Peptides having antioxidant activity and compositions containing the same

Abstract

The present invention provides a functional peptide with excellent antioxidant activity, which has excellent biocompatibility and significantly better antioxidant activity than conventional GSH, thereby overcoming the limitations of weak antioxidant activity of existing peptides. The peptide of the present invention can promote antioxidant activity or effectively repair biological tissue or cell damage caused by reactive oxygen species generated due to oxidative stress. Therefore, the peptide with such antioxidant activity effectively acts on cell damage caused by oxidative stress, and can therefore be used to prevent and repair cell damage.

Description

Technical Field

[0001] The present invention relates to a biocompatible peptide material having antioxidant activity, in particular to a peptide having free radical scavenging activity and having the function of promoting the expression of Nrf2 as an antioxidant transcription factor and promoting the expression of SOD2 as an antioxidant enzyme, and a composition containing the peptide. Background Art

[0002] Typically, cells produce harmful oxygen, such as reactive oxygen species (ROS) or free radicals, when they are heavily exposed to stress caused by intrinsic factors (e.g., respiration and energy metabolism in the body) generated by biological processes and external factors (e.g., pollutants, toxic substances, ultraviolet light, invasion of pathogens).

[0003] Normal cells use antioxidant enzymes to remove harmful oxygen through metabolic processes. However, continuous or severe stress leads to the accumulation of harmful oxygen in cells instead of removing it, causing inflammatory reactions or cell damage, which in turn leads to the onset of various diseases.

[0004] In addition, harmful oxygen may cause DNA damage, protein denaturation, enzyme inactivation, etc. in cells, and lipid peroxides diffuse into cells or move in the bloodstream, which promotes the generation of new free radicals, thereby promoting arteriosclerosis, cancer, myocardial infarction, stroke, heart disease, rheumatoid arthritis, chronic neurodegenerative diseases such as amyotrophic lateral sclerosis or Parkinson's disease (PD), diabetes, hepatitis, nephritis, atopic diseases, autoimmune diseases, inflammatory responses, aging, and similar phenomena in various tissues (Alam, MN et al., Saudi Pharm. J. 2013; Curr. Opin. Neurol., 9(4): 260-264, 1996).

[0005] Furthermore, such stress in cells is known to be the main cause of wrinkle formation or skin-related diseases such as atopic dermatitis, acne or skin cancer because it damages cells, cuts connective tissue of the dermis layer of the skin or causes cross-linking.

[0006] To this end, the body's antioxidant system can remove continuously generated free radicals and heal damaged cells. For example, glutathione (GSH) combines three amino acids (L-glutamic acid, L-cysteine ​​and L-glycine). Glutathione is synthesized by glutamine cysteine ​​ligase (GCL) and glutathione synthetase (GSS). It is known that glutathione is repeatedly oxidized and reduced by utilizing the activity of antioxidant enzymes such as glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST) and quinone oxidoreductase (NADPH: quinone oxidoreductase 1 (NQO1)), thereby exhibiting antioxidant efficacy or removing toxic substances. (Mates, JM et al., Clinical biochemistry. 1999; Vrankovic, J. et al., Turkish Journal of Biology. 2012).

[0007] In addition, the main biological defense mechanism in vivo against oxidative stress is based on the Nrf2 (nuclear factor erythroid 2-related factor 2)-ARE (antioxidant response element) signaling pathway present in cells, and the Nrf2-ARE signaling pathway promotes the synthesis of glutathione and antioxidant enzymes. Nrf2 is a transcription factor, which is known to remove reactive oxygen species by synthesizing antioxidant enzymes such as GPx, GR, GST, superoxide dismutase (SOD), catalase, NQO1 and heme oxygenase-1 (HO-1) under oxidative stress, or by regulating the expression of enzymes such as GCL and GS that synthesize glutathione antioxidants to increase the content of glutathione in cells. As a result, reactive oxygen species are removed or converted into non-erosive substances (Lee, JM et al., J.Biochem.Mol.Biol.2004).

[0008] In addition, Nrf2 increases the ability of cells to resist free radicals in various tissues. Therefore, when the expression of Nrf2 is promoted in various tissues, cell protection can be expected based on antioxidation (Baird, L. et al., Arch. Toxicol. 2011).

[0009] The most important factor in effectively maintaining the body's antioxidant defense mechanism is to normalize the production and reaction rate of the SOD enzyme, which mainly neutralizes superoxide anion free radicals, which are the strongest reactive oxygen species. However, with age, the production and reaction rate of the SOD enzyme in the human body decrease, so it is important to consume foods that supplement it. Among the antioxidant enzymes, SOD plays an important role in the intracellular antioxidant defense system, which reduces free radicals that are normally produced during cellular energy metabolism. In mammals, SOD2 is also called "mitochondrial Mn-SOD."

[0010] However, the normal function of the biological antioxidant system of humans is gradually lost due to metabolic syndrome or adult diseases, which are caused by various factors, including environmental stress, such as pollution or ultraviolet radiation or biological stress, aging and diseases associated with aging, such as degenerative diseases, economic growth, dietary changes, etc. In this case, it is necessary to apply antioxidants, that is, substances that can remove reactive oxygen by activating Nrf2 electronic factors, so as to provide antioxidants to prevent the antioxidant system from malfunctioning. In response to this requirement for the administration of antioxidants, efforts to administer antioxidants or antioxidant foods and drugs to thereby prevent or treat the above-mentioned diseases or delay aging have been made in various ways. So far, antioxidants that can meet these requirements have not yet been developed.

[0011] Under this background, due to repeated efforts to find food ingredients having an antioxidant effect without causing any side effects, the present inventors identified a peptide having a strong antioxidant effect based on the function of removing active oxygen by promoting the expression of transcription factor Nrf2, the function of removing active oxygen by promoting the expression of antioxidant enzyme SOD2, and the function of removing active oxygen by promoting the expression of both Nrf2 and SOD2. Based on this discovery, the present invention was completed. Summary of the invention

[0012] Technical issues

[0013] Therefore, the present invention has been completed in order to solve the above problems, and an object of the present invention is to provide a peptide that has no autoimmunity, degrades harmlessly after biological recovery, and exhibits excellent antioxidant activity, thereby improving free radical scavenging ability, promoting the expression of Nrf2 and SOD2, and enhancing antioxidant activity. Peptides with antioxidant activity can be used as therapeutic agents for diseases associated with oxidative damage to cells, and have antioxidant activity against oxidative stress by effectively acting on biological tissues or cells thereof.

[0014] Another object of the present invention is to provide an antioxidant composition and a method for preparing the same, wherein the antioxidant composition comprises at least one peptide having antioxidant activity and having an amino acid sequence represented by Formula 1 or Formula 2.

[0015] Another object of the present invention is to provide a pharmaceutical composition or a pharmaceutical composition for animals other than humans to prevent, treat or alleviate oxidative damage to cells containing the peptide as an active ingredient.

[0016] Another object of the present invention is to provide a method for preventing or treating oxidative damage to cells, comprising orally administering the peptide to humans or animals other than humans.

[0017] Another object of the present invention is to provide a novel use of the peptide in preparing a medicine or veterinary drug for preventing or treating oxidative damage to cells.

[0018] Technical Solution

[0019] According to one aspect of the present invention, the above and other objects can be achieved by providing an antioxidant composition comprising a peptide having antioxidant activity and having an amino acid sequence represented by Formula 1 or Formula 2:

[0020] [General formula 1]

[0021] γ-Glu-Xaa 1

[0022] [General formula 2]

[0023] γ-Glu-Xaa 2 -Xaa 3

[0024] in

[0025] XA 1 any one of alanine (A), cysteine ​​(C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), leucine (L), asparagine (N), proline (P), glutamine (Q), arginine (R), threonine (T), valine (V), valine amide (V-NH2), valinol (2-amino-3-methyl-1-butanol) (V-ol), tryptophan (W), tyrosine (Y), ornithine (ORN), methionine sulfoxide (M(O)), cysteine ​​in which the hydrogen of the sulfhydryl group is substituted with a methyl group (C(S-Me)), cysteine ​​(S-Me) (O2) in which the hydrogen of the sulfhydryl group is substituted with a methyl group, and 2-aminobutyric acid (Abu);

[0026] XA 2 any one of cysteine ​​(C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), leucine (L), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), valine amide (V-NH2), valinol (2-amino-3-methyl-1-butanol) (V-ol), tryptophan (W), tyrosine (Y), ornithine (ORN), methionine sulfoxide (M(O)), cysteine ​​in which the hydrogen of the sulfhydryl group is substituted with a methyl group (C(S-Me)), cysteine ​​(S-Me) (O2) in which the hydrogen of the sulfhydryl group is substituted with a methyl group, and 2-aminobutyric acid (Abu); and

[0027] XA 3 It is any one amino acid selected from alanine (A), cysteine ​​(C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), lysine (K), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), valine (V) and ornithine (ORN).

[0028] The peptide may include at least one peptide selected from: γ-GLU-ALA, γ-GLU-CYS, γ-GLU-CYS(S-Me), γ-GLU-CYS(S-Me)(O2), γ-G LU-ASP, γ-GLU-GLU, γ-GLU-PHE, γ-GLU-GLY, γ-GLU-HIS, γ-GLU-ILE, γ-GLU-LEU, γ-GLU-MET( O), γ-GLU-ASN, γ-GLU-ORN, γ-GLU-PRO, γ-GLU-GLN, γ-GLU-ARG, γ-GLU-THR, γ-GLU-VAL, γ-GL U-VAL-NH2, γ-GLU-TRP, γ-GLU-TYR, γ-GLU-VAL-ol, γ-GLU-GLN-GLU, γ-GLU-GLU-GLU, γ-GLU- ILE-GLY, γ-GLU-PRO-GLY, γ-GLU-GLN-GLN, γ-GLU-SER-GLY, γ-GLU-THR-GLY, γ-GLU-VAL-GLY , γ-GLU-VAL-ALA, γ-GLU-VAL-CYS, γ-GLU-VAL-ASP, γ-GLU-VAL-GLU, γ-GLU-VAL-PHE, γ-GLU- VAL-HIS, γ-GLU-VAL-LYS, γ-GLU-VAL-MET, γ-GLU-VAL-ASN, γ-GLU-VAL-ORN, γ-GLU-VAL-PRO , γ-GLU-VAL-ARG, γ-GLU-VAL-VAL, γ-GLU-CYS(S-Me)-GLY, γ-GLU-Abu-GLY and γ-GLU-LEU-GLY.

[0029] The peptide may include at least one dipeptide selected from the following: γ-GLU-ALA, γ-GLU-CYS, γ-GLU-CYS(S-Me), γ-GLU-CYS(S-Me)(O2), γ-GLU-GLY, γ-GLU-HIS, γ-GLU-ILE, γ-GLU-LEU, γ-GLU-ASN, γ-GLU-PRO, γ-GLU-GLN, γ-GLU-THR, γ-GLU-VAL-NH2, γ-GLU-TRP, γ-GLU-TYR, and γ-GLU-VAL-ol.

[0030] The said peptide can include at least one kind of peptide: γ-GLU-CYS、γ-GLU-CYS(S-Me)、γ-GLU-CYS(S-Me)(O2), γ-GLU-GLY、γ-GLU-HIS、γ-GLU-ILE、γ-GLU-GLN、γ-GLU-TRP and γ-GLU-TYR。

[0031] The peptide may include at least one tripeptide selected from the following: γ-GLU-GLN-GLU, γ-GLU-ILE-GLY, γ-GLU-PRO-GLY, γ-GLU-SER-GLY, γ-GLU-THR-GLY, γ-GLU-VAL-CYS, γ-GLU-VAL-ASP, γ-GLU-VAL-GLU, γ-GLU-VAL-PHE, γ-GLU-VAL-MET, γ-GLU-VAL-ASN, and γ-GLU-VAL-ARG.

[0032] The peptide may include at least one selected from the following: γ-GLU-CYS(S-Me)(O2), γ-GLU-HIS, γ-GLU-CYS, γ-GLU-CYS(S-Me), γ-GLU-GLY, γ-GLU-ILE, γ-GLU-GLN, γ-GLU-TRP, γ-GLU-TYR, γ-GLU-GLN-GLU, γ-GLU-ILE-GLY, γ-GLU-PRO-GLY, γ-GLU-SER-GLY, γ-GLU-THR-GLY, γ-GLU-VAL-CYS, γ-GLU-VAL-ASP, γ-GLU-VAL-GLU, γ-GLU-VAL-PHE, γ-GLU-VAL-MET, γ-GLU-VAL-ASN, and γ-GLU-VAL-ARG.

[0033] The peptide may include at least one selected from the group consisting of γ-GLU-CYS(S-Me)(02), γ-GLU-HIS, γ-GLU-ILE, γ-GLU-GLN-GLU, γ-GLU-THR-GLY and γ-GLU-MAL-CYS.

[0034] According to another aspect of the present invention, there is provided a method for preparing an antioxidant composition, the method comprising:

[0035] a) adding enzymes to the yeast suspension and performing hydrolysis; and

[0036] b) recovering a peptide fraction having a molecular weight of 500 Da or less from the yeast hydrolysate obtained after the hydrolysis.

[0037] Step a) may include: a-1) adding 0.5 wt % to 1.5 wt % of alkaline endoprotease (Alcalase) relative to the solid content of the yeast suspension, and performing primary hydrolysis for 3 to 5 hours;

[0038] a-2) adding 0.5 wt % to 1.5 wt % of a complex flavor enzyme (Flavourzyme) relative to the solid content of the primary hydrolysate, and performing secondary hydrolysis for 5 to 15 hours; and

[0039] a-3) adding 0.1 to 0.5 wt % of alkaline endoprotease and complex flavor protease relative to the solid content of the secondary hydrolysate, and performing secondary hydrolysis for 20 to 30 hours.

[0040] According to another aspect of the present invention, a pharmaceutical composition, a food composition, a feed composition and a cosmetic composition for treating oxidative damage to cells are provided, wherein the composition comprises at least one peptide selected from the amino acid sequences shown in Formula 1 and Formula 2 as an active ingredient.

[0041] The generation of free radicals or reactive oxygen species (ROS) may lead to oxidative damage to cells.

[0042] According to another aspect of the present invention, there is provided a use of at least one peptide selected from the amino acid sequences represented by Formula 1 and Formula 2 above in the preparation of a medicament for preventing or treating oxidative damage to cells.

[0043] According to another aspect of the present invention, a method for treating oxidative damage to cells is provided, the method comprising orally administering to a human or non-human animal at least one peptide selected from the amino acid sequences shown in the above general formula 1 and general formula 2.

[0044] The generation of free radicals or reactive oxygen species (ROS) may lead to oxidative damage to cells.

[0045] Beneficial Effects

[0046] The present invention relates to a functional peptide having excellent antioxidant activity, and is provided in the form of a peptide having excellent biocompatibility and antioxidant activity, which is significantly superior to conventional GSH peptides, thereby overcoming the limitation of low antioxidant activity of the prior art. The peptide of the present invention effectively promotes the recovery of damage to biological tissues or cells caused by free radicals caused by oxidative stress, or promotes antioxidant activity. Therefore, this peptide with antioxidant activity can be used to prevent and restore cell damage by effectively acting on cell damage caused by oxidative stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1a is a graph showing DPPH scavenging ability, Figure 1b The reducing ability of each yeast hydrolysate (A4, AF10, AF24) is shown, wherein the control is a yeast hydrolysate without enzyme treatment, A2 is a yeast hydrolysate lysed with alkaline endoprotease for 2 hours, A4 is a yeast hydrolysate lysed with alkaline endoprotease for 4 hours, AF10 is a yeast hydrolysate lysed with alkaline endoprotease for 4 hours and then with complex flavor protease for 10 hours, and AF24 is a yeast hydrolysate lysed with alkaline endoprotease for 4 hours, then with complex flavor protease for 10 hours, and further with complex flavor protease for 24 hours.

[0048] Figure 2a is a graph showing the ability to remove DPPH, Figure 2b It is a graph showing the reducing ability of each yeast hydrolysate fraction (Preparation Examples 1 to 4), wherein the control is a yeast hydrolysate not treated with the enzyme, 10 kDa or more is the fraction of the yeast hydrolysate of Preparation Example 1, 10 kDa to 5 kDa is the fraction of the yeast hydrolysate of Preparation Example 2, 5 kDa to 1 kDa is the fraction of the yeast hydrolysate of Preparation Example 3, and 1 kDa or less is the fraction of the yeast hydrolysate of Preparation Example 4.

[0049] Figure 3 : is a graph showing secondary fractions (I, II and III) according to molecular weight determined based on the molecular weight distribution of the filtrate of the yeast hydrolyzate of 1 kDa or less selected from Experimental Example 2, in which peptides having antioxidant activity were screened using high performance liquid chromatography (HPLC).

[0050] Figures 4a to 4d The figure shows the MS / MS spectra and predicted structures of representative peptides identified from the secondary fraction II having the best antioxidant activity in yeast hydrolysate, wherein A is γ-Glu-Cys-Gly (GSH), B is γ-Glu-Val-Gly, C is γ-Glu-Cys, and D is γ-Glu-Val.

[0051] Figure 5a to Figure 5c is a graph showing the results of real-time PCR for detecting the expression level of Nrf2 mRNA in 100 peptides identified from yeast hydrolysate.

[0052] Figure 6 is a graph showing the results of real-time PCR for detecting the expression level of SOD2 mRNA in 100 peptides identified from yeast hydrolysate.

[0053] Best Mode for Carrying Out the Invention

[0054] Hereinafter, the present invention will be described in detail.

[0055] Representative amino acids and their abbreviations are as follows: alanine (Ala, A), isoleucine (Ile, I), leucine (Leu, L), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), tryptophan (Trp, W), valine (Val, V), asparagine (Asn, N), cysteine ​​(Cys, C), glutamine (Gln, Q), glycine (Gly, G), serine (Ser, S), threonine (Thr, T), tyrosine (Try, Y), aspartic acid (Asp, D), glutamic acid (Glu, E), arginine (Arg, R), histidine (His, H) and lysine (Lys, K).

[0056] In addition, the modified amino acids and their abbreviations are as follows: valine amide (V-NH2), valinol (2-amino-3-methyl-1-butanol) (V-ol), ornithine (ORN), methionine sulfoxide (M(O)), cysteine ​​(C(S-Me)) in which the hydrogen of the sulfhydryl group is replaced by a methyl group, cysteine ​​(S-Me) (O2) in which the hydrogen of the sulfhydryl group is replaced by a methyl group and has a sulfoxide structure, and 2-aminobutyric acid (Abu). In particular, (O2) in the above formula represents a sulfoxide structure.

[0057] In the present invention, a peptide refers to a linear molecule formed by amino acid residues being bonded to each other via peptide bonds.

[0058] The peptides of the present invention can be prepared according to chemical synthesis methods known in the art, particularly solid phase synthesis techniques (Merrifield, J. Amer. Chem. Soc. 85: 2149-54 (1963); Stewart et al., Solid Phase Peptide Synthesis, 2nd. ed., Pierce Chem. Co.: Rockford, 111 (1984)).

[0059] As used herein, the term "antioxidant activity" refers to the property of inhibiting cellular oxidation caused by highly reactive free radicals or reactive oxygen species when exposed to oxidative stress due to environmental stress or biological stress such as intracellular metabolism or ultraviolet rays, including the property of removing free radicals or reactive oxygen species to reduce the damage to cells caused thereby.

[0060] As used herein, the term "antioxidant composition" refers to a composition comprising antioxidant peptides in an amount of 20 wt % to 50 wt %, preferably 30 wt % to 40 wt %, relative to the total weight of the composition. The composition may further comprise a pharmaceutically acceptable carrier, excipient or diluent, which will be apparent to those skilled in the art.

[0061] In one aspect, the present invention relates to an antioxidant composition comprising at least one peptide selected from the group consisting of the amino acid sequences represented by the following general formula 1 and general formula 2:

[0062] [General formula 1]

[0063] γ-Glu-Xaa 1

[0064] [General formula 2]

[0065] γ-Glu-Xaa 2 -Xaa 3

[0066] in

[0067] XA 1 any one of alanine (A), cysteine ​​(C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), leucine (L), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), valinol (2-amino-3-methyl-1-butanol) (V-ol), tryptophan (W), tyrosine (Y), ornithine (ORN), methionine sulfoxide (M(O)), cysteine ​​in which the hydrogen of the sulfhydryl group is substituted with a methyl group (C(S-Me)), cysteine ​​(S-Me) (O2) in which the hydrogen of the sulfhydryl group is substituted with a methyl group, and 2-aminobutyric acid (Abu);

[0068] XA 2any one of cysteine ​​(C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), leucine (L), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), valinol (2-amino-3-methyl-1-butanol) (V-ol), tryptophan (W), tyrosine (Y), ornithine (ORN), methionine sulfoxide (M(O)), cysteine ​​in which the hydrogen of the sulfhydryl group is substituted with a methyl group (C(S-Me)), cysteine ​​(S-Me) (O2) in which the hydrogen of the sulfhydryl group is substituted with a methyl group, and 2-aminobutyric acid (Abu); and

[0069] XA 3 It is any one amino acid selected from alanine (A), cysteine ​​(C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), lysine (K), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), valine (V) and ornithine (ORN).

[0070] In the present invention, general formulas are provided to illustrate the peptide structures of the present invention, and it is obvious to those skilled in the art that modified peptides thereof also fall within the scope of the present invention.

[0071] In one embodiment, the N-terminus or C-terminus of the peptide of the present invention can be modified by a hydroxyl (-OH) or an amino (-NH2). The modification of the amino acid can greatly improve the stability of the peptide of the present invention. Here, the term "stability" refers to in vivo stability and storage stability (e.g., room temperature storage stability). The protecting group can protect the peptide of the present invention from the attack of the in vivo protease.

[0072] Yeast hydrolysate is obtained through a series of enzymatic hydrolysis steps, peptides are separated from the yeast hydrolysate through primary and secondary separation and purification, and then the effects of promoting the expression of Nrf2 and SOD2 mRNA between the peptides are compared, thereby obtaining the peptides with antioxidant activity according to the present invention. The peptide has higher Nrf2-mRNA and SOD2-mRNA promoting activity than natural GSH peptides, and despite having a short sequence (composed of 2 to 3 amino acid residues), it still shows significantly excellent antioxidant effects (ability to scavenge DPPH, reducing ability, ability to promote the expression of Nrf2-mRNA, and ability to promote the expression of SOD2-mRNA), and can therefore be used in various fields requiring antioxidant activity.

[0073] Therefore, the peptide with antioxidant activity according to the present invention can be provided in the form of a composition for removing reactive oxygen and preventing or treating oxidative damage to cells, tissues or organs caused by free radicals caused by oxidative stress. In addition, the antioxidant composition can be provided as a feed, feed additive or food additive, which has the effect of preventing or treating cell damage caused by reactive oxygen.

[0074] The peptide having the amino acid sequence represented by Formula 1 or Formula 2 according to the present invention has a short sequence of 2 to 3 amino acids by sequentially bonding amino acid residues to the γ chain of L-glutamic acid.

[0075] Specifically, the peptide having the amino acid sequence represented by Formula 1 or Formula 2 may have antioxidant activity and include at least one selected from the group consisting of γ-GLU-ALA, γ-GLU-CYS, γ-GLU-CYS(S-Me), γ-GLU-CYS(S-Me)(O2), γ-GLU-ASP, γ-GLU-GLU, γ-GLU-PHE, γ-GLU-GLY, γ-GLU-HIS, γ-GLU-ILE, γ- GLU-LEU, γ-GLU-MET(O), γ-GLU-ASN, γ-GLU-ORN, γ-GLU-PRO, γ-GLU-GLN, γ-GLU-ARG, γ-GLU-THR, γ -GLU-VAL, γ-GLU-VAL-NH2, γ-GLU-TRP, γ-GLU-TYR, γ-GLU-VAL-ol, γ-GLU-GLN-GLU, γ-GLU-ALA-GLY , γ-GLU-GLU-GLU, γ-GLU-ILE-GLY, γ-GLU-PRO-GLY, γ-GLU-GLN-GLN, γ-GLU-SER-GLY, γ-GLU-THR-G LY, γ-GLU-VAL-GLY, γ-GLU-VAL-ALA, γ-GLU-VAL-CYS, γ-GLU-VAL-ASP, γ-GLU-VAL-GLU, γ-GLU-VAL -PHE, γ-GLU-VAL-HIS, γ-GLU-VAL-LYS, γ-GLU-VAL-MET, γ-GLU-VAL-ASN, γ-GLU-VAL-ORN, γ-GLU-V AL-PRO, γ-GLU-VAL-ARG, γ-GLU-VAL-VAL, γ-GLU-CYS(S-Me)-GLY, γ-GLU-Abu-GLY and γ-GLU-LEU-GLY.

[0076] In one embodiment, the peptide is preferably a peptide of Formula 1, wherein Xaa 1It is any one of the amino acids selected from alanine (A), cysteine ​​(C), glycine (G), histidine (H), isoleucine (I), leucine (L), asparagine (N), proline (P), glutamine (Q), threonine (T), valine amide (V-NH2), valinol (2-amino-3-methyl-1-butanol) (V-ol), tryptophan (W), tyrosine (Y), cysteine ​​in which the hydrogen of the sulfhydryl group is substituted by a methyl group (C(S-Me)), and cysteine ​​in which the hydrogen of the sulfhydryl group is substituted by a methyl group and has a sulfoxide structure (S-Me) (O2).

[0077] Specifically, the peptide of general formula 1 may include at least one dipeptide selected from the group consisting of γ-GLU-ALA, γ-GLU-CYS, γ-GLU-CYS(S-Me), γ-GLU-CYS(S-Me)(O2), γ-GLU-GLY, γ-GLU-HIS, γ-GLU-ILE, γ-GLU-LEU, γ-GLU-ASN, γ-GLU-PRO, γ-GLU-GLN, γ-GLU-THR, γ-GLU-VAL-NH2, γ-GLU-TRP, γ-GLU-TYR and γ-GLU-VAL-ol, and has an antioxidant effect and an effect of promoting the expression of Nrf2 mRNA and SOD2 mRNA, and although its sequence is shorter than that of conventional GSH with an antioxidant effect, it has a comparable or higher effect and is therefore still more useful in synthesis, preparation and storage.

[0078] More preferably, the peptide is a peptide of Formula 1, wherein Xaa 1 It is any one of the amino acids selected from cysteine ​​(C), glycine (G), histidine (H), isoleucine (I), glutamine (Q), tryptophan (W), tyrosine (Y), cysteine ​​(C(S-Me)) in which the hydrogen of the sulfhydryl group is replaced by a methyl group, and cysteine ​​(S-Me)(O2) in which the hydrogen of the sulfhydryl group is replaced by a methyl group and has a sulfoxide structure. Compared with GSH, which is well known to have an antioxidant effect, the peptide has a significantly improved effect of promoting the expression of Nrf2 and SOD2, in particular, at least one dipeptide selected from γ-GLU-CYS, γ-GLU-CYS(S-Me), γ-GLU-CYS(S-Me)(O2), γ-GLU-GLY, γ-GLU-HIS, γ-GLU-ILE, γ-GLU-GLN, γ-GLU-TRP and γ-GLU-TYR.

[0079] In one embodiment, in the peptide of Formula 2, Xaa 2 is any one of the amino acids selected from glycine (G), isoleucine (I), proline (P), serine (S), threonine (T) and valine (V), Xaa 3It is any one of amino acids selected from cysteine ​​(C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), methionine (M), asparagine (N) and arginine (R). Compared with GSH, the peptide has a significantly improved effect of promoting the expression of both Nrf2 and SOD2, and the peptide is particularly selected from at least one tripeptide of γ-GLU-GLN-GLU, γ-GLU-ILE-GLY, γ-GLU-PRO-GLY, γ-GLU-SER-GLY, γ-GLU-THR-GLY, γ-GLU-VAL-CYS, γ-GLU-VAL-ASP, γ-GLU-VAL-GLU, γ-GLU-VAL-PHE, γ-GLU-VAL-MET, γ-GLU-VAL-ASN and γ-GLU-VAL-ARG.

[0080] In one embodiment, the peptide having a significantly improved effect of promoting the expression of both Nrf2 and SOD2 compared to GSH, more preferably, is characterized in that Xaa in the general formula 1 1 is any one of amino acids selected from cysteine ​​(C), glycine (G), histidine (H), isoleucine (I), glutamine (Q), tryptophan (W), tyrosine (Y), cysteine, cysteine ​​in which the hydrogen of the sulfhydryl group is replaced by a methyl group (C(S-Me)), and cysteine ​​(S-Me)(O2) in which the hydrogen of the sulfhydryl group is replaced by a methyl group and has a sulfoxide structure, wherein Xaa in the general formula 2 2 is any one of the amino acids selected from isoleucine (I), proline (P), glutamine (G), serine (S), threonine (T) and valine (V), Xaa 3 It is any one amino acid selected from the group consisting of cysteine ​​(C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), methionine (M), asparagine (N) and arginine (R). Specifically, the peptide preferably includes at least one selected from the group consisting of: γ-GLU-CYS(S-Me)(O2), γ-GLU-HIS, γ-GLU-CYS, γ-GLU-CYS(S- Me), γ-GLU-GLY, γ-GLU-ILE, γ-GLU-GLN, γ-GLU-TRP, γ-GLU-TYR, γ-GLU-GLN-GLU, γ-GLU- ILE-GLY, γ-GLU-PRO-GLY, γ-GLU-SER-GLY, γ-GLU-THR-GLY, γ-GLU-VAL-CYS, γ-GLU-VAL-ASP, γ-GLU-VAL-GLU, γ-GLU-VAL-PHE, γ-GLU-VAL-MET, γ-GLU-VAL-ASN, and γ-GLU-VAL-ARG.

[0081] Most preferably, in Formula 1, Xaa 1 is any one of histidine (H), isoleucine (I) and cysteine ​​in which the hydrogen of the sulfhydryl group is replaced by a methyl group and has a sulfoxide structure (S-Me)(O2). In Formula 2, Xaa 2 is any one of the amino acids selected from glutamine (Q), threonine (T) and valine (V), Xaa 3 It is any one amino acid selected from the group consisting of cysteine ​​(C), glutamic acid (E) and glycine (G).

[0082] Specifically, the peptide may include at least one peptide selected from the group consisting of γ-GLU-CYS (S-Me) (O2), γ-GLU-HIS, γ-GLU-ILE, γ-GLU-GLN-GLU, γ-GLU-THR-GLY and γ-GLU-VAL-CYS. The peptide has an antioxidant effect (reducing ability, DPPH scavenging ability and Nrf2 mRNA activity), and an effect of increasing the expression levels of Nrf2 mRNA and SOD2 mRNA to about 2 to 3 times that of the positive control GSH.

[0083] Meanwhile, γ-GLU-HIS had a significant effect of increasing the mRNA expression levels of Nrf2 and SOD2 by 2 to 8 times compared with GSH.

[0084] The antioxidant composition of the present invention can be used as a pharmaceutical composition, a health food composition, a feed, a feed additive, a cosmetic composition, or a pharmaceutical composition for animals other than humans.

[0085] As used herein, the term "antioxidant activity" refers to the property of inhibiting cellular oxidation caused by highly reactive free radicals or reactive oxygen species due to exposure to environmental stress or biological stress caused by intracellular metabolism or oxidative stress caused by external factors (such as ultraviolet rays), including the activity of scavenging free radicals or reactive oxygen species to reduce the damage caused to cells thereby. The effects related thereto are confirmed by in vitro experiments.

[0086] The antioxidant composition comprises antioxidant peptides in an amount of 20 wt % to 50 wt %, preferably 30 wt % to 40 wt %, relative to the total weight of the composition. The composition may further comprise a pharmaceutically acceptable carrier, excipient or diluent, which is obvious to those skilled in the art.

[0087] In another aspect, the present invention relates to a method for preparing an antioxidant composition comprising a peptide fraction, the method comprising:

[0088] a) adding enzymes to the yeast suspension and performing hydrolysis; and

[0089] b) recovering a peptide fraction having a molecular weight of 500 Da or less from the yeast hydrolysate obtained after the hydrolysis.

[0090] Step a) preferably comprises: a-1) adding 0.5 wt% to 1.5 wt% of alkaline endoprotease relative to the solid content of the yeast suspension, and performing primary hydrolysis for 3 to 5 hours;

[0091] a-2) adding 0.5 wt % to 1.5 wt % of a complex flavor protease relative to the solid content of the primary hydrolysate, and performing secondary hydrolysis for 5 to 15 hours; and

[0092] a-3) adding 0.1 wt % to 0.5 wt % of alkaline endoprotease and complex flavor protease relative to the solid content of the secondary hydrolyzate, and performing secondary hydrolysis for 20 to 30 hours. When yeast hydrolyzate is prepared by the above method, a fraction containing a large amount of peptides having excellent antioxidant effects can be obtained.

[0093] The hydrolysis is preferably performed at a temperature of 40° C. to 70° C. When the hydrolysis is performed at a temperature below 40° C., the hydrolysis does not proceed well, and the secretion of antioxidant metabolites that resist physical stress cannot be sufficiently induced, so that sufficient peptides cannot be obtained. When the temperature range exceeds 70° C., the problem of apoptosis may occur.

[0094] The pH during hydrolysis is preferably between 6 and 7.

[0095] In step b), specifically, the yeast hydrolysate obtained after the hydrolysis is fractionated based on molecular weight to recover a peptide fraction having a molecular weight of 500 Da or less.

[0096] The method may further comprise, prior to step (b), removing residues from the yeast hydrolysate, and the removal of residues may be performed by known methods for removing solids from a mixture, such as centrifugation and filtration. Preferably, the method is centrifugation.

[0097] Prior to step (b), the method of the present invention may further include inactivating the enzymes contained in the yeast hydrolysate. The inactivation is performed before and / or after the removal of the residue, and is preferably performed before step (b) by inactivating the enzymes contained in the yeast hydrolysate, removing the residue by centrifugation, and separating the supernatant. The inactivation is preferably performed by heat treatment, and may be performed by keeping the hydrolysate at a temperature of 100° C. to 120° C. for 10 to 30 minutes.

[0098] In step (b), a peptide fraction having a molecular weight of 500 Da or less is obtained, and the fraction can be obtained by a known method for separating substances based on molecular weight, such as dialysis, electrophoresis, various column chromatography, etc. The chromatography for removing unwanted substances can be ion exchange chromatography, gel permeation chromatography, HPLC, reverse phase HPLC, affinity column chromatography, ultrafiltration or a combination thereof. Preferably, the method for obtaining the fraction of the present invention can be carried out by removing substances falling outside the target molecular weight range by dialysis using a dialysis membrane.

[0099] The molecular weight of the obtained fraction is 1000 Da or less, preferably 100 Da to 700 Da. More preferably, the molecular weight of the fraction may be 500 Da or less.

[0100] In addition, the peptide having antioxidant activity may be present in an amount of 70 to 85 weight % relative to the total weight of the obtained fraction.

[0101] In another aspect, the present invention relates to a pharmaceutical composition for treating oxidative damage to cells, the pharmaceutical composition containing at least one peptide selected from the amino acid sequences represented by Formula 1 and Formula 2 above as an active ingredient.

[0102] In another aspect, the present invention relates to a pharmaceutical composition or a pharmaceutical composition for administration to animals other than humans to prevent, treat or reduce oxidative damage to cells, wherein the pharmaceutical composition contains at least one peptide selected from the amino acid sequences represented by general formula 1 and general formula 2 above as an active ingredient.

[0103] In another aspect, the present invention relates to a method for preventing or treating oxidative damage to cells, which comprises orally administering to a human or an animal other than a human at least one peptide selected from the amino acid sequences represented by the above general formula 1 and general formula 2.

[0104] In another aspect, the present invention relates to a novel use of at least one peptide selected from the amino acid sequences represented by Formula 1 and Formula 2 above in the preparation of a medicament or veterinary drug for preventing or treating oxidative damage to cells.

[0105] As used herein, the term "oxidative damage to cells" refers to cell damage caused by highly reactive free radicals or reactive oxygen species generated due to exposure to environmental stress or biological stress caused by intracellular metabolism or oxidative stress caused by external factors such as ultraviolet rays.

[0106] As used herein, the term "containing as an active ingredient" means containing the peptide of the present invention in an amount sufficient to achieve the efficacy or activity of improving, treating or preventing oxidative damage to cells.

[0107] As used herein, the term "prevention" may refer to any effect of inhibiting or delaying the onset of oxidative damage to cells by administering a composition according to the present invention to a subject. As used herein, the term "treatment" may refer to any effect of improving or alleviating symptoms caused by oxidative damage to cells by administering a composition according to the present invention to a subject suspected of suffering from oxidative damage to cells.

[0108] The types of diseases caused by oxidative damage to cells that can be prevented or treated by the present invention are not limited, and are preferably diseases associated with damage to cells caused by internal or external factors such as intracellular metabolism or external factors such as ultraviolet rays due to highly reactive free radicals or active oxygen generated when exposed to oxidative stress.

[0109] Diseases associated with oxidative damage to cells include at least one selected from the group consisting of: neurodegenerative diseases such as multiple sclerosis, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and muscular dystrophy; ocular diseases such as retinopathy, macular degeneration, and cataracts; ischemic diseases such as myocardial infarction and cerebral infarction; inflammatory diseases selected from the group consisting of arthritis, vasculitis, glomerulonephritis, and lupus erythematosus; and atherosclerosis.

[0110] In the present invention, it was found that at least one peptide selected from the amino acid sequences represented by the above-mentioned general formula 1 and general formula 2 promoted and increased the expression of Nrf2 mRNA and SOD2 mRNA, which is an antioxidant defense mechanism for cells subjected to oxidative stress by tBHP (direct-acting oxidative stress inducer) treatment. In addition, the peptide can be reduced by tBHP, significantly increasing cell viability. That is, it was found that the added pharmaceutical composition of the present invention increased the enzymatic activity of the antioxidant, indicating that the pharmaceutical composition induces the expression of the antioxidant, which is essentially an enzyme, thereby enabling cell adaptation.

[0111] At least one peptide selected from the amino acid sequences represented by Formula 1 and Formula 2 has the remarkable advantages of increasing cellular antioxidant activity, inhibiting cell damage caused by oxidative stress due to internal or external factors, and being non-cytotoxic and stable at the administered dose.

[0112] In addition to the peptide as an active ingredient, the "drug", "pharmaceutical composition for animals (veterinary composition)" or "drug for animals (veterinary drug)" may also contain suitable additives, carriers, excipients or diluents commonly used in the preparation of pharmaceutical compositions. Based on the total weight of the composition, the pharmaceutically acceptable additive, carrier, excipient or diluent according to the present invention may be present in an amount of 0.1 to 90 parts by weight, but is not limited thereto.

[0113] The additives that can be used in the present invention can be starch, gelatinized starch, microcrystalline cellulose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, toffee, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, opary, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, white sugar, glucose, sorbitol, talc, etc.

[0114] The term "carrier" refers to a compound that facilitates the delivery of a compound into cells or tissues. The term "diluent" refers to a compound that stabilizes the biologically active state of a target compound and dissolves in water to dilute the compound.

[0115] The carrier, excipient or diluent is not particularly limited and may be, for example, lactose, glucose, sugar, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc.

[0116] The dosage of the drug, veterinary composition or veterinary drug may vary depending on the age, sex and weight of the patient or animal in need of treatment, and will vary depending on the condition of the subject in need of treatment, the specific species or type of disease to be treated, the route of administration and the nature of the therapeutic agent used.

[0117] In actual clinical administration, the pharmaceutical composition of the present invention can be administered in any of various oral and parenteral preparations. Preparations can be prepared using diluents or excipients such as fillers, extenders, adhesives, wetting agents, disintegrants and surfactants. Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc., and may be prepared by mixing the peptide with at least one excipient such as starch, calcium carbonate, sucrose, lactose or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, oral liquids and solutions, emulsions, syrups, etc. In addition to commonly used simple diluents water and liquid paraffin, liquid preparations may also include various excipients such as wetting agents, sweeteners, spices and preservatives. Preparations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. As non-aqueous solvents and suspending agents, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate can be used. As bases for suppositories, Witepsol, polyethylene glycol, Tween 61, cocoa butter, lauric acid esters, glycerin gelatin, etc. can be used.

[0118] The pharmaceutical composition of the present invention can be administered orally or parenterally according to the desired method, and parenteral administration is preferably external application to the skin, intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection.

[0119] The dosage of the pharmaceutical composition of the present invention varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of the disease. Based on the amount of the peptide of the present invention, the daily dosage is 0.0001 mg / kg to 100 mg / kg, preferably 0.001 mg / kg to 10 mg / kg, and the pharmaceutical composition can be administered 1 to 6 times a day.

[0120] The pharmaceutical composition of the present invention can be used alone or in combination with surgery, radiotherapy, hormone therapy, chemotherapy and methods using biological response modifiers.

[0121] The method of administering the pharmaceutical composition of the present invention is not particularly limited, and may be any invasive or non-invasive administration method, including oral administration, injection, etc., and may be suppository administration or transdermal administration. The active ingredient may be administered in the form of a conventional pharmaceutical preparation together with a solid or liquid pharmaceutical carrier suitable for an administration method such as oral administration and injection. Examples of such preparations include solid preparations such as tablets, granules, powders, and capsules; liquid preparations such as solutions, suspensions, and emulsions; and lyophilized preparations. These preparations can be prepared by conventional formulation methods. In addition, the pharmaceutical composition of the present invention may optionally contain any pharmaceutically or physiologically acceptable solid or liquid carrier, additive, etc.

[0122] Examples of carriers include glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glycerides, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid esters, gelatin, albumin, amino acids, water, physiological saline, etc. In addition, if necessary, conventional additives such as stabilizers, wetting agents, emulsifiers, binders and isotonic agents can be appropriately added to the pharmaceutical composition of the present invention.

[0123] The method of treatment comprises orally or parenterally administering the composition to a human or an animal other than a human, particularly a mammal, for example, orally administering the composition to a subject in need of treatment, wherein the cell damage is caused by internal or external oxidative stress, or wherein the cell damage is caused by other factors.

[0124] The dosage, method of administration and administration of the treatment may be determined with reference to the dosage, method of administration and number of administrations of the pharmaceutical composition, medicament, veterinary composition or veterinary drug.

[0125] In another aspect, the present invention relates to an antioxidant food composition comprising at least one peptide selected from the amino acid sequences represented by Formula 1 and Formula 2 above as an active ingredient.

[0126] The peptide can be used to prepare a food composition in the form of a capsule, tablet, powder, granule, liquid, pill, flake, paste, syrup, gel, jelly or stick preparation. Alternatively, a general food can be prepared by adding the food composition to food materials such as beverages, tea, spices, chewing gum, candy, etc. Food has a specific effect on health when consumed, but unlike general medicines, since food is used as a raw material, there are no side effects that may occur when taking medicine for a long time, which is an advantage.

[0127] The food composition is very useful because it can be eaten every day. Although the amount of peptides cannot be consistently defined relative to the type of target food, the amount of peptides that can be added to such food compositions is generally 0.01% to 50% by weight, preferably 0.1% to 20% by weight, of the target food, without changing the original taste of the food. In addition, foods provided in the form of capsules, tablets, powders, granules, liquids, pills, flakes, pastes, syrups, gels, jellies or sticks are generally added in an amount of 0.1% to 100% by weight, preferably 0.5% to 80% by weight.

[0128] The food composition may comprise at least one peptide represented by Formula 1 or Formula 2 as an active ingredient, and ingredients commonly used in food production, such as proteins, carbohydrates, fats, nutrients, seasonings and flavoring agents. Examples of carbohydrates include: monosaccharides, such as glucose and fructose; disaccharides, such as maltose, sucrose and oligosaccharides; polysaccharides, such as common sugars, such as dextrin and cyclodextrin, and sugar alcohols, such as xylitol, sorbitol and erythritol.

[0129] As flavoring agents, natural flavoring agents (sweet protein, stevia extracts (such as rebaudioside A and glycyrrhizin) and synthetic flavoring agents (such as saccharin and aspartame) can be used. For example, when the food composition of the present invention is produced in the form of beverages and drinks, it can also contain citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, various plant extracts, etc. in addition to the peptide of the present invention.

[0130] The food compositions of the present invention include human food, beverages as well as animal feed and feed additives.

[0131] In addition, in an animal feed additive containing at least one peptide represented by Formula 1 or Formula 2 obtained by the present invention as an active ingredient, the peptide includes a polysaccharide and is produced in a powder form to produce an animal feed additive, and the animal feed additive is additionally added to an animal feed. At this time, the animal feed can be selected from animal feed powder, calf milk replacer and piglet feed, and the peptide content based on dry weight is 1g to 2g (about 0.1%) relative to 1kg of feed.

[0132] The food and health food composition of the present invention may contain the peptide of the present invention alone or in combination with other foods or food ingredients, and may be appropriately used according to conventional methods.

[0133] In another aspect, the present invention relates to an antioxidant cosmetic composition comprising at least one peptide selected from the group consisting of the amino acid sequences represented by Formula 1 and Formula 2 as an active ingredient.

[0134] The preparation of the antioxidant cosmetic composition can be selected from skin external ointment, cream, softening lotion, nourishing lotion, packaging, essence, hair tonic, shampoo, rinse, conditioner, hair cream, gel, skin care lotion, skin softener, skin toner, lotion, milk emulsion, water emulsion, nutrition emulsion, massage cream, nutrition cream, eye cream, moisturizing cream, hand cream, mask, foundation, nutrition essence, sunscreen, soap, cleaning foam, cleaning milk, cleaning cream, body lotion and body cleanser, but not limited to this. The composition of each preparation can include various substrates and additives necessary and appropriate for preparing the preparation, and those skilled in the art can easily select the type and amount of these components.

[0135] When the preparation of the present invention is a paste, cream or gel, animal fiber, plant fiber, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicon dioxide, talc or zinc acid can be used as a carrier component.

[0136] When the preparation of the present invention is a powder or spray, lactose, talc, silicon dioxide, aluminum hydroxide, calcium silicate or polyamide powder can be used as a carrier component. In particular, when the preparation is in the form of a spray, a propellant such as chlorofluorocarbon, propane / butane or dimethyl ether may also be included.

[0137] When the preparation of the present invention is a solution or emulsion, a solvent, a solubilizer or an emulsifier, such as water, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol oil, aliphatic glycerides, polyethylene glycol or fatty acid esters of sorbitan, etc. are used as carrier components.

[0138] When the preparation of the present invention is a suspension, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitan ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth and the like may be used as a carrier component.

[0139] When the preparation of the present invention is in the form of a surfactant-containing cleanser, aliphatic alcohol sulfates, aliphatic alcohol ether sulfates, sulfosuccinic acid monoesters, isethionates, imidazolines Derivatives, methyl taurates, sarcosinates, fatty acid amide ether sulfates, alkylaminobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glyceryl fatty acid esters can be used as carrier components. DETAILED DESCRIPTION

[0140] Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. However, it will be apparent to those skilled in the art that the following embodiments are provided only to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0141] Experimental Example 1: Screening of low molecular weight peptides with antioxidant activity from yeast hydrolysate

[0142] 1. Preparation of yeast hydrolysate

[0143] The pH value of yeast (Saccharomyces cerevisiae) cells was adjusted to 7.0, heated to 60°C, and an endopeptidase (alkaline endoprotease) was added in an amount of 1% relative to the solid content, followed by enzymatic hydrolysis for 2 hours or 4 hours ("A2" or "A4" sample). After adding 1% of exopeptidase (complex flavor protease) to the sample (A4) cleaved with alkaline endoprotease compared to the solid content, enzymatic hydrolysis was performed for 10 hours ("AF10" sample). After a total of 10 hours of cleavage, 0.125% of alkaline endoprotease and complex flavor protease relative to the solid content were added thereto at 55°C, followed by enzymatic hydrolysis for 24 hours ("AF24" sample). The cleaved supernatant was inactivated at 85°C for 30 minutes, and peptides with a molecular weight of less than 10 kDa were separated by an ultrafiltration process. The alkaline endoprotease and complex flavor protease used herein were purchased from Novozymes.

[0144] The antioxidant efficacy (DPPH) and reducing capacity of each yeast hydrolysate (A4, AF10, AF24) were measured.

[0145] 2. DPPH (2,2-diphenyl-1-picryl-hydrazide-hydrate) determination method

[0146] The antioxidant activity due to the DPPH scavenging ability of yeast hydrolysates was measured using the methods of Choi Chang-seop et al. (2003) and Shekhar et al. (2014). 20 μl of sample (1 μg / ml to 5000 μg / ml) dissolved in distilled water was added to 180 μl of ethanol solution containing DPPH (0.111 mM). The resulting mixture was reacted at 25°C for 30 minutes, and the absorbance was measured at 517 nm. A standard curve was drawn with ascorbic acid, and the DPPH scavenging ability (%) was calculated and expressed as [((absorbance without sample - absorbance of sample) / absorbance without sample] × 100.

[0147] 3. Reducing power determination method

[0148] The reducing power of yeast hydrolysate was measured using the method of Oyaizu (1986). Specifically, a sample (1 μg / ml to 5000 μg / ml, 60 μl) dissolved in distilled water was mixed with a phosphate buffer (150 μl, 0.2 M, pH 6.6) and potassium ferricyanide (K3Fe(CN)6, 150 μl, 1%), and then reacted at 50°C for 20 minutes. In addition, 150 μl of trichloroacetic acid (10%) was added thereto, followed by centrifugation at 2000 g for 10 minutes. 150 μl of the supernatant containing the sample was added to 150 μl of DW and FeCl3 (30 μl, 0.1%). The resulting mixture was reacted at 25°C for 10 minutes, and the absorbance was measured at 700 nm.

[0149] 4. Conclusion

[0150] Figure 1a is a graph showing DPPH scavenging ability, Figure 1b The reducing capacity (b) of each yeast hydrolysate (A4, AF10 and AF24) is shown, wherein the control is a yeast hydrolysate not treated with enzymes, A2 is a yeast hydrolysate lysed with alkaline endoprotease for 2 hours, A4 is a yeast hydrolysate lysed with alkaline endoprotease for 4 hours, AF10 is a yeast hydrolysate lysed with alkaline endoprotease for 4 hours and then with complex flavor protease for 10 hours, and AF24 is a yeast hydrolysate lysed with alkaline endoprotease for 4 hours, then with complex flavor protease for 10 hours, and further with complex flavor protease for 24 hours.

[0151] like Figure 1a and 1b As shown in Figure 2, it was found that the AF24 sample treated with alkaline endoprotease and complex flavor protease in sequence and cleaved at multiple stages showed the best DPPH scavenging ability and reducing ability. That is, it can be seen that the DPPH scavenging ability ( Figure 1a ) and reduction capability ( Figure 1b ) may vary depending on the enzyme and time. Therefore, in the present invention, the AF24 sample was used to screen for peptides having an antioxidant effect.

[0152] Experimental Example 2: Preliminary separation of yeast hydrolysate based on molecular weight and analysis of antioxidant activity of each fraction

[0153] In order to isolate and select peptides with excellent antioxidant activity from AF24 samples and the best antioxidant activity among yeast hydrolysates, fractions were prepared from AF24 yeast hydrolysates based on their molecular weight (size), and their oxidative potency (DPPH) and reducing ability were measured and compared.

[0154] 1. Preparation of yeast hydrolysate fractions

[0155] Specifically, the AF24 yeast hydrolysate was filtered through ultrafiltration membranes having sizes of 10 kDa, 5 kDa and 1 kDa, water was removed from the filtrate of each fraction at 105°C for 48 hours, and the dry weight was measured to obtain the contents shown in the following Table 1. As can be seen from Table 1, most fractions of the yeast hydrolysate were substances having a size of 1 kDa or less.

[0156] Table 1

[0157] project Molecular weight of yeast fraction (Daltons) content(%) Preparation Example 1 10000 or more 0.3 Preparation Example 2 10000 to 5000 1.2 Preparation Example 3 5000 to 1000 15.2 Preparation Example 4 1000 or less 83.3

[0158] 2. Antioxidant activity test of yeast hydrolysate fractions based on molecular weight (size)

[0159] Using the same method as that for measuring the DPPH scavenging ability and reducing ability in Experimental Example 1, the DPPH scavenging ability and reducing ability of each yeast hydrolyzate fraction were measured (Preparation Examples 1 to 4).

[0160] Figure 2a is a graph showing the ability to remove DPPH, Figure 2b It is a graph showing the reducing power (b) of each yeast hydrolyzate fraction (Preparation Examples 1 to 4), wherein the control is a yeast hydrolyzate not treated with the enzyme, 10 kDa or more is the fraction of the yeast hydrolyzate of Preparation Example 1, 10 kDa to 5 kDa is the fraction of the yeast hydrolyzate of Preparation Example 2, 5 kDa to 1 kDa is the fraction of the yeast hydrolyzate of Preparation Example 3, and 1 kDa or less is the fraction of the yeast hydrolyzate of Preparation Example 4.

[0161] from Figure 2a and Figure 2bIt can be seen that the fraction of 5 kDa or less showed a DPPH scavenging ability of 6% or more, and the fraction of 1 kDa or less (Preparation Example 4) showed the highest antioxidant activity, especially 3 to 8 times higher antioxidant activity. It can be seen that similar to the DPPH scavenging ability, the fraction of 1 kDa or less (Preparation Example 4) also showed significantly excellent reducing ability (antioxidant activity), especially 4 times or more higher reducing ability (antioxidant activity). Therefore, in the present invention, the yeast hydrolyzate filtrate of 1 kDa or less was selected to screen peptides with antioxidant effects.

[0162] Experimental Example 3: Secondary separation of yeast hydrolysate filtrates of 1 kDa or less based on molecular weight

[0163] After the yeast hydrolyzate filtrate of 1 kDa or less was subjected to secondary separation according to molecular weight, the antioxidant activity was measured to select the final sample for screening of peptides with antioxidant activity.

[0164] 1. HPLC analysis

[0165] The molecular weight distribution of the yeast hydrolyzate filtrate of 1 kDa or less selected in Experimental Example 2 was determined, and secondary separation was performed based on the molecular weight distribution. For this purpose, the molecular weight distribution of the yeast hydrolyzate filtrate of 1 kDa or less was detected using high performance liquid chromatography (HPLC, Agilent 1200 series) and GPC method system software. The HPLC conditions are shown in Table 2 below.

[0166] Specifically, after 10 μl of 1 kDa or less yeast hydrolyzate filtrate was injected into an Agilent PLaquagel-OH 20 column, HPCL was performed at a flow rate of 1 ml per minute for 20 minutes. The mobile phase used here was a mixture of water, acetonitrile and trifluoroacetic acid in a ratio of 50:50:0.1, and the sample was detected at a wavelength of 220 nm.

[0167] Table 2

[0168] column Agilent PL aquagel-OH 20 (5μm, 7.5mm ID x 300mm L) Mobile phase <![CDATA[Water∶CH3CN∶TFA = 50∶50∶0.1]]> Injection volume 10μl Flow rate 1.0ml / min wavelength 220nm

[0169] 2. Conclusion

[0170] Figure 3 : is a graph showing secondary fractions (I, II and III) obtained according to molecular weight based on the molecular weight distribution of the yeast hydrolyzate filtrate of 1 kDa or less selected in Experimental Example 2, wherein detection was performed using high performance liquid chromatography (HPLC) to screen for peptides having antioxidant activity.

[0171] like Figure 3As shown, the total molecular weight of the yeast hydrolyzate filtrate of 1 kDa or less selected in Experimental Example 2 is 1 kDa (1000 Dalton) or less. It was found that the yeast hydrolyzate filtrate contained 15.1% of peptides with a molecular weight of 500 Dalton or more (secondary fraction I), 81.9% of peptides with a molecular weight of 500 Dalton or less (secondary fraction II), and 3.0% of amino acids (secondary fraction III). Therefore, the secondary fraction II with the greatest antioxidant activity was finally selected to screen peptides with antioxidant activity, and peptides were identified from the secondary fraction II.

[0172] Experimental Example 4: Identification of 100 peptides with antioxidant activity from secondary fraction II

[0173] In order to screen antioxidant functional peptides from the finally selected secondary fraction II, peptides present in the secondary fraction II were identified and the antioxidant activities were compared among them.

[0174] 1. Peptide identification

[0175] The amino acid sequence of the peptide was analyzed using a mass spectrometer LC MS / MS system (Waters, USA), and the conditions and method are shown in Table 3 below.

[0176] Specifically, 5 μl of the final selected secondary fraction II was injected into the LC MS / MS system and separated using a BEH C4 column. 100% solvent A was injected as the mobile phase for 1 minute at a flow rate of 0.2 ml / min, and then solvent A was adjusted from 100% to 99% within 9 minutes. Then, solvent A was reduced from 99% to 0% within 0.5 minutes, and solvent A was adjusted to 100% again within the last 4.5 minutes. Mass spectrometry was performed using electrospray ionization in the positive mode in the range of 50 m / z to 1200 m / z. Its working coefficients were as follows: ion source temperature: 120°C; ion source temperature: 120°C; desolvation temperature: 400°C; MassLynx 4.1 software (Waters, USA) was used for sample analysis.

[0177] Table 3

[0178]

[0179] 2. Conclusion

[0180] Figures 4a to 4d The figure shows the MS / MS spectra and predicted structures of representative peptides identified from the secondary fraction II having the best antioxidant activity in yeast hydrolysate, wherein A is γ-Glu-Cys-Gly (GSH), B is γ-Glu-Val-Gly, C is γ-Glu-Cys, and D is γ-Glu-Val.

[0181] like Figures 4a to 4d As shown, the results indicated that a total of 100 peptides were identified from the secondary fraction II obtained from yeast hydrolysate with the best antioxidant activity.

[0182] Experimental Example 5: Comparison of Nrf2 mRNA expression levels among 100 peptides

[0183] 1. Synthesis of 100 peptides

[0184] The 100 peptides identified were prepared using solid phase synthesis technology and analyzed using an LC-MS / MS mass spectrometer system (Waters, USA). Then, in order to accurately determine whether each synthesized peptide has the same structure as each identified and designed peptide, the molecular weight was extracted in XIC mode based on the monoisotopic value of the peptide indicator material (1.0 ppm or less), and two or more product ions corresponding to the MS / MS phase were found. All samples and indicators were measured in full scan and positive mode.

[0185] 2. Analysis of the antioxidant activity (Nrf2 mRNA expression level) of 100 peptides

[0186] In the present invention, the antioxidant activities among 100 peptides were compared, and the changes in Nrf2 mRNA expression were measured by PCR to select the final peptides.

[0187] (1) Cell culture and treatment of 100 peptides

[0188] HEK293 cells (kidney cells) were cultured at 6.6×10 5 Cells were seeded in DMEM (Dulbecco's modified Eagle's medium) medium on a 6-well plate at a density of 100 cells / well and cultured at 37°C for 24 hours under 5% CO2. The next day, the medium was changed to a serum-free and amino acid-free medium, and the cells were further cultured for 20 hours or longer. The cells were treated with Tert-BHP (300 μM) and 500 μM 100 peptides and then cultured for 18 hours.

[0189] (2) RNA extraction

[0190] RNA was extracted from cells treated with the 100 peptides described in (1) using TRIzol (Thermo Fisher Scientific). TMcDNA was synthesized from RNA using the Reverse Transcription System (Promega, WI, USA) by annealing at 25°C for 5 min, extending at 42°C for 60 min, and denaturing at 70°C for 15 min with 1 μg RNA, 0.5 μg oligo dt primer, 0.5 μg random primers, and RT-PCR premix.

[0191] (3) cDNA synthesis and PCR

[0192] Using Maxime TM PCR was performed using PCR PreMix (i-StarTaq) (Intron Biotechnology, Korea). TM Universal Green Supermix (Bio-Rad, USA) was used for real-time PCR. 100 ng cDNA, 2 μM (pmol / μl) primers, 10 μl 2X SsoAdvanced TM Universal Green Supermix and DW were added to the PCR plate wells. Then, the temperature cycle was adjusted to 95°C for 3 min, 95°C for 10 sec, and 58°C for 40 sec, for a total of 40 cycles. TM The PCR reaction was performed using a real-time PCR detection system (Bio-Rad, CA, USA), and the fluorescence intensity was measured at the end of each cycle. The primer sequences used for PCR analysis to detect Nrf2 expression are shown in Table 4 below.

[0193] Table 4

[0194]

[0195] 3. Conclusion

[0196] Figure 5a to Figure 5c is a graph showing the results of real-time PCR for detecting the expression level of Nrf2 mRNA in 100 peptides identified from yeast hydrolysate. Figures 5a to 5c Controls, treatment with tBHP only (a direct acting oxidative stress inducer) as a negative control, treatment with tBHP followed by GSH peptide as a positive control and the names of the 100 peptides corresponding thereto are shown (see Tables 5 and 6).

[0197] A total of 100 peptides obtained in Experimental Example 4 were identified. The Nrf2 mRNA expression levels among the peptides were compared. As a result, a total of 48 peptides with increased Nrf2 mRNA amounts were selected, and their Nrf2 mRNA expression levels were quantitatively measured. The results are shown in Figures 5a to 5c The chemical formulas and structural formulas of the 48 selected peptides and the remaining unselected peptides are shown in Tables 5 and 6 below. Table 5 shows the sequences of the 48 selected peptides. Table 6 shows the sequences of the unselected peptides.

[0198] Table 5

[0199]

[0200]

[0201] Table 6

[0202]

[0203]

[0204] In Table 6, α-GLU is glutamic acid, β-GLU is β-glutamic acid, Nle is norleucine, Nva is norvaline, tL or tLEU is tert-leucine, and βA or βALA is β-alanine. In addition, the sequence represented by γ-GLU means that the amino acid residues are sequentially bonded to the γ chain of L-glutamic acid.

[0205] That is, a total of 100 peptides were identified from yeast hydrolysate, but a total of 48 peptides having antioxidant activity that promoted Nrf2 mRNA expression were detected.

[0206] The growth of normal cells treated with tBHP as an oxidant was inhibited by oxidative stress. When the cells treated with tBHP were treated with a peptide having an antioxidant effect, the oxidation and growth inhibition of the cells caused by tBHP were reduced.

[0207] like Figure 5a to Figure 5cAs shown, 48 peptides (γ-GLU-ALA, γ-GLU-CYS, γ-GLU-CYS(S-Me), γ-GLU-CYS(S-Me)(O2), γ-GLU-ASP, γ-GLU-GLU, γ-GLU-PHE, γ-GLU-GLY, γ-GLU-HIS, γ-GLU-ILE, γ-GLU-LEU, γ-GLU-MET(O), γ-GLU-ASN, γ-GLU-ORN, γ-GLU-PRO, γ-GLU-GLN, γ-GLU-ARG, γ-GLU-THR, γ-GLU-VAL, γ-GLU-VAL-NH2, γ-GLU-TRP, γ-GLU-TYR, γ-GLU-VAL-ol, γ-GLU-GLN-GLU, γ-GLU-ALA-GLY, γ-GLU-GLU-GLU, γ-GLU-I LE-GLY, γ-GLU-PRO-GLY, γ-GLU-GLN-GLN, γ-GLU-SER-GLY, γ-GLU-THR-GLY, γ-GLU-VAL-GLY, γ-GLU-VAL-ALA, γ-GLU-VAL-CYS, γ-GLU-VAL-ASP, γ-GLU-VAL-GLU, γ-GLU-VAL-PHE, γ-GLU-VAL-HIS, γ-GLU-VAL-LYS, γ-GLU-VAL-MET, γ-GLU-VAL-ASN, γ-GLU-VAL-ORN, γ-GLU-VAL-PRO, γ-GLU-VAL-ARG, γ-GLU-VAL-VAL, γ-GLU-CYS(S-Me)-GLY, γ-GLU-Abu-GLY and γ-GLU-LEU-GLY) can increase the expression of Nrf2 mRNA.

[0208] In addition, among the 48 peptides, there is an increase in Nrf2 mRNA expression to make it equivalent or better to the positive GSH control peptide was identified as γ-GLU-ALA、γ-GLU-CYS、γ-GLU-CYS(S-Me)、γ-GLU-CYS(S-Me)(O2), γ-GLU-ASP、γ-GLU-GLU、γ-GLU-PHE、γ-GLU-GLY、γ-GLU-HIS、γ-GLU-ILE、γ-GLU -LEU、γ-GLU-ASN、γ-GLU-ORN、γ-GLU-PRO、γ-GLU-GLN、γ-GLU-ARG、γ-GLU-THR、γ-GLU-VAL、γ-GLU-VAL-NH2、γ-GLU-TRP、γ-GLU-TYR、γ-GLU-VAL-ol,although the sequence is shorter than GSH,but still shows comparable or better effect to GSH.

[0209] In addition, among the 48 peptides, the tripeptides that were identified as having a significantly improved Nrf2 mRNA expression effect compared to the positive control GSH were γ-GLU-GLN-GLU, γ-GLU-ILE-GLY, γ-GLU-PRO-GLY, γ-GLU-THR-GLY, γ-GLU-VAL-CYS, γ-GLU-VAL-ASP, γ-GLU-VAL-GLU, γ-GLU-VAL-PHE, γ-GLU-VAL-MET, γ-GLU-VAL-ASN and γ-GLU-VAL-ARG. More preferably, it can be seen that the same concentration of γ-GLU-CYS (S-Me) (O2) or γ-GLU-HIS increases the expression level of Nrf2 mRNA by four times or more than four times compared to the positive control GSH (the expression of Nrf2 mRNA is restored to normal levels). In particular, α-GLU-HIS and β-GLU-HIS had no effect compared to γ-GLU-HIS.

[0210] Experimental Example 6: Analysis of SOD2 mRNA expression levels of 100 peptides

[0211] The expression of SOD2 mRNA as another antioxidant factor was determined. Cell line culture, RNA extraction, cDNA synthesis and PCR experiments were performed in the same manner as in Experimental Example 5 above. A detailed description is given below.

[0212] 1. Analysis of the antioxidant activity (SOD2 mRNA expression level) of 100 peptides in Experimental Example 5

[0213] (1) Cell culture and treatment of 100 peptides

[0214] HEK293 cells (kidney cells) were cultured at 6.6×10 5 Cells were seeded in DMEM (Dulbecco's modified Eagle's medium) medium on a 6-well plate at a density of 100 cells / well and cultured at 5% CO2 and 37°C for 24 hours. The next day, the medium was changed to a serum-free and amino acid-free medium, and the cells were further cultured for 20 hours or longer. The cells were treated with Tert-BHP (300 μM) and 500 μM 100 peptides and then cultured for 18 hours.

[0215] (2) RNA extraction

[0216] RNA was extracted from cells treated with the 100 peptides described in (1) using TRIzol (Thermo Fisher Scientific). TMcDNA was synthesized from RNA using the Reverse Transcription System (Promega, WI, USA). cDNA was synthesized by annealing at 25°C for 5 min, extending at 42°C for 60 min, and denaturing at 70°C for 15 min with 1 μg RNA, 0.5 μg oligo dt primer, 0.5 μg random primer, and RT-PCR premix.

[0217] (3) cDNA synthesis and PCR

[0218] Using Maxime TM PCR was performed using PCR PreMix (i-StarTaq) (Intron Biotechnology, Korea). TM Universal Green Supermix (Bio-Rad, USA) was used for real-time PCR. 100 ng cDNA, 2 μM (pmol / μl) primers, 10 μl 2X SsoAdvanced TM Universal GreenSupermix and DW were added to the PCR plate wells. Then, the temperature cycle was adjusted to 95°C for 3 min, 95°C for 10 sec, and 58°C for 40 sec, for a total of 40 cycles. TM The PCR reaction was performed using a real-time PCR detection system (Bio-Rad, California, USA), and the fluorescence intensity was measured at the end of each cycle. The primer sequences used for PCR analysis to detect SOD2 mRNA expression are shown in Table 7 below.

[0219] Table 7

[0220]

[0221] 2. Conclusion

[0222] Figure 6 is a graph showing the results of real-time PCR for detecting the expression level of SOD2 mRNA in 100 peptides identified from yeast hydrolysate. Figure 6 A control and a treatment with tBHP followed by GSH peptide as a positive control are shown. The names of the 48 peptides are shown in Table 5.

[0223] like Figure 6As shown, the relative SOD2 mRNA expression induced by oxidative stress was determined and the antioxidant efficacy between the peptides was compared. RT-PCR was performed on a total of 100 peptides obtained from yeast hydrolysate. The results identified the following 39 peptides with increased SOD2 mRNA expression: γ-GLU-CYS(S-Me)(O2), γ-GLU-HIS, γ-GLU-ILE-GLY, γ-GLU-PRO-GLY, γ-GLU-GLN-GLU, γ-GLU-THR-GLY, γ-GLU-VAL-CYS, γ-GLU-VAL-ASP, γ-GL U-VAL-GLU, γ-GLU-VAL-PHE, γ-GLU-VAL-MET, γ-GLU-VAL-ASN, γ-GLU-VAL-ARG, γ-GLU-ALA, γ-GLU-CYS, γ-GLU-CYS(S-Me), γ-GLU-GLY, γ-GLU-ILE, γ-GL U-LYS, γ-GLU-LEU, γ-GLU-MET, γ-GLU-MET(O), γ-GLU-ASN, γ-GLU-PRO, γ-GLU-GLN, γ-GLU-THR, γ-GLU-VAL-NH2, γ-GLU-VAL-ol, γ-GLU-TRP, γ-GLU-TYR, γ-GLU-Abu-GLY, γ-GLU-GLN-GLN, γ-GLU-VAL-ALA, γ-GLU-VAL-GLY, γ-GLU-VAL-HIS, γ-GLU-VAL-LYS, γ-GLU-VAL-ORN, γ-GLU-VAL-PRO, and γ-GLU-VAL-VAL. Except for 39 peptides, other peptides had little effect on increasing SOD2 mRNA expression.

[0224] As a result of quantitative analysis of SOD2 mRNA expression among 39 peptides, the dipeptides identified as γ-GLU-ALA, γ-GLU-CYS, γ-GLU-CYS(S-Me), γ-GLU-CYS(S-Me)(O2), γ-GLU-GLY, γ-GLU-HIS, γ-GLU-ILE, γ-GLU-LYS, γ-GLU-LEU, γ-GLU-MET, γ-GLU-MET(O), γ-GLU-ASN, γ-GLU-PRO, γ-GLU-GLN, γ-GLU-THR, γ-GLU-VAL-NH2, γ-GLU-TRP, γ-GLU-TYR and γ-GLU-VAL-ol had SOD2 mRNA expression levels comparable to or higher than that of the positive control GSH, which exhibited effects comparable to or better than that of GSH despite having shorter sequences than GSH.

[0225] In addition, tripeptides with significantly superior SOD2 mRNA expression compared to the positive control GSH were identified as γ-GLU-GLN-GLU, γ-GLU-Abu-GLY, γ-GLU-ILE-GLY, γ-GLU-PRO-GLY, γ-GLU-SER-GLY, γ-GLU-THR-GLY, γ-GLU-VAL-CYS, γ-GLU-VAL-ASP, γ-GLU-VAL-GLU, γ-GLU-VAL-GLY, γ-GLU-VAL-HIS, γ-GLU-VAL-PHE, γ-GLU-VAL-MET, γ-GLU-VAL-ASN and γ-GLU-VAL-ARG.

[0226] Preferably, γ-GLU-CYS(S-Me)(02), γ-GLU-HIS, γ-GLU-ILE, γ-GLU-MET(0), γ-GLU-GLN-GLU, γ-GLU-THR-GLY and γ-GLU-VAL-CYS show a significantly higher effect of increasing SOD2 mRNA expression to about 2 to 3 times the normal level (SOD2 mRNA expression is much higher than the normal level (control)).

[0227] In summary, the test results demonstrate that yeast (Saccharomyces cerevisiae) cells are heated to 60°C and treated with alkaline endoprotease for 4 hours to perform cell lysis, compound flavor protease is added thereto for enzymatic hydrolysis for 10 hours, and alkaline endoprotease and compound flavor protease are added thereto for enzymatic hydrolysis for 24 hours, and the supernatant is collected to prepare yeast hydrolysate. First, a fraction having a molecular weight of 1 kDa or less is separated from the yeast hydrolysate thus prepared by an ultrafiltration membrane, and then, a fraction having a molecular weight ([M+H] + Then, a total of 100 peptides were identified therefrom.

[0228] Antioxidant activity of 100 peptides(Nrf2,SOD2 mRNA) analysis showed that a total of 48 peptides (γ-GLU-ALA, γ-GLU-CYS, γ-GLU-CYS(S-Me), γ-GLU-CYS(S-Me)(O2), γ-GLU-ASP, γ-GLU-GLU, γ-GLU-PHE, γ-GLU-GLY, γ-GLU-HIS, γ-GLU-ILE, γ-GLU-LEU, γ-GLU-MET(O), γ-GLU-ASN, γ-GLU-ORN, γ-GLU-PRO, γ-GLU-GLN, γ-GLU-ARG, γ-GLU-THR, γ-GLU-VAL, γ-GLU-VAL-NH2, γ-GLU-TRP, γ-GLU-TYR, γ-GLU-VAL-ol, γ-GLU-GLN-GLU, γ-GLU-ALA-GLY, γ-GLU-GLU-GLU, γ -GLU-ILE-GLY, γ-GLU-PRO-GLY, γ-GLU-GLN-GLN, γ-GLU-SER-GLY, γ-GLU-THR-GLY, γ-GLU-VAL-GLY, γ-GLU-VAL-ALA, γ-GLU-VAL-CYS, γ-GLU-VAL-ASP, γ-GLU-VAL-GLU, γ-GLU-VAL-PHE, γ-GLU-VAL-HIS, γ-GLU-VAL-LYS, γ-GLU-VAL-MET, γ-GLU-VAL-ASN, γ-GLU-VAL-ORN, γ-GLU-VAL-PRO, γ-GLU-VAL-ARG, γ-GLU-VAL-VAL, γ-GLU-CYS(S-Me)-GLY, γ-GLU-Abu-GLY and γ-GLU-LEU-GLY) have antioxidant activity.

[0229] Among the dipeptides having a shorter sequence length than the positive control GSH, γ-GLU-ALA, γ-GLU-CYS, γ-GLU-CYS(S-Me), γ-GLU-CYS(S-Me)(O2), γ-GLU-GLY, γ-GLU-HIS, γ-GLU-ILE, γ-GLU-LEU, γ-GLU-ASN, γ-GLU-PRO, γ-GLU-GLN, γ-GLU-THR, γ-GLU-VAL-NH2, γ-GLU-TRP, γ-GLU-TYR and γ-GLU-VAL-ol were found to have significantly excellent activity in promoting the expression of Nrf2 and SOD2 comparable to or better than GSH. Among them, the dipeptides with statistically significant activity in promoting the expression of Nrf2 and SOD2 compared with GSH were identified as γ-GLU-CYS, γ-GLU-CYS(S-Me), γ-GLU-CYS(S-Me)(O2), γ-GLU-GLY, γ-GLU-HIS, γ-GLU-ILE, γ-GLU-GLN, γ-GLU-TRP and γ-GLU-TYR.

[0230] In addition, it has been found that among the tripeptides having the same sequence length as the positive control GSH, γ-GLU-GLN-GLU, γ-GLU-ILE-GLY, γ-GLU-PRO-GLY, γ-GLU-SER-GLY, γ-GLU-THR-GLY, γ-GLU-VAL-CYS, γ-GLU-VAL-ASP, γ-GLU-VAL-GLU, γ-GLU-VAL-PHE, γ-GLU-VAL-MET, γ-GLU-VAL-ASN and γ-GLU-VAL-ARG have statistically significant activities in promoting the expression of Nrf2 and SOD2 compared with GSH.

[0231] That is, a total of 21 peptides were identified that have better effects on promoting Nrf2 expression and SOD2 expression than GSH, which is conventional and recognized as having an antioxidant effect, and they were determined to be γ-GLU-CYS(S-Me)(O2), γ-GLU-HIS, γ-GLU-CYS, γ-GLU-CYS(S-Me), γ-GLU-GLY, γ-GLU-ILE, γ-GLU-GLN, γ-GLU-TRP, γ-GLU-TYR, γ-GLU-GLN-GLU, γ-GLU-ILE-GLY, γ-GLU-PRO-GLY, γ-GLU-SER-GLY, γ-GLU-THR-GLY, γ-GLU-VAL-CYS, γ-GLU-VAL-ASP, γ-GLU-VAL-GLU, γ-GLU-VAL-PHE, γ-GLU-VAL-MET, γ-GLU-VAL-ASN and γ-GLU-VAL-ARG.

[0232] It was found that 6 of the peptides (γ-GLU-CYS(S-Me)(O2), γ-GLU-HIS, γ-GLU-ILE, γ-GLU-GLN-GLU, γ-GLU-THR-GLY and γ-GLU-VAL-CYS) had antioxidant effects (reducing ability, DPPH scavenging ability and Nrf2 mRNA activity were comparable to or better than GSH), and had the effect of promoting SOD2 mRNA expression to 2 to 3 times that of the positive control GSH, while γ-GLU-HIS was shown to have the effect of promoting Nrf2 and SOD2 mRNA expression to 2 to 8 times that of GSH.

Claims

1. Use of at least one peptide selected from the group consisting of the amino acid sequences γ-GLU-GLN and γ-GLU-GLN-GLU in the preparation of a composition for anti-oxidation.

2. Use of at least one peptide selected from the group consisting of the amino acid sequences γ-GLU-GLN and γ-GLU-GLN-GLU in the preparation of a feed composition for antioxidant activity.

3. Use of at least one peptide selected from the group consisting of the amino acid sequences γ-GLU-GLN and γ-GLU-GLN-GLU in the preparation of a cosmetic composition for anti-oxidation.

4. Use of at least one peptide selected from the group consisting of the amino acid sequences γ-GLU-GLN and γ-GLU-GLN-GLU for the preparation of a medicament for preventing or treating oxidative damage to cells.

5. The use according to claim 4, wherein the oxidative damage to the cells is caused by the generation of free radicals or reactive oxygen species (ROS).

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