NO: 323, SEQ ID NO: 327, SEQ ID NO: 337.

In one aspect of the invention, the peptide may be derived from human telomerase.

According to another aspect of the invention, a polynucleotide encoding a peptide having antioxidant activity, a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 340 or a peptide having a sequence homology of 80% or more with the amino acid sequence Or a polynucleotide encoding a peptide that is a fragment thereof.

In another aspect of the invention, the peptide may be composed of up to 30 amino acids.

In another aspect of the invention, the peptide may be composed of an amino acid sequence comprising SEQ ID NO: 1.

In another aspect of the invention, the peptide may be composed of any one amino acid sequence of SEQ ID NO: 1 to 340.

In another aspect of the invention, the peptide may be derived from human telomerase.

According to another aspect of the present invention, a peptide comprising an amino acid sequence of any one of SEQ ID NOs: 1 to 340, a peptide having a sequence homology of 80% or more with the amino acid sequence, or a fragment thereof as a active ingredient Antioxidant compositions are provided.

In another aspect of the invention, the composition may be for the treatment or prevention of diseases due to peroxidation or free radicals.

In another aspect of the present invention, the disease caused by the peroxidation action or free radicals may be a brain nervous system or cardiovascular disease.

In another aspect of the invention, the cerebral nervous system or cardiovascular disease may include one or more of Alzheimer's disease, Lou Gehrig's disease, Parkinson's disease, Creutzfeldt-Jakob disease, stroke, cerebral infarction and cerebral hemorrhage.

In another aspect of the invention, the composition may be a pharmaceutical composition.

In another aspect of the invention, the composition may be for skin aging or whitening.

In another aspect of the invention, the composition may be a cosmetic composition.

In another aspect of the invention, the composition may be a food composition.

According to another aspect of the present invention, there is provided a method of treating or preventing a disease due to peroxidation or free radicals, characterized by administering the aforementioned antioxidant composition.

A peptide having a sequence of any one of SEQ ID NOs: 1 to 340 according to the present invention or a peptide or fragment having a sequence having 80% homology with the sequence has excellent antioxidant activity. Therefore, the composition comprising the peptide of the present invention can be used as a pharmaceutical composition or a cosmetic composition for the antioxidant effect, and even a food composition, which is widely used for the prevention or treatment of diseases that may occur due to the inability of the human antioxidant system to function normally. Can be.

1 is a graph showing the effect of hydrogen peroxide (H ₂ O ₂ ) according to the concentration on the cell activity of the PC12 cell line. As the concentration of hydrogen peroxide increased, we analyzed the decrease of PC12 cell activity. (A) is the result of analysis of viability activity using MTT assay, and (B) is the cell using CCK-8 assay. Results of the activity analysis (* indicates p <0.05 when compared to the control group (control group)).

FIG. 2 is a graph showing the degree of cellular activity when PEP 1 was treated at each concentration in PC12 cells whose cell activity was reduced by hydrogen peroxide. When PEP 1 was treated to PC12 cells whose cell activity was decreased by hydrogen peroxide, the results showed that the cell activity was not reduced (* indicates p <0.05 when compared to the control group).

3 is a graph showing the effect of hydrogen peroxide on the cell activity of neural stem cells according to the concentration. As the concentration of hydrogen peroxide increases, the result of analyzing the decrease of neural stem cell activity is shown (* indicates p <0.05 compared with the control group).

Figure 4 is a graph confirming the degree of recovery of cell activity reduced by increasing the concentration of PEP 1 in order to see the effect of PEP 1 in the neural stem cells cell activity decreased by hydrogen peroxide. (A) shows the results of MTT assay and CCK assay, and (B) shows the results of cytotoxicity. (* Mark is 200 μM when p <0.05 compared with control group) P <0.05 compared to the group treated with H ₂ O ₂ only).

5 is a graph showing the degree of cellular activity when PEP 1 was treated at different concentrations in neural stem cells damaged by β-amyloid. The result shows that the proliferative activity of the neural stem cells is restored by increasing the concentration of PEP 1 (* indicates p <0.05 when compared to a control group, and # indicates that 200 μM H ₂ O _{2 was} treated only). Compared to the group when p <0.05).

6 is a graph showing the degree of cell death when PEP 1 was treated according to each concentration in neural stem cells damaged by hydrogen peroxide. The result shows that the apoptosis of neural stem cells damaged by hydrogen peroxide is effectively reduced in proportion to the concentration of PEP 1 (* denotes ₂₀₀ μM H ₂ when p <0.05 when compared to a control group). P <0.05 compared to the group treated with only O ₂ ).

FIG. 7 is a graph showing the degree of cell migration when PEP 1 is treated according to respective concentrations in neural stem cells damaged by hydrogen peroxide. The cell migration activity of the neural stem cells damaged by hydrogen peroxide is restored in proportion to the concentration of PEP 1 (* indicates p <0.05 when compared to the control group, and # indicates ₂₀₀ μM H ₂ O ₂ only). P <0.05 compared to treated group).

FIG. 8 is a photograph showing the extent of reactive oxygen species (ROS) generation when PEP 1 is treated according to each concentration in neural stem cells damaged by hydrogen peroxide. The damage caused by hydrogen peroxide increased the concentration of free radicals in neural stem cells in proportion to the concentration of PEP 1 (* indicates ₂₀₀ μM H ₂ O when p <0.05 compared to the control group). P <0.05 compared to _20,000 treated group).

Figure 9 is a photograph of 2D electrophoresis analysis of the effect of PEP 1 protein expression level control effect in the hydrogen stem cells damaged by hydrogen peroxide.

10 is a graph showing the effect of PEP 1 on the change in protein expression level of neural stem cells damaged by hydrogen peroxide and photographs of 2D electrophoresis analysis results.

FIG. 11 is a graph comparing the effect of PEP 1 between cellular activities of neural stem cells and cortical neurons. In MTT assay results, PEP 1 shows a higher induction of cellular activity in neural stem cells than brain cortical neurons (* denotes ₂₀₀ μM H ₂ O when p <0.05 compared to control group). P <0.05 compared to _20,000 treated group).

12 to 32 show the results of ROS inhibition activity of peptides using the DCF-DA method.

The present invention may be variously modified and may have various embodiments. Hereinafter, the present invention will be described in more detail. However, this is not intended to limit the present invention to specific embodiments, it should be understood to include all transformations, equivalents, and substitutes included in the spirit and scope of the present invention. In the following description of the present invention, if it is determined that the detailed description of the related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

Oxygen that enters the body through respiration can become oxidizing free radicals through the oxidation process, which damages DNA, destroys red blood cells, and kills cells. Can be.

Our body has such a defense against free radicals, for example, superoxide dismutase (SOD), catalase, or peroxidase, such as lactoperoxidase, glutathione peroxidase, etc. ) Can be minimized by removing free radicals by the action of antioxidant enzymes such as) and low molecular weight antioxidants such as vitamin C (ascorbic acid), vitamin E (tocopherol), and uric acid. have. However, when these biological defenses fail or are exposed to excessive ROS, they may not be able to easily break down or repair the residual or excess oxygen species, or they may be subjected to oxidative stress. do.

These oxidative stresses result in excessive free radicals and reactive oxygen species such as hydrogen peroxide, which can cause fatal oxygen toxicity in the living body, as well as non-selective and irreversible effects on the cellular components of substrates, proteins, sugars, and DNA. It is known to cause various diseases such as aging, cancer, Alzheimer's disease, brain diseases such as Parkinson's disease, heart disease, ischemia, arteriosclerosis, skin disease, digestive disease, rheumatism and autoimmune diseases by inducing destruction. , CE et al Ann Intern Med 1987 , 107:......... 526-45; Adelman, R. et al, Proc Natl Acad Sci USA, 1988, 85 (8): 2706-8, Moran Benhar et al. EMBO Reports , 2002, 3 (5): 420-425).

One aspect of the present invention provides a pharmaceutical composition for antioxidant, specifically for preventing cell peroxidation or preventing cell death by peroxidation, comprising as an active ingredient at least one peptide selected from the peptides of SEQ ID NOs: 1 to 340. A composition comprising as an active ingredient one or more peptides of the peptides set forth in SEQ ID NOs: 1 to 340 of one aspect of the present invention prevents and protects the death of cells damaged by free radicals, thereby exhibiting an excellent antioxidant effect, and furthermore, a biological It can have anti-aging, skin whitening, skin regeneration, skin moisturizing and skin wrinkle improvement. The pharmaceutical composition according to one aspect of the present invention has an excellent antioxidant effect by including at least one peptide selected from the peptides of SEQ ID NOs: 1 to 340 as an active ingredient, thereby preventing various diseases and symptoms caused by peroxidation or free radicals. Or can be treated. In another aspect of the invention, diseases caused by peroxidation or free radicals include cerebral nervous or cardiovascular diseases. In another aspect of the invention, the neurological or cardiovascular diseases include, but are not limited to, one or more of Alzheimer's disease, Lou Gehrig's disease, Parkinson's disease, Creutzfeldt-Jakob disease, stroke, cerebral infarction and cerebral hemorrhage.

Peptides described in SEQ ID NO: 1 to SEQ ID NO: 340 are shown in Tables 1 to 6 below. SEQ ID NO: 341 is the sequence of human telomerase full length protein. SEQ ID NO: 1 is a peptide derived from telomerase and consists of 16 amino acids. The peptide of SEQ ID NO: 2 to SEQ ID NO: 77 is a peptide comprising the sequence of SEQ ID NO: 1. Peptides of SEQ ID NO: 78 to 179 are fragments of the peptide of SEQ ID NO: 1. Peptides of SEQ ID NO: 180 to 340 are fragments of SEQ ID NO: 341 which are peptides derived from telomerase.

The "names" in Tables 1 to 7 below are named to distinguish peptides. In another aspect of the present invention, at least one of the peptides set forth in SEQ ID NOs: 1 to 340 comprises a "synthetic peptide" synthesized by selecting peptides of the corresponding positions among the peptides contained in telomerase. As used herein, the term "pep" refers to a peptide having any one of SEQ ID NOs: 1 to 340, or a peptide having 80% or more sequence homology with the sequence, or a fragment thereof.

TABLE 1

One aspect of the present invention is a peptide having an antioxidant activity, a peptide comprising an amino acid sequence of any one or more of SEQ ID NOS: 1 to 340, a peptide having a sequence homology of 80% or more with the amino acid sequence, or a fragment thereof It provides a polynucleotide encoding. The polynucleotides can be used to mass produce peptides. For example, a large amount of peptides can be produced by culturing a host cell containing a vector containing a polynucleotide encoding a peptide.

Peptides disclosed herein can include peptides having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology. In addition, the peptides disclosed herein, peptides or fragments thereof comprising SEQ ID NO: 1 and one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, six or more amino acids Or peptides with seven or more amino acids changed.

In one aspect of the invention, amino acid changes belong to a property that allows the physicochemical properties of the peptide to be altered. For example, amino acid changes can be made, such as improving the thermal stability of the peptide, altering substrate specificity, changing the optimal pH, and the like.

In one aspect of the present invention, a peptide comprising an amino acid sequence of any one or more of SEQ ID NOs: 1 to 340, a peptide having a sequence homology of 80% or more with the amino acid sequence, or a peptide thereof is 30 or less It may consist of amino acids.

In one aspect of the invention, a peptide having a sequence of SEQ ID NOs: 1 to 340, a peptide that is a fragment of SEQ ID NOs: 1 to 340, or a peptide having at least 80% sequence homology with the peptide sequence is a telomerase, specifically human ( Homo sapiens ) includes peptides derived from telomerase.

As used herein, "amino acid" includes not only the 22 standard amino acids that are naturally incorporated into the peptide, but also D-isomers and modified amino acids. Accordingly, in one aspect of the invention the peptide may be a peptide comprising D-amino acids. Meanwhile, in another aspect of the present invention, the peptide may include a non-standard amino acid or the like which has been post-translational modified. Examples of post-translational modifications include phosphorylation, glycosylation, acylation (including, for example, acetylation, myristoylation and palmitoylation), alkylation ), Carboxylation, hydroxylation, glycation, biotinylation, ubiquitinylation, changes in chemical properties (e.g., beta-elimination deimidization) , Deamidation) and structural changes (eg, formation of disulfide bridges). It also includes changes in amino acids, such as changes in amino groups, carboxy groups or side chains, caused by chemical reactions that occur during the linkage with crosslinkers to form peptide conjugates.

Peptides disclosed herein can be wild-type peptides identified and isolated from a natural source. On the other hand, the peptides disclosed herein may be artificial variants, comprising an amino acid sequence in which one or more amino acids are substituted, deleted and / or inserted compared to peptides that are fragments of SEQ ID NO: 1. Amino acid changes in the wild type polypeptide as well as in artificial variants include conservative amino acid substitutions that do not significantly affect the folding and / or activity of the protein. Examples of conservative substitutions include basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, valine and methionine), aromatic amino acids (phenylalanine, Tryptophan and tyrosine), and small amino acids (glycine, alanine, serine and threonine). Amino acid substitutions that generally do not alter specific activity are known in the art. The most common exchanges are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly, and vice versa. Other examples of conservative substitutions are shown in the following table.

TABLE 2

Substantial modifications in the biological properties of the peptide include (a) their effect on maintaining the structure of the polypeptide backbone, eg, a sheet or helical conformation, within the substitution region, (b) the charge of the molecule at the target site. Or their effect in maintaining hydrophobicity, or (c) their effect in maintaining the bulk of the side chains, is carried out by selecting significantly different substitutions. Natural residues are divided into the following groups based on common side chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues affecting chain orientation: gly, pro; And

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will be made by exchanging a member of one of these classes for another class. Any cysteine residue that is not involved in maintaining the proper conformation of the peptide can generally be substituted with serine to improve the oxidative stability of the molecule and to prevent abnormal crosslinking. Conversely, cysteine bond (s) can be added to the peptide to improve its stability.

Another type of amino acid variant of the peptide is a change in the glycosylation pattern of the antibody. By change is meant the deletion of one or more carbohydrate residues found in the peptide and / or the addition of one or more glycosylation sites that are not present in the peptide.

Glycosylation of peptides is typically either N-linked or O-linked. N-linked refers to a carbohydrate moiety attached to the side chain of an asparagine moiety. Tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for enzymatic attachment of carbohydrate moieties to asparagine side chains. Thus, the presence of one of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation means attaching one of the sugars N-acetylgalactosamine, galactose or xylose to hydroxyamino acids, most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine You can also use

The addition of glycosylation sites to the peptide is conveniently performed by changing the amino acid sequence to contain one or more of the above mentioned tripeptide sequences (for N-linked glycosylation sites). Such changes may also be made by adding or replacing one or more serine or threonine residues with the sequence of the original antibody (for O-linked glycosylation sites).

In one aspect of the invention, the polynucleotide may be a naturally occurring or artificial DNA or RNA molecule as a nucleic acid molecule, and may be single stranded or double stranded. The nucleic acid molecule may be one or more, which may be nucleic acid molecules of the same type (eg, having the same nucleotide sequence), or may be nucleic acid molecules of other types. Including, but not limited to, one or more of DNA, cDNA, decoy DNA, RNA, siRNA, miRNA, shRNA, stRNA, snoRNA, snRNA, PNA, antisense oligomer, plasmid and other modified nucleic acids It is not.

In addition, a peptide having a sequence of SEQ ID NOs: 1 to 340, a peptide that is a fragment of the sequence of SEQ ID NOs: 1 to 340, or a peptide having a sequence homology of 80% or more with the peptide sequence according to an aspect of the present invention has low intracellular toxicity It has the advantage of high in vivo stability.

In one aspect of the present invention, an antioxidant comprising a peptide comprising an amino acid sequence of any one or more of SEQ ID NOs: 1 to 340, a peptide having a sequence homology of 80% or more with the amino acid sequence, or a fragment thereof as an active ingredient To provide a composition.

Antioxidant composition according to an aspect of the present invention is 0.01g of a peptide comprising a peptide sequence of any one or more of SEQ ID NO: 1 to 340, a peptide having a sequence homology of 80% or more with the amino acid sequence or a fragment thereof / L to 1kg / L, specifically 0.1g / L to 100g / L, more specifically 1g / L to 10g / L may be included in the content. When included in the above range is not only suitable for showing the intended effect of the present invention, it can satisfy both the stability and safety of the composition, it may be appropriate to include in the above range in terms of cost-effectiveness.

The composition according to one aspect of the present invention can be applied to all animals including humans, dogs, chickens, pigs, cattle, sheep, guinea pigs or monkeys.

In one aspect of the invention the composition is a peptide having a sequence selected from the group consisting of SEQ ID NOs: 1 to 340, a peptide that is a fragment of any one of SEQ ID NOs: 1 to 340, or at least 80% sequence homology with the peptide sequence. Provided is a pharmaceutical composition for antioxidant, specifically, for preventing cell peroxidation or preventing cell death by peroxidation, comprising a peptide as an active ingredient. The pharmaceutical composition for antioxidant according to one aspect of the present invention, specifically for preventing cell peroxidation or preventing cell death by peroxidation may be oral, rectal, transdermal, intravenous, intramuscular, intraperitoneal, intramedullary, intradural or subcutaneous. May be administered.

Formulations for oral administration may be, but are not limited to, tablets, pills, soft or hard capsules, granules, powders, solutions or emulsions. Formulations for parenteral administration may be, but are not limited to, injections, drops, lotions, ointments, gels, creams, suspensions, emulsions, suppositories, patches or sprays.

Pharmaceutical compositions according to one aspect of the invention may include additives such as diluents, excipients, lubricants, binders, disintegrants, buffers, dispersants, surfactants, colorants, flavoring or sweetening agents as needed. Pharmaceutical compositions according to one aspect of the invention may be prepared by conventional methods in the art.

The active ingredient of the pharmaceutical composition according to one aspect of the present invention will vary depending on the age, sex, weight, pathology and severity of the subject to be administered, the route of administration or the judgment of the prescriber. Dosage determination based on these factors is within the level of one of skill in the art and its daily dosage may be, for example, 10 ng / kg / day to 10 mg / kg / day, specifically 0.1 μg / kg / day to 1 mg / kg / day, More specifically 1 μg / kg / day to 100 μg / kg / day, and more specifically 2 μg / kg / day to 50 μg / kg / day, but is not limited thereto. The pharmaceutical composition according to one aspect of the present invention may be administered once to three times a day, but is not limited thereto.

In one aspect, the present invention provides an antioxidant comprising a peptide comprising an amino acid sequence of any one or more of SEQ ID NOs: 1 to 340, a peptide having a sequence homology of 80% or more with the amino acid sequence, or a fragment thereof as an active ingredient. Provided is a skin external preparation composition.

According to another aspect of the present invention, a peptide comprising an amino acid sequence of any one or more of SEQ ID NOs: 1 to 340, a peptide having a sequence homology of 80% or more with the amino acid sequence, or a fragment thereof is included as an active ingredient. It provides a cosmetic composition for antioxidant, specifically for preventing cell peroxidation or preventing cell death by peroxidation.

The topical skin composition or cosmetic composition according to one aspect of the present invention may be provided in any formulation suitable for topical application. For example, it may be provided in the form of a solution, an emulsion obtained by dispersing an oil phase in an aqueous phase, an emulsion obtained by dispersing an aqueous phase in an oil phase, a suspension, a solid, a gel, a powder, a paste, a foam, or an aerosol. Such formulations may be prepared according to conventional methods in the art.

The cosmetic composition according to one aspect of the present invention may include other ingredients that can give a synergistic effect to the main effect, preferably within the range of not impairing the main effect. In addition, the cosmetic composition according to an aspect of the present invention may further include a moisturizer, an emulsifier, a surfactant, a UV absorber, a preservative, a bactericide, an antioxidant, a pH adjuster, an organic or inorganic pigment, a perfume, a cooling agent, or a restriction agent. It may include. The blending amount of the above components can be easily selected by those skilled in the art within the range that does not impair the object and effect of the present invention, the blending amount is 0.01 to 5% by weight, specifically 0.01 to 3% by weight based on the total weight of the cosmetic composition Can be.

In one aspect of the invention the composition comprises a peptide having a sequence selected from the group consisting of SEQ ID NOs: 1 to 340, a peptide which is a fragment of the sequence of SEQ ID NO: 1 or a peptide having a sequence homology of at least 80% with the peptide sequence It provides a food composition for antioxidant comprising.

The formulation of the food composition according to one aspect of the present invention is not particularly limited, but may be, for example, formulated into tablets, granules, powders, solutions, solid preparations, and the like. Each formulation may be appropriately selected and formulated by those skilled in the art according to the formulation or purpose of use, in addition to the active ingredient, and may be synergistic when applied simultaneously with other raw materials.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term without the number before the noun is not intended to limit the quantity but rather to the presence of one or more of the mentioned noun articles. The terms "comprising", "having", and "comprising" are interpreted as open terms (ie, meaning "including but not limited to").

Note that referring to a range of numbers is simply an alternative to referring to each individual number within the range individually, unless it is stated that each individual number is referred to individually in the specification. Is incorporated herein as is. The end values of all ranges fall within that range and can be combined independently.

All methods mentioned herein may be performed in the proper order unless otherwise specified or clearly contradicted by context. The use of one embodiment and all embodiments or example language (eg, “such as”) is merely intended to better describe the invention, unless it is within the scope of the claims, and covers the scope of the invention. It is not intended to be limiting. No language in the specification should be construed as essential to the practice of the invention as to any unclaimed elements. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Preferred embodiments of the invention include the most optimal mode known to the inventors for carrying out the invention. Variations of the preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. The inventors expect those skilled in the art to make appropriate use of such variations, and the inventors expect the invention to be practiced in a manner different from that described herein. Accordingly, the invention includes all modifications and equivalents of the subject matter referred to in the appended claims, as permitted by patent law. Moreover, any combination of the abovementioned elements within all possible variations is included in the invention unless expressly stated to the contrary or apparently contradictory in context. While the invention has been particularly shown and described with reference to exemplary embodiments, those skilled in the art will understand that various changes in form and detail may be made without departing from the spirit and scope of the invention as defined by the following claims.

Hereinafter, the configuration and effects of the present invention will be described in more detail with reference to Examples and Experimental Examples. However, the following Examples and Experimental Examples are provided only for the purpose of illustration in order to help the understanding of the present invention, but the scope and scope of the present invention is not limited thereto.

Example 1 also increased after the hydrogen peroxide treatment of PEP in one neural stem cell survival Measure

The antioxidant effect of PEP 1 was used to demonstrate the effect of preventing the death of neural stem cells induced by hydrogen peroxide. That is, after culturing neural stem cells, treatment with hydrogen peroxide induced neurotoxicity, and to determine whether PEP 1 has a neuroprotective effect on neurotoxicity due to hydrogen peroxide treatment, the treated cells were treated with PEP 1, PI3K signaling pathway was analyzed.

실험예Experimental Example 1-1. 펩티드 PEP 1(서열번호 1)의 합성 1-1. Synthesis of Peptide PEP 1 (SEQ ID NO: 1)

A peptide consisting of 16 amino acids having the structural formula of Formula 1 having the following SEQ ID NO: 1 (PEP 1) selected from human telomerase was synthesized.

Peptide PEP 1 of SEQ ID NO: 1 could be prepared according to the known solid phase peptide synthesis method. Specifically, peptides were synthesized by coupling amino acids one by one from the C-terminus through Fmoc solid phase synthesis (SPPS) using ASP48S (Peptron, Inc., Daejeon, Korea). As follows, the first amino acid at the C-terminus of the peptides was attached to the resin. For example:

NH ₂ -Lys (Boc) -2-chloro-Trityl Resin

NH ₂ -Ala-2-chloro-Trityl Resin

NH ₂ -Arg (Pbf) -2-chloro-Trityl Resin

All amino acid feedstocks used for peptide synthesis were protected with N-term Fmoc and all residues removed from acid Trt, Boc, t-Bu (t-butylester), Pbf (2,2,4,6, 7-pentamethyl dihydro-benzofuran-5-sulfonyl) and the like were used. For example:

Fmoc-Ala-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc- Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Trp (Boc) -OH, Fmoc-Met-OH, Fmoc -Asn (Trt) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ahx-OH, Trt-Mercaptoacetic acid.

Coupling reagent is HBTU [2- (1H-Benzotriazole-1-yl) -1,1,3,3-tetamethylaminium hexafluorophosphate] / HOBt [N-Hydroxxybenzotriazole] / NMM [4-Methylmorpholine] It was. Fmoc removal was performed using piperidine in DMF in 20% of DMF. The cleavage cocktail (TFA (trifluoroacetic acid) / TIS (triisopropylsilane) / EDT (ethanedithiol) / H ₂ O = 92.5 / 2.5 / 2.5 / 2.5] was used to isolate the synthesized peptide from Resin and remove the protecting group of residues. It was.

Each peptide was synthesized by repeating a process of reacting the amino acids with each other, washing with a solvent, and then deprotecting the amino acid using the state in which the amino acid protecting group was bound to the solid support. The synthesized peptide was separated from the resin and then purified by HPLC, and confirmed by MS and lyophilized.

The specific synthesis process of PEP 1 is described as follows.

1) coupling

Amino acid (8 equivalents) protected by NH ₂ -Lys (Boc) -2-chloro-Trityl Resin and coupling reagent HBTU (8 equivalents) / HOBt (8 equivalents) / NMM (16 equivalents) were dissolved in DMF and added. Reaction was performed at room temperature for 2 hours, and washed with DMF, MeOH, and DMF in that order.

2) Fmoc deprotection

Piperidine in DMF at 20% was added thereto, reacted twice at room temperature for 5 minutes, and washed in the order of DMF, MeOH, and DMF.

3) Repeated reaction of 1 and 2, peptide basic skeleton NH ₂ -E (OtBu) -AR (Pbf) -PALLT (tBu) -S (tBu) -R (Pbf) LR (Pbf) -FIPK (Boc) -2-chloro-Trityl Resin).

4) Cleavage: The cleavage cocktail was added to the synthesized peptide Resin to separate the peptide from the resin.

5) Cooling diethyl ether is added to the mixture, followed by centrifugation to precipitate the peptide.

6) After purification by Prep-HPLC, the molecular weight was confirmed by LC / MS and frozen to prepare a powder.

실험예Experimental Example 1-2. PC12 세포 및 1-2. PC12 cells and 신경줄기세포의Neural stem cell 준비 Preparations

As normal cells, PC12 cells (pheochromocytoma cells) are derived from the rat adrenal cortex and induced differentiation of neurons as a nerve growth factor. The neural stem cells were isolated from the cerebral cortex from the head of the rat embryo on the 13th day of gestation, and then treated with basic fibroblast growth factor (bFGF) for 1 week.

More specifically, the PC-12 cell line derived from rat adrenal gland phaeochromocytoma used 88022401 Sigma, and the culture medium was RPMI 1640 + 2 mM Glutamine + 10%. Horse Serum + 5% FBS (Foetal Bovine Serum) was used. PC12 cells are 50-70% confluent for differentiation. Differentiation was done in DMEM-Hi containing 15% FBS supplemented with 50 ng / ml NGF and replaced with fresh NGF-containing medium every 2-3 days. Differentiation is completed in 5-7 days.

For neural stem cells, cerebral cortex was isolated from rat embryos on day 13 of gestation and treated with bFGF (Basic Fibroblast Growth Factor) for one week under the following culture conditions: Ca ² ⁺ / Mg ² ⁺ -free Fibronectin (1 μg / ml, Sigma, for at) after coating with HBSS (Hank's balanced salt solution), poly-ornithine (5 μg / ml, Sigma, St. Louis, MO at 37 ° C overnight) 12 mm glass coverslips (Bellco, Vineland, NJ) coated with polyornithine-fibronectin pretreated and coated with at least 2 h before use.

Experimental Example 1-3. Neurotoxicity Assessment of PEP 1 in PC12 Cells Damaged by Hydrogen Peroxide

In order to observe the neuroprotective effect of PEP 1 on neurotoxicity of hydrogen peroxide, the concentration of hydrogen peroxide was selected using MTT and CCK-8 (see FIG. 1), 200 μM hydrogen peroxide treatment, and 0.01 to 500 uM concentration. After treatment with PEP 1 at various concentrations for 48 hours, cell viability and killing were evaluated using MTT assay, CCK-8 assay, LDH assay, and TUNEL assay. In addition, the degree of proliferation of cells was analyzed using BrdU assay.

MMT Assay at 10 ⁴ in a 200 ml PBS - 10 were used ⁶⁹⁶ well plates containing the cells. 20 ml MTT solution was added and mixed well. Incubated for 4 hours at 37 ℃ temperature in the dark. Aliquots were prepared for analysis and mixed with 200 ml of acidic isopropanol. After further incubation for 1 hour at 37 ° C. in the dark, the OD value was analyzed at 570 nm in an ELISA plate reader (standard wavelength is 630 nm).

CCK-8 assays include WST-8 [2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium, monosodium salt] and 1- An assay for counting viable cell counts by binding methoxy PMS. That is, 10 μl of reagent kit was treated in the cell and incubated for 3 hours in a 96 well plate. Cell viability was analyzed at 450 nm in ELISA plate reader. All measurements normalized the optical density (OD) values compared to plates without cell culture.

In the LDH assay, LDH was quantified using a colorimetric assay kit (Roche Boehringer-Mannheim, Ind., USA), which was secreted from cultured neural stem cells. Cell viability was analyzed at 490 nm in ELISA plate reader and standard wavelength was 690 nm. All measurement results normalized the optical density (OD) values in comparison to plates without cell culture.

Apoptotic cell death was measured using TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling, Roche Boehringer-Mannheim, IN, USA) assay. To observe intact, compressed, fragmented nuclei, TUNEL stained cells were prepared using 4 ', 6-diamidino-2-phenylindole (DAPI, Sigma, Saint Louis, MO, USA; 100 μg / ml in PBS). Counterstained for 20 minutes and washed several times with PBS. The glass slides were then mounded using Moviol 4-88 solution (Calbiochem, Merck Biosciences, Darmstadt, Germany). Observation was made under fluorescence microscopy of Olympus under excitation wavelengths appropriate for TUNEL staining.

In the BrdU assay, cell proliferation was measured. Neural stem cells were collected for 48 hours of exposure to 20 μM of Aβ _25-35 (amyloid β 20-35 fragment) with different concentrations of PEP 1. The neural stem cells were incubated for 5 hours in BrdU-label medium (10 μM BrdU). Thereafter, cell proliferation was measured using BrdU Labeling and Detection Kit (Roche Boehringer-Mannheim, Ind., USA). Cell proliferation was analyzed at 370 nm in ELISA plate reader and standard wavelength was 492 nm. All measurements normalized the optical density (OD) values compared to plates without cell culture.

The effect of PEP 1 on inhibiting PC12 cell death by hydrogen peroxide was treated with 500uM hydrogen peroxide in various concentrations of PEP 1, and the survival rate of neurons was analyzed by CCK-8 assay. The increase was not confirmed (see Figure 2).

Experimental Example 1-4. Evaluation of Neurotoxicity of PEP 1 in Neuronal Stem Cells Damaged by Hydrogen Peroxide

Based on Experimental Examples 1-3, neurotoxicity was evaluated in the same manner except for changing the cells to confirm that the same results as PC12 cells in neural stem cells.

As described above, the concentration of hydrogen peroxide was selected as 200uM from CCK-8 and MTT assay (see FIG. 3), and 200uM hydrogen peroxide and various concentrations of PEP 1 were used to confirm whether PEP 1 inhibited neural stem cell death by hydrogen peroxide. After treatment, CCK-8 and MTT assays were used to confirm cell viability. As a result, the cell viability was increased depending on the concentration of 1 uM PEP 1. (See FIG. 4A.) Another method was performed LDH assay to evaluate the degree of cell death. As a result, it was confirmed that the apoptosis increased by hydrogen peroxide was effectively reduced by PEP 1, and the effect appeared from 1 μM concentration. . (See Figure 4B)

Through the BrdU assay, it was confirmed how the cell proliferation rate, which was reduced by oxidative damage, was changed by the co-treated PEP 1, and the results confirmed that the cell proliferation rate, which was reduced by the hydrogen peroxide treatment, was recovered by the treatment of PEP 1. (See Figure 5)

To confirm whether the increase in cell viability was related to cytotoxicity, TUNEL staining confirmed that the cytotoxicity increased by 200 uM hydrogen peroxide was reduced in a concentration-dependent manner. (See Figure 6)

실험예Experimental Example 1-5. PEP 1의 신경세포 이동성 평가 1-5. Neuronal Mobility Assessment of PEP 1

Cell mobility is a very important part of neural stem cells. After treatment with hydrogen peroxide and PEP 1 under various conditions, migration assays were performed to measure changes in cell mobility. Experimental results on cell mobility confirmed that the cell migration reduced by hydrogen peroxide was recovered by PEP 1 treatment, and at 10 μM concentration, the increased mobility was compared to that of the control group. It has been shown that there may be more effective results. (See Figure 7)

실험예Experimental Example 1-6. PEP 1의 항산화효과 평가 1-6. Evaluation of the Antioxidant Effect of PEP 1

The change in development after hydrogen peroxide and PEP 1 treatment was observed using DCF-DA stained samples (Molecular Probes Inc., Eugene, OR, USA). It was confirmed that free radicals were increased by 200 μM of hydrogen peroxide and decreased free radicals in the group treated with PEP 1. (See Figure 8)

Experimental Example 1-7. Change of expression level of protein related to PI3K pathway to confirm neuroprotective mechanism of PEP 1 that inhibits damage caused by hydrogen peroxide

To confirm the mechanism of action, the protein analysis (proteomics) showed that the proteins related to TCA cycle, cell migration, and GPCR signaling pathway in the group treated with PEP 1 were compared with the group treated with hydrogen peroxide only. It was confirmed that the regulation of the expression level of them was changed in a significantly positive direction, showing a neuroprotective effect. (See FIG. 9)

The protein analysis (proteomics) method is as follows.

단백질 샘플 준비Protein Sample Preparation

The cultured neural stem cell pellets were washed twice with cold PBS and sample lysis solution (7M urea, 2M Thiourea containing 4% (w / v) 3-[(3-cholamidopropy) dimethyammonio] -1 sonicated for 10 seconds in propanesulfonate (CHAPS), consisting of 1% (w / v) dithiothreitol (DTT), 2% (v / v) pharmalyte, and 1 mM benzamidine (using Sonoplus (Bandelin electronic, Germany)). ).

Protein was extracted by vortexing (vortexing) at room temperature for 1 hour. After centrifugation at 15,000 × g, 15 ° C. for 1 hour, insoluble components were removed and only the dissolved fractions were used for 2D gel electrophoresis (2D PAGE). Protein concentration can be analyzed by the Bradford method.

2D PAGE2D PAGE

IPG dry strips (4-10 NL IPG, 24 cm, Genomine, Korea) were treated with 2% CHAPS (3-[(3-cholamidopropy) as 7M urea, 2M thiourea for 12-16 hours. ) Equilibrated with solvent containing dimethyammonio] -1-propanesulfonate), 1% dithiothreitol (DTT), and 1% pharmalyte, and 200 μg of each sample was loaded. Isoelectric focusing (IEF) was performed using a Multiphor II electrophoresis unit and an EPS 3500 XL power supply (Amersham Biosciences) at 20 ° C. For the IEF, the sample was linearly increased from 150 to 3,500 volts over 3 hours and kept constant at 3,500 volts. When 96kVh was exceeded, it was made to end. Prior to two-dimensional electrophoresis, the strips were incubated for 10 minutes in equilibration buffer (containing 50 mM Tris-Cl, pH6.8, 6M urea, 2% SDS and 30% glycerol). The first was done with 1% DTT and the second with 2.5% iodoacetamide. Equilibrated strips were injected into SDS-PAGE gels (20 × 24 cm, 10-16%) and SDS-PAGE was performed using a Hoefer DALT 2D system (Amersham Biosciences). The 2D gel was operated at 1,700Vh at 20 ° C.

이미지 분석(Image analysis)Image analysis

Quantitative analysis of the images was performed using PDQuest (version 7.0, BioRad) software. Each spot was normalized by the total valid spot intensity.

PMFPMF (Peptide Mass Fingerprinting) (Peptide Mass Fingerprinting)

For protein identification through mass protein fingerprinting, protein spots were cut using trypsin, digested (Promega, Madison, WI) and CHCA (α cyano-4-hydroxycinnamic acid) was added to 50% acetonitrile / Mixed with 0.1% TFA, MALDI-TOF analysis (Microflex LRF 20, BrukerDaltonics) was performed as described by Fernandes J et al. Spectra were collected within 300-3000 m / z at 600 shots / spectrum and calibrated by two point internal calibration using the trypsin auto digestion peak (m / z 842.5099.2211.1046). Peak lists were generated using Flex Analysis 3.0. The threshold values used for peak-picking are as follows: the minimum resolution of monoisotopic mass is 500 and the S / N is 5. The MASCOT (http://www.matrixscience.com/), a search program developed by Matrixscience, is used. It was used for protein identification via protein fingerprinting. The following parameters are input factor values used for database searches: trypsin for cleavage enzyme, a maximum of one missed cleavage, iodoacetamide (Cys) for complete modification, Partial modifications include oxidation, monoisotopic masses, and mass error ± 0.1 Da. Probability scoring was used as the PMF acceptance criterion.

항체 Antibodies 마이크로어레이Microarray

Antibody microarrays were performed using a Panorama Antibody Microarray-Cell Signaling kit (Sigma-Aldrich). Briefly, 1.5 × 10 ⁷ cells were placed in a 100 mm dish and treated with 20 μM Aβ25-35 (beta amyloid 25-35) and 10 μM PEP 1 for 48 hours.

Collected cells and protein samples were prepared according to the manufacturer's protocol. These protein samples were labeled with either Cy3 or Cy5 (Amersham Biosciences, UK) and subjected to antibody microarray (Sigma-Aldrich) analysis. Array slides were scanned with a GenePix Personal 4100A scanner (Molecular Devices) and data analyzed with GenePix Pro 5.0 (Molecular Devices).

웨스턴Weston 블랏Blot (Western blot) 분석Western blot analysis

Ki67, PI3K (p85α phosphatidylinositol 3-kinase), pAkt (phosphorylated Akt, Ser473), GSK-3β (phosphorylated glycogen synthase kinase-3β, Ser9), HSTF) -1 (heat shock transcription factor-1), Bcl-2 ( B cell lymphoma-2), cytoplasmic high-mobility group protein 1 (HMGB-1), Bax, cytosolic cytochrome c and cleaved caspase-3 (Asp175) were analyzed by Western blot. Western blots of various intracellular signals were performed immediately after 48 hours of treatment. Briefly, 5 × 10 ⁶ cells were washed twice in cold PBS and lysed in lysis buffer ([50 mM Tris, pH 8.0), 150 mM NaCl, 0.02% sodium azide, 0.2% SDS, 100 μg on ice for 30 minutes. / ml phenyl methyl sulfonyl fluoride (PMSF), 50 μl / ml aprotinin, 1% Igepal 630, 100 mM NaF, 0.5% sodium deoxy choate, 0.5 mM EDTA, 0.1 mM EGTA] The nuclei were agglomerated by 2000 xg centrifugation for 10 minutes, and lysates were removed by 10,000 xg HMGB-1 cytoplasm and cytosol cytochrome c levels to assess mitochondria and cytos as directed by the manufacturer. The sol fraction was isolated using the Mitochondra / Cytosol Fractionation Kit (Abcam, UK) Briefly, 20 μM Aβ25-35 was treated with PEP 1 at various concentrations for 48 hours, then neural stem cells were collected and ice-cold PBS Washed once with DTT (dithiothreitol) and Re-mixed with 1.0 mL of 1 × cytosol extraction buffer containing protease inhibitors After incubating for 10 minutes on ice and above, the cell suspension was homogenized with a syringe for 30 to 50 hours. The silver was centrifuged at 3,000 rpm for 10 minutes at 4 ° C. The supernatants were again centrifuged at 13,000 rpm for 30 minutes to fractionate the mitochondrial fraction (pellets) and the cytosol fraction (supernatant). After washing once with isolation buffer, it was dissolved in mitochondrial cord files containing DTT and protease inhibitors.Samples containing the equivalent amounts of protein (20 μg) were 10% SDS-PAGE (sodium docecyl sulfate- It was digested with polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK). The nitrocellulose membrane was blocked with 5% skim milk and then incubated with certain primary antibodies. Antibodies used were anti-Ki67 (1: 200, Abcam, UK), anti-p85α PI3K (1: 1000, Millipore), anti-pAkt (Ser473) (1: 500, Cell Signaling, Beverly, MA, USA), anti-pGSK-3β (Ser9) (1: 1000, Santa Cruz Biotech, Santa Cruz, CA, USA), anti-HSTF-1 (H-311) (1: 1000, Santa Cruz, CA, USA), anti- Bcl-2 (1: 1000, Cell Signaling), anti-HMGB-1 (1: 500, Cell Signaling), anti-BAX (1: 1000, Cell Signaling), anti-cytochrome c (1: 200, Cell Signaling) , and anti-cleaved caspase-3 (Asp 175) (1: 1000, Cell signaling, Beverly, MA, USA). Membranes were washed with Tris-buffered saline containing 0.05% Tween-20 (TBST), and after ECL detection (Amersham Pharmacia Biotech), HRP-conjugated anti-rabbit antibody or anti-mouse antibody (Amersham Pharmacia Biotech, Piscataway, NJ, USA) The blots were quantified with an image analyzer (GE Healthcare, ImageQuant LAS 4000).

Based on the above analysis results, the effect of PEP 1 on Phosphatidylinositol-3-kinase (PI3K) / AKT signaling pathway, which plays a critical role in the growth and survival of neurons, was confirmed. The PI3K signaling pathway is activated by a variety of growth factors and regulators and is involved in the normal regulation of neuronal growth and survival. AKT signaling pathways inactivate several proapoptotic factors and inhibit GSK3β, a well known representative cell death signal.

Therefore, Western Experiment was carried out to confirm the effects of hydrogen peroxide and PEP 1 on the PI3K pathway in Experimental Example 1-7, the treatment of PEP 1 compared with the treatment of hydrogen peroxide alone Ki67, pAKT, PI3K, HSTF -1, Bcl-2 was increased, and apoptosis signal caspase-3, cytochrome-c, Bax and inflammatory response signal COX-2 were decreased. (See FIG. 10)

Experimental Example 1-8. Comparison of PEP 1 Neuroprotective Effects on Neural Stem Cells and Cortical Neurons

On the basis of the positive effects of PEP 1 obtained from neural stem cells, we tried to determine whether the same effect was seen in other neurons. Hydrogen peroxide and PEP 1 were treated in neural stem cells and primary cultured cortical neurons and the results were compared.

Similar to neural stem cells, the concentration of survival was increased in cortical neurons. In these two cells, PEP 1 was found to have an effect of inhibiting neuronal cell death (see FIG. 11).

실시예Example 2: 펩티드의 2: of peptide 활성산소종Reactive oxygen species 생성 produce 억제여부Suppression 측정 Measure

실험예Experimental Example 2-1. 2-1. 세포내Intracellular 활성 activation 산소종Oxygen species ((Reactive Oxygen Species )측정방법((Reactive Oxygen Species) Measurement Method

The DCF-DA method was used to determine if the production of reactive oxygen species (ROS) in the cell line is inhibited.

Jurkat cells (Jurkat, Clone E6-1 (ATCC ^® TIB-152 ™), a human T lymphocyte cell line, were injected into a 96-well plate with 1x10 ⁵ cells, followed by 1uM peptide and antioxidant NAC (N-acetyl-cysteine). After treatment for 1 hour at 2mM concentration, the medium was removed, washed two and three times with PBS, and then reacted with DCF-DA (Molecular Probes Inc., Eugene, OR, USA) 25mM at 37 ° C. After 30 minutes, Induce ROS with 4 mM H ₂ O ₂ for 30 minutes Neutralize with 100 ul ROS lysis buffer (2.5 mM KH 2 PO 4, 0.1 M EDTA, 0.1% Triton X-100) using Infinte M200 Tecan (Tecan Trading AG, Switzerland) analyzer ROS generation was measured at 485 nm (emission) / 535 nm (exitation).

Experimental Example 2-2. Intracellular Reactive Oxygen Species by Peptides in Human T Lymphocyte Cell Lines Impact on Development of Reactive Oxygen Species

In order to determine the effect on the antioxidant activity of peptides, H ₂ O ₂ was treated to peptide-treated cells using the same method as Experimental Example 2-1, and ROS was induced by intracellular reactive oxygen species.

As a result, it was observed that PEP 1 inhibited ROS induction by about 40% compared to cells treated with H ₂ O ₂ , while NAC, an antioxidant, was inhibited by about 30%. Accordingly, as a result of screening by measuring the antioxidant effect of the RIA series peptides against ROS based on PEP 1, peptides that inhibit ROS activity could be selected.

As a result of the ROS measurement, SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 24, sequence SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44 SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: SEQ ID NO: 75, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 85, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 101, SEQ ID NO: 108, SEQ ID NO: 109 , SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 12 5, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 186, SEQ ID NO: 189, SEQ ID NO: 192, SEQ ID NO: 197, SEQ ID NO: 200, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 210, SEQ ID NO: 240, SEQ ID NO: 272, SEQ ID NO: 273, SEQ ID NO: 284, SEQ ID NO: 286, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: No. 290, SEQ ID NO: 291, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO: 310, SEQ ID NO: 323, SEQ ID NO: 327, SEQ ID NO: 330, SEQ ID NO: 337 is stimulated by H ₂ O ₂ It was confirmed that there is an effect of lowering the ROS of the cells.

In addition, as a control, NAC, a well-known antioxidant, was compared, and as a result, SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 180, SEQ ID NO: 186, SEQ ID NO: 189, SEQ ID NO: 192, SEQ ID NO: 197, SEQ ID NO: 200, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 210, SEQ ID NO: 272, SEQ ID NO: 273, SEQ ID NO: 284, SEQ ID NO: 286, SEQ ID NO: 295, SEQ ID NO: 310, SEQ ID NO: 323, SEQ ID NO: 327, and SEQ ID NO: 337 were found to have an excellent effect of lowering ROS than NAC. In addition, it was confirmed that the effect may be used as an excellent antioxidant.

Claims

As a peptide having antioxidant activity,

A peptide comprising an amino acid sequence of any one or more of SEQ ID NOs: 1 to 340, a peptide having a sequence homology of 80% or more with the amino acid sequence, or a fragment thereof.
The peptide of claim 1, wherein the fragment is a fragment consisting of three or more amino acids.
The peptide of claim 1, wherein the peptide consists of up to 30 amino acids.
The method of claim 1. The peptide is a peptide consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 340.
The method of claim 1. The peptide is SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 24, SEQ ID NO: 25 , SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, sequence SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75 , SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 85, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 101, SEQ ID NO: 108, SEQ ID NO: 109, sequence SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 125, Western SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 186, SEQ ID NO: 189, SEQ ID NO: 192, SEQ ID NO: 197, SEQ ID NO: 200, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 210, SEQ ID NO: 240, SEQ ID NO: 272, SEQ ID NO: 273, SEQ ID NO: 284, SEQ ID NO: 286, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO: 310, SEQ ID NO: 323, SEQ ID NO: 327, SEQ ID NO: 330, SEQ ID NO: 337 Peptides comprising an amino acid sequence.
The method of claim 5. The peptide is SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 42 , SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 78, sequence SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 141 , SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 180, SEQ ID NO: 186, SEQ ID NO: 189, SEQ ID NO: 192, SEQ ID NO: 197, SEQ ID NO: 200, sequence SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 210, SEQ ID NO: 272, SEQ ID NO: 273, SEQ ID NO: 284, SEQ ID NO: 286, A peptide comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 295, SEQ ID NO: 310, SEQ ID NO: 323, SEQ ID NO: 327, SEQ ID NO: 337.
As a polynucleotide encoding a peptide having antioxidant activity,

A polynucleotide encoding a peptide comprising an amino acid sequence of any one of SEQ ID NOs: 1 to 340 or a peptide having a sequence homology of 80% or more with the amino acid sequence or a fragment thereof.
The polynucleotide of claim 7 wherein the peptide consists of up to 30 amino acids.
The polynucleotide of claim 7, wherein the peptide consists of an amino acid sequence of any one of SEQ ID NOs: 1-340.
An antioxidant composition comprising a peptide according to any one of claims 1 to 6 as an active ingredient as a composition for inhibiting oxidation or aging.
The antioxidant composition of claim 10, wherein the peptide consists of the amino acid sequence of any one of SEQ ID NOs: 1-340.
The antioxidant composition according to claim 10, wherein the composition is used for the treatment or prevention of diseases caused by peroxidation or free radicals.
The antioxidant composition of claim 10 wherein the composition is a pharmaceutical composition.
The antioxidant composition according to claim 10, wherein the disease due to peroxidation or free radicals is a cerebral nervous system or a cardiovascular disease.
The antioxidant composition of claim 14, wherein the cerebral nervous system or cardiovascular disease comprises one or more of Alzheimer's disease, Lou Gehrig's disease, Parkinson's disease, Creutzfeldt-Jakob disease, stroke, cerebral infarction and cerebral hemorrhage.
The antioxidant composition according to claim 10, wherein the composition is a cosmetic composition for preventing skin aging.
The antioxidant composition according to claim 10, wherein the composition is a cosmetic composition for skin whitening.
The antioxidant composition according to claim 10, wherein the composition is a health promotion food composition for preventing or inhibiting cellular aging and cellular oxidation in the human body.
A method of treating or preventing a disease caused by peroxidation or free radicals, characterized by administering the antioxidant composition according to any one of claims 10 to 15.
A method of preventing or inhibiting the oxidation or aging of cells due to peroxidation or active oxygen, characterized by administering the antioxidant composition according to claim 10.