JP4348436B2 - Catechin binding peptide - Google Patents

Description

  The present invention relates to a peptide capable of binding to catechins or a peptide having antioxidant activity. The peptide of the present invention is derived from the 67 kDa laminin receptor protein having a binding activity to catechins. The peptides provided by the present invention are useful in the field of food (including beverages) or pharmaceuticals.

  Of the catechins, epigallocatechin gallate (EGCG) is the main ingredient occupying about 50% of tea catechins. Other teas include epigallocatechin, epicatechin gallate, epicatechin (hereinafter, EGC, ECG, EC), and the like.

  Tea has a long history of being used as a medicine since ancient times, and the relationship between its efficacy and ingredients has been analyzed since modern times. Among them, EGCG was discovered in 1947 by A. Bradfield et al. Physiological actions of tea catechins including EGCG include antioxidant, anticancer, plasma cholesterol elevation inhibition, blood pressure elevation inhibition, platelet aggregation inhibition, blood sugar elevation inhibition, dementia prevention, anti-ulcer, anti-inflammatory, anti-allergy, antibacterial・ Anti-cavities, anti-virus, detoxification, intestinal flora improvement, deodorization, etc. have been reported.

  Of these, an anticancer effect has been reported very often, and has an antimutation effect, an anticarcinogenic promotion effect, an antitumor growth inhibitory effect, an antiinvasion / metastasis inhibitory effect, and an antiangiogenesis inhibitory effect. Recent reports report that EGCG inhibits DNA synthesis of leukemia cells and induces apoptosis, and EGCG suppresses the growth of breast cancer cell lines. Furthermore, it has been reported that EGCG strongly suppresses the growth of cancer cells compared to normal cells. Regarding invasion and metastasis, catechins have been reported to suppress the invasion of highly metastatic cells in an invasion test using Matrigel, and that EGCG inhibits the adhesion of cancer cells to fibronectin and laminin. .

  Molecular analysis of these catechin actions has recently been reported. For example, EGCG inhibits the growth of Her-2 antigen-high-expressing cells, which have been suggested to be associated with cancer, in a concentration-dependent manner. Its mechanism of action has been reported to be inhibition of its downstream signaling by suppressing Her-2 phosphorylation. It has also been reported that catechins such as EGCG inhibit angiogenesis that is closely related to tumor growth. The mechanism is shown to be that catechin suppresses phosphorylation of VEGF receptor VEGFR-1 which is a growth factor of vascular endothelial cells. It has been reported that this does not depend on the antioxidant / antiradical activity of catechins. Similarly, it has been reported that catechins suppress the phosphorylation of PDGF-Rbeta in vascular smooth muscle cells by PDGF-BB, which is another growth factor, thereby suppressing vascular thickening. Furthermore, it has been reported that EGCG inhibits angiogenesis and endothelial cell proliferation in vivo by EGF-2.

  In addition to antitumor effects, catechins have revealed various physiological actions at the molecular level. EGCG has been reported to have antidiabetic effects by suppressing glucose production in hepatocytes and promoting tyrosine phosphorylation of insulin receptors and IRS-1. In addition, EGCG is strongly expected to suppress many neurological disorders from reports showing strong neuroprotective action in Parkinson model mice. EGCG and its methylated form suppress the expression of Fc epsilon RI in basophils, which is an allergic factor, and also the expression of COX-2 and NO synthase 2 induced by IL-1 beta in cartilage EGCG has been reported to suppress.

  Bacteria type II fatty acid synthase (Non-patent document 1), Bcl-2 (Non-patent document 2), DNA methyltransferase (Non-patent document 3), 67kDa laminin receptor (non-patent document) A protein such as Patent Document 4) has been reported. However, each of these proteins is known to bind to other substances, and none of them is a substance that specifically binds only to EGCG. There have been no reports of substances that specifically bind only to EGCG.

  On the other hand, the 67 kDa laminin receptor (hereinafter 67LR) described above is a homodimer in which a 37 kDa precursor protein translated from mRNA encoding 295 amino acids undergoes an acylation polymerization reaction with fatty acids in cells. Or it is a protein which became 67 kDa by heterodimerization, and it functions as a laminin receptor only after it moves to the cell membrane surface with integrins (Non-patent document 5, Non-patent document 6, Non-patent document 7). Due to the high expression of this laminin receptor in many cancer cells, it is regarded as a tumor fetal antigen as an immunogen of T cells as a pan-tumor specific transplant antigen. A dozen or so laminin receptors have already been reported in addition to 67LR, among which 67LR is strongly associated with cancer. Expression of 67LR ligand laminin has no effect on prognosis, but 67LR expression has been shown to have negative outcomes.

  Based on these findings, several experiments have been reported that were conducted in anticipation of exhibiting an antitumor effect by suppressing the expression of 67LR. The 67LR low-expressing cell line prepared by introducing 67LR antisense RNA into a cancer cell line has a significant decrease in tumor growth ability and metastatic ability in vivo in mice compared to the original parent cell line. It has been reported that the survival rate of mice is improved. Furthermore, it has been reported that this low 67LR-expressing cell line has lower tumor angiogenesis and production of VEGF, which is a pro-angiogenic factor, than the parent line. Similarly, in the tumor metastasis experiment using an antibody against anti-67LR, the same effect as that of the antisense experiment is recognized.

  Several reports have been made on the function of 67LR even in non-tumor relations. Laminin-derived peptides (cysteine-aspartic acid-proline-glycine-tyrosine-isoleucine-glycine-serine-arginine) and EGF-derived peptides (cysteine-valine It is reported to be suppressed by isoleucine-glycine-tyrosine-serine-glycine-aspartic acid-arginine-cysteine). ENOS expression and NO production, which are implicated in arteriosclerosis induced by shear force on vascular endothelial cells, are suppressed by laminin-derived pentapeptide (tyrosine-isoleucine-glycine-serine-arginine) that binds to 67LR It has been reported.

  Recently, 67LR acts as a receptor for prion protein, which is thought to be the etiology of Creutzfeldt-Jakob disease, and prion binds and is internalized. Mutant 67LR lacks a membrane domain in its binding and internalization. It has been reported to be inhibited by the secretion of. Further, it has been reported that the binding site when the prion protein binds to the 67LR receptor is a portion of amino acid numbers 161-179 (Non-patent Document 8).

  67LR has also been reported to be expressed in CD4 + CD8- or CD4-CD8 + subsets of memory cells of CD45RO + / CD45RA-, a subset of T cells, suggesting that 67LR acts on the immune system. Yes.

  There are also several reports on 67LR mRNA expression. It has been reported that its expression is suppressed by p53 which is a tumor suppressor and TNF-α and IFN-γ which are anticancer factors.

Prior art documents

Y. Zhang et al .: J. Biol. Chem., 2004, 279: 30994-31001 M. Leone et al .: Cancer Res., 2003, 63: 8118-8121 M. Fang et al .: Cancer Res., 2003, 63: 7563-7570 H. Tachibana et al .: Nat. Struc. Mol. Biol., 2004, 11: 380-381 Yow, H. K. et al .: Proc. Nat. Acad. Sci. 1988, 85: 6394-6398 T. H. Landowski et al .: Biochemistry, 1995, 34: 11276-11287 S. Buto et al .: J. Cell. Biochem, 1998, 69: 244-251 C. Hundt et al .: EMBO J., 2001, 20: 5876-5886

  The inventor has conducted research on the bioregulatory function of food ingredients. Then, in the 67LR protein, a place involved in binding with EGCG was identified, and a peptide (SEQ ID NO: 15) having binding activity with EGCG was found. Furthermore, it has been found that such a peptide itself or a variant thereof has an antioxidant activity, and the present invention has been completed.

I. Catechin binding peptides:
The present invention provides the following catechin-binding peptides (a), (b) or (c):
(A) a peptide consisting of the amino acid sequence of SEQ ID NO: 15;
(B) The amino acid sequence of SEQ ID NO: 15, consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added, and binding to ECG, EGCG or EGCGMe (preferably EGCG) An active peptide; or (c) part of the amino acid sequence of the 67 kDa laminin receptor (67LR),
A portion derived from at least amino acid numbers _{161 to 170} : A ₁₆₁ -A ₁₆₂ -A ₁₆₃ -A ₁₆₄ -A ₁₆₅ -A ₁₆₆ -A ₁₆₇ -A ₁₆₈ -A ₁₆₉ -A ₁₇₀
(In the array:
A ₁₆₁ is an amino acid having a non-polar aliphatic side chain, preferably an amino acid selected from the group consisting of glycine, alanine, valine, leucine, isoleucine and proline, more preferably isoleucine;
A ₁₆₂ is an amino acid having a non-polar aliphatic side chain, preferably an amino acid selected from the group consisting of glycine, alanine, valine, leucine, isoleucine and proline, more preferably proline;
A ₁₆₃ is an amino acid having a polar uncharged side chain, preferably an amino acid selected from the group consisting of serine, threonine, cysteine, methionine, asparagine and glutamine, more preferably cysteine;
A ₁₆₄ is an amino acid having a polar uncharged side chain, preferably an amino acid selected from the group consisting of serine, threonine, cysteine, methionine, asparagine and glutamine, more preferably asparagine;
A ₁₆₅ is an amino acid having a polar uncharged side chain, preferably an amino acid selected from the group consisting of serine, threonine, cysteine, methionine, asparagine and glutamine, more preferably asparagine;
A ₁₆₆ is an amino acid having a positively charged side chain, preferably an amino acid selected from the group consisting of lysine, arginine, and histidine, more preferably lysine;
A ₁₆₇ is an amino acid having a non-polar aliphatic side chain, preferably an amino acid selected from the group consisting of glycine, alanine, valine, leucine, isoleucine and proline, more preferably glycine;
A ₁₆₈ is an amino acid having a non-polar aliphatic side chain, preferably an amino acid selected from the group consisting of glycine, alanine, valine, leucine, isoleucine and proline, more preferably alanine;
A ₁₆₉ is an amino acid having a positively charged side chain, preferably an amino acid selected from the group consisting of lysine, arginine, and histidine, more preferably histidine;
A ₁₇₀ is an amino acid having a polar uncharged side chain, preferably an amino acid selected from the group consisting of serine, threonine, cysteine, methionine, asparagine, and glutamine, more preferably serine)
And an amino acid sequence that may contain a part to which one or several amino acids adjacent to it may be substituted, deleted, inserted and / or added; and ECG, EGCG or EGCGMe (preferably A peptide having binding activity to (EGCG). However, from the scope of the present invention, known peptides, for example, peptides consisting of the amino acid sequence of amino acids 161 to 179 of 67LR described in Non-Patent Document 8 are excluded.

  In this specification, the number of amino acids to be substituted when “one or several amino acids are substituted, deleted, inserted, and / or added” with respect to the peptide is the number of amino acids that the peptide comprising the amino acid sequence is desired. Although it does not specifically limit as long as it has a function, For example, it is about 1-9 pieces or 1-4 pieces. For example, substitution with an amino acid having similar properties (charge and / or polarity) will not lose the desired function even if a large number of amino acids are substituted.

  The amino acid substitution or the like may be a substitution that does not cause an electrostatic change in the binding property to catechins, for example, a substitution with an amino acid having a similar charge and / or polarity. Such substitution includes, for example, an amino acid (glycine, alanine, valine, leucine) having a non-polar aliphatic side chain in the side chain (sometimes expressed as R group) near physiological pH (7.0). , Isoleucine, proline, etc.), substitution of amino acids with polar uncharged side chains (serine, threonine, cysteine, methionine, asparagine, glutamine, etc.), side chains have a positive charge near physiological pH There are substitutions between amino acids (such as lysine, arginine, and histidine), substitutions between polar amino acids, and substitutions between nonpolar amino acids.

  In the study of the present inventors, all of the peptides having the sequence of the amino acid numbers 161 to 170 of the 67 kDa laminin receptor or a part thereof (SEQ ID NOs: 15 and 18-30) except the peptide of SEQ ID NO: 23 In the MS analysis, a peak indicating binding to EGCG was observed. From these results, it is considered that K of amino acid number 166 is important for binding. In addition, in the peptide having the sequence of amino acid numbers 161 to 170, the peptide (SEQ ID NOs: 31 and 32) in which the 166th K is substituted with R or H, which is the same basic amino acid, is similarly EGCG. And a binding peak was observed. Furthermore, when the binding with EGCG was examined for each of the 20 major amino acids constituting the protein, binding peaks were observed only for the basic amino acids K, H, and R. On the other hand, the observation of a binding peak by MS analysis does not necessarily mean that it has a high binding activity. Therefore, in order for a peptide to bind to EGCG, the presence of basic amino acids in the peptide is first important, and the presence of other amino acids is important in order to bring high binding activity or specificity to the peptide. It is believed that there is.

  Further, in the study of the present inventors, the peptide having the sequence of 67LR amino acid numbers 161 to 170 (SEQ ID NO: 15) was found to have neutralizing ability against the cell growth inhibitory action of EGCG, but its C-terminal was observed. Both of the peptide lacking one amino acid (SEQ ID NO: 18) and the peptide lacking one N-terminal amino acid (SEQ ID NO: 19) neutralize the cell growth inhibitory action of EGCG. Noh was no longer seen (Example 1). Furthermore, in the peptide having the sequence of the amino acid numbers 161 to 170 described above, the peptide in which K is substituted with R or H (SEQ ID NOs: 31 and 32) shows the same neutralizing ability as that before substitution. Nevertheless, the neutralizing ability disappeared from the peptide in which K and H were substituted with S (SEQ ID NO: 33) (Example 5).

From this point of view, further variants can be designed based on the above-mentioned catechin-binding peptides, and further variants can also be included in the scope of the present invention as equivalents.
In the present specification, when “catechin” is used in a broad sense, or “catechin” is used, it means a polyoxy derivative of 3-oxyflavan unless otherwise specified. This includes catechins, gallocatechins, and their 3-galloyl forms, and their isomers and racemates, and their methylated forms. Specifically, catechin, gallocatechin, catechin gallate, gallocatechin gallate (gallocatechin-3- O -gallate; GCG); epicatechin (EC), which is the main green tea catechin, epigallocatechin (EGC), Epicatechin gallate (epicatechin-3- O -gallate; ECG) and epigallocatechin gallate (epigallocatechin-3- O -gallate; EGCG); epigallocatechin methyl gallate (epigallocatechin-3- O − gallate; EGCGMe), specifically, epigallocatechin-3- O- (3- O- methyl) gallate; EGCG3 "Me, epigallocatechin-3- O- (4- O- methyl) gallate; EGCG4" Me . In the present specification, among catechins, those that are 3-galloyl bodies are particularly referred to as “galloylcatechins”.

  In the inventor's MS analysis, the peptide of SEQ ID NO: 7 or SEQ ID NO: 15 bound to EGCG (Example 2) and also bound to ECG, GCG, EGCG3 "Me and EGCG4" Me. On the other hand, it did not bind to EC and EGC. In Non-patent Document 4, no interaction between 67LR-transfected cells and EC or EGC was observed. Accordingly, the catechin-binding peptides of the present invention are particularly galloylcatechins (specifically CG, ECG, GCG, EGCG, EGCG3 "Me, EGCG4" Me, more specifically ECG, GCG, EGCG, EGCG3 "Me, EGCG4" Me, More specifically, it can bind to EGCG). In the present specification, the binding activity of the peptide of the present invention to catechins may be described with particular reference to the binding activity to EGCG. The same applies to catechins.

  Whether or not a certain peptide has binding activity to catechins is determined by confirming the presence of a complex of catechins and a candidate peptide, for example, as described in Example 2 of this specification. Can do. Moreover, the presence or absence of the binding activity of a certain peptide with catechins and the height of the binding activity are determined by, for example, neutralizing (blocking) the activity of catechins for the target peptide according to the description in Example 1 of the present specification. Judgment can also be made by evaluating performance.

  The catechin-binding peptide of the present invention is composed of an extremely short peptide chain as compared with the 67LR protein. The preparation of 67LR protein is extremely difficult both quantitatively and qualitatively, whereas the catechin-binding peptide of the present invention can be chemically synthesized and can be easily prepared in large quantities and with high purity. Have

  Preferred examples of the catechin-binding peptides of the present invention include a comparison of the degree of neutralization of EGCG activity at the same concentration determined by the method described in Example 2 of the present specification, and complete neutralization of EGCG activity. Neutralization of EGCG activity and / or EGCG binding activity higher than 67LR by comparison of concentration or Kd value obtained by the method using surface resonance plasmon described in Non-Patent Document 4. Noh is higher than 67LR.

A preferred example of the catechin-binding peptide of the present invention is a peptide consisting of the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 31 or SEQ ID NO: 32.
The catechin-binding peptide of the present invention can be easily produced by those skilled in the art using conventional methods.

II. Antioxidant peptides:
The present invention also provides an antioxidant peptide which is the following (a), (b) or (c):
(A) a peptide consisting of the amino acid sequence of SEQ ID NO: 15;
(B) a peptide consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 15, and having antioxidant activity; or (c) 67LR Amino acid sequence derived from amino acid numbers 161-170: A ₁₆₁ -A ₁₆₂ -A ₁₆₃ -A ₁₆₄ -A ₁₆₅ -A ₁₆₆ -A ₁₆₇ -A ₁₆₈ -A ₁₆₉ -A ₁₇₀
(In the array:
A ₁₆₁ is an amino acid having a non-polar aliphatic side chain, preferably an amino acid selected from the group consisting of glycine, alanine, valine, leucine, isoleucine and proline, more preferably isoleucine;
A ₁₆₂ is an amino acid having a non-polar aliphatic side chain, preferably an amino acid selected from the group consisting of glycine, alanine, valine, leucine, isoleucine and proline, more preferably proline;
A ₁₆₃ is an amino acid having a polar uncharged side chain, preferably an amino acid selected from the group consisting of serine, threonine, cysteine, methionine, asparagine and glutamine, more preferably cysteine;
A ₁₆₄ is an amino acid having a polar uncharged side chain, preferably an amino acid selected from the group consisting of serine, threonine, cysteine, methionine, asparagine and glutamine, more preferably asparagine;
A ₁₆₅ is an amino acid having a polar uncharged side chain, preferably an amino acid selected from the group consisting of serine, threonine, cysteine, methionine, asparagine and glutamine, more preferably asparagine;
A ₁₆₆ is an amino acid having a positively charged side chain, preferably an amino acid selected from the group consisting of lysine, arginine, and histidine, more preferably lysine;
A ₁₆₇ is an amino acid having a non-polar aliphatic side chain, preferably an amino acid selected from the group consisting of glycine, alanine, valine, leucine, isoleucine and proline, more preferably glycine;
A ₁₆₈ is an amino acid having a non-polar aliphatic side chain, preferably an amino acid selected from the group consisting of glycine, alanine, valine, leucine, isoleucine and proline, more preferably alanine;
A ₁₆₉ is an amino acid having a positively charged side chain, preferably an amino acid selected from the group consisting of lysine, arginine, and histidine, more preferably histidine;
A ₁₇₀ is an amino acid having a polar uncharged side chain, preferably an amino acid selected from the group consisting of serine, threonine, cysteine, methionine, asparagine and glutamine, more preferably serine) Or a peptide _consisting of 3 or more sequences (preferably 4 or more, more preferably 5 or more, more preferably 6 or more) which is a part and contains at least A ₁₆₄ or A ₁₆₇ and has antioxidant activity . However, from the scope of the present invention, known peptides, for example, peptides consisting of the amino acid sequence of amino acids 161 to 179 of 67LR described in Non-Patent Document 8 are excluded.

  In the present specification, the term “antioxidant activity” (also referred to as “antioxidant ability”) refers to an activity or ability to suppress oxidation, which includes TBARS elimination activity (Example 3 of the present specification). And DPPH radical scavenging activity (see Example 4 herein). The “antioxidant activity” of the peptide of the present invention can be evaluated by one or both of TBARS scavenging activity and DPPH radical scavenging activity. If it has either one, it can be said that it has “antioxidant activity”.

Preferred examples of the antioxidant peptide of the present invention include SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24. SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 or peptide consisting of the amino acid sequence of SEQ ID NO: 32, or amino acid sequence : A ₁₆₄ -A ₁₆₅ -A ₁₆₆ (wherein A ₁₆₄ is asparagine; A ₁₆₅ is asparagine; A ₁₆₆ is lysine).

  Those skilled in the art can easily produce the antioxidant peptide of the present invention using conventional methods.

III. Use:
The present invention provides useful uses of the following peptides (a), (b) or (c) derived from 67LR protein:
(A) a peptide consisting of the amino acid sequence of SEQ ID NO: 15;
(B) The amino acid sequence of SEQ ID NO: 15, consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added, and binding to ECG, EGCG or EGCGMe (preferably EGCG) A peptide having activity; or (c) a portion derived from at least amino acid numbers ₁₆₁ to 170 in the partial amino acid sequence of 67 kDa laminin receptor (67LR): A ₁₆₁ -A ₁₆₂ -A ₁₆₃ -A ₁₆₄ -A ₁₆₅ -A ₁₆₆ -A ₁₆₇ -A ₁₆₈ -A ₁₆₉ -A ₁₇₀ (wherein A _{161 to} A ₁₇₀ are as defined above), and one or several amino acids adjacent thereto are substituted, deleted, A peptide comprising an amino acid sequence which may contain a part which may be inserted and / or added; and a peptide having binding activity to ECG, EGCG or EGCGMe (preferably EGCG).

  The peptides defined above have binding activity with galloylcatechins (preferably ECG, EGCG or EGCGMe, more preferably EGCG). Therefore, like an antibody, for detection of galloylcatechins (preferably ECG, EGCG or EGCGMe, more preferably EGCG), they can be labeled and / or immobilized as necessary. . Specifically, the peptide is galloated in a sample, for example, a natural product, a processed product (food, medicine, quasi-drug, processed product raw material, processed intermediate product, etc.), body fluid (blood, urine, etc.). It can be used for specific detection and / or quantification of ircatechins (Example 7), and can be an element in the form of reagents, kits, test papers, sensors and the like.

  Conventional methods can be applied to labeling and immobilizing peptides. For example, it can be labeled by binding FITC (fluorescein iso-thiocyanate) or biotin to the N-terminus of the peptide, and the biotinylated peptide can be immobilized on an avidin-binding carrier (Example 6). The peptide of the present invention can also be immobilized on a carrier by a covalent bond. Covalent immobilization is preferable from the viewpoint that there are few restrictions on the conditions for applying and elution of a sample to an immobilization carrier (for example, HPLC column).

  The present invention also includes a step of concentrating and / or purifying galloylcatechins (preferably ECG, EGCG or EGCGMe, more preferably EGCG), characterized in that the peptide as defined above is used. A method for producing catechins is also provided. In this method, the peptide can be immobilized on a carrier and used as a ligand for affinity column chromatography.

  The present invention also provides a method for immobilizing galloylcatechins (preferably ECG, EGCG or EGCGMe, more preferably EGCG), characterized by using the peptide defined above. Specifically, the peptide can be used for immobilizing catechins in an antibacterial filter using catechins, a formaldehyde scavenger, or the like. Various embodiments including conjugates of peptides and galloylcatechins are included within the scope of the present invention.

  The present invention also provides a method for taste correction of galloylcatechins (preferably ECG, EGCG or EGCGMe, more preferably EGCG), characterized by using a peptide as defined above, and galloylcatechins comprising the peptide (Preferably ECG, EGCG or EGCGMe, more preferably EGCG) and a food (beverage) containing the peptide and galloylcatechins (preferably ECG, EGCG or EGCGMe, more preferably EGCG) Or a pharmaceutical composition. Catechins, especially galloylcatechins, have a specific bitter taste, and therefore have a limited amount when added to foods and the like. However, the bitter taste of catechins can be masked by adding a peptide capable of binding to the catechins of the present invention.

  Specifically, the food of the present invention containing galloylcatechins and peptides includes nutritional functional foods, foods for specified health use, health foods, nutritional supplements, drinks, tea drinks, soft drinks, carbonated drinks, sports drinks Or the like.

  The pharmaceutical composition of the present invention comprising galloylcatechins and a peptide comprises catechins having antioxidant, anticancer, plasma cholesterol elevation inhibition, blood pressure elevation inhibition, platelet aggregation inhibition, blood glucose elevation inhibition, dementia prevention, antiulcer , Anti-inflammatory, anti-allergic, antibacterial / anti-cavitary, antiviral, detoxification, intestinal flora improvement, deodorization, etc. (tea function, edited by Keiichiro Muramatsu et al., Academic Publishing Center, 2002) Can be used for the treatment of In particular, EGCG has an anticancer effect, specifically an antimutation effect, an anticarcinogenic promotion effect, an antitumor growth inhibitory effect, an antiinvasion / metastasis inhibitory effect, an antiangiogenesis inhibitory effect; more specifically, it inhibits DNA synthesis Induced apoptosis of leukemia cells (Int. J. Mol. Med., 2001, 7: 645-652, DM Smith et al.); Breast cancer cell growth inhibitory action (J. Cell. Biochem., 2001, 82: 387) -398, KT Kavanagh et al .; Selective growth inhibition of cancer cells (Arch. Biochem. Biophys, 2000, 376: 338-346, N. Ahmad et al.); Invasion of highly metastatic cells in relation to cancer invasion and metastasis Inhibitory action, inhibiting cancer cell adhesion to fibronectin and laminin (Cancer Lett., 1995, 98: 27-31, M. Suzuka et al., Cell Biol. Int., 1993, 17: 559-564, M. Isemura et al., Cancer Lett., 2001, 173: 15-20, Y. Suzuki et al.); Her-2 antigen high-expressing cells by inhibiting its downstream signaling by inhibiting Her-2 phosphorylation Antiproliferative effect (Cancer Res., 2002, 62: 652-655, S. Pianetti et al.); Angiogenesis inhibitory effect by suppressing phosphorylation of VEGF R-1 which is a receptor for VEGF (Cancer Res, 2002, 62: 381-385, S. Lamy et al.) PDGF-BB suppresses PDGF-R beta phosphorylation in vascular smooth muscle cells (FASEB J, 2002, 16: 893-895, A. Sachinidis et al .; EGF-2 can be used to treat diseases or conditions associated with angiogenesis and endothelial cell proliferation inhibitory action (Nature, 1999, 389: 381, Y. Cao et al.). It also inhibits hepatocyte glucose production and promotes tyrosine phosphorylation of insulin receptor and IRS-1 (J. Biol. Chem., 2002, 277: 34933-34940, ME Waltner). -Law et al.); Inhibition of neuropathy, more particularly neuroprotection in neurological diseases (eg Parkinson's disease) (J. Biol. Chem., 2002, 277: 30574-30580, Y. Levites et al.); Allergy Suppresses the expression of Fc epsilon RI in basophils (J. Agric. Food Chem, 2002, 50: 5729-5734, Y. Fujimura et al.); COX induced by IL-1 beta in cartilage -2 and NO synthase 2 expression (Free Radical Biology & Medicin, 2002, 33: 1097-2002, S. Ahmed et al.). Furthermore, the function of 67LR, specifically the function that has a negative outcome in cancer (Breast Cancer Research and Treatment, 1998, 52: 137-145, S. Menard et al.); Tumor growth and metastatic potential (British) Journal of Cancer, 1999, 80: 1115-1122, K. Satoh et al.); Tumor angiogenesis regulation function and VEGF production regulation function (Cancer Letters, 2000, 153: 161-168, M. Tanaka et al.); Tumor metastasis regulation Function (Jpn. J. Cancer Res., 1999, 90: 425-431, K. Narumi et al.); Neovascular growth-regulating function induced in ischemic disease (Am. J. Pathol, 2002, 160: 307- 313, D. Gebarowska et al .; eNOS expression and inhibition of NO production regulatory function that has been pointed out to be involved in shear-induced arteriosclerosis on vascular endothelial cells (J. Biol. Chem., 1999, 274: 15996- 16002, T. Gloe et al .; functions as a receptor for prion protein and regulates prion binding and internalization (EMBO J, 2001, 20: 5863-5875, S. Gauc zynski et al .; Immunoregulatory function (J. Immunol, 1999, 163: 3430-3440, SM Canfield et al.); 67LR mRNA expression by tumor suppressor p53, anticancer factors TNF-α and IFN-γ It can also be used to treat diseases or conditions associated with cancer-related functions based on the suppression of (Biochem. Biophys. Res. Commun., 1998, 251: 564-569, N. Clausse et al.). As used herein, the term “treatment” for a disease or condition, except for special cases, includes treating, preventing, and stopping progression of the disease or condition. This includes coping treatment that suppresses symptoms and fundamental treatment.

  The pharmaceutical composition of the present invention containing galloylcatechins and a peptide is also useful as a quasi-drug, and can also be used in the form of lotion, soap, shampoo, wet tissue or the like.

  Since the peptide defined above also has an action of promoting the antioxidant action of catechins (Example 8), the present invention further comprises galloylcatechins (preferably characterized by using the peptide defined above) Is a method for promoting the antioxidant activity of ECG, EGCG or EGCGMe, more preferably EGCG), the antioxidant activity of galloylcatechins (preferably ECG, EGCG or EGCGMe, more preferably EGCG) containing the peptide An accelerator is also provided.

  The invention further provides an antioxidant comprising a peptide as defined above. The antioxidant of the present invention can be in the form of food or pharmaceuticals. When used as a pharmaceutical, it can be used for the treatment of diseases or conditions associated with antioxidant activity.

  Catechin is both an antioxidant and a property as an oxidant, and beverages containing catechins such as green tea beverages contain a large amount of hydrogen peroxide derived from catechins. In such a case, the presence of the peptide of the present invention that binds to the galloylcatechins of the present invention makes it possible to retain the antioxidant effect while suppressing the oxidizing action of EGCG. The function of promoting the antioxidant activity of such galloylcatechins possessed by the peptide of the present invention is also observed in basic amino acids (Example 9).

  There is an example of using catechin as a protective agent for cells and organs. In this case, the protective action of catechin is due to the antioxidant action. The peptide of the present invention can also be used effectively to enhance such action. .

  The peptide defined above overlaps with the sequence of amino acid numbers 161 to 179 (non-patent document 8), which is the binding site when pathogenic prion binds to 67LR. Therefore, since the peptide defined above can be used as an anti-prion substance and the infection with pathogenic prion can be prevented by inhibiting the binding to 67LR, the peptide or gallocatechin body defined above can be used as a prion. It can be used as an inhibitor. Accordingly, the present invention also provides an anti-67LR ligand agent (more specifically, an anti-prion agent), as well as galloylcatechins (preferably ECG, EGCG or EGCGMe, more preferably EGCG) containing the peptide defined above. A pharmaceutical composition for treating prion diseases (eg, bovine sea surface encephalopathy (BSE), Creutzfeldt-Jakob disease (CJD), juvenile-onset variant Creutzfeldt-Jakob disease (vCJD)) is provided.

  The peptide of the present invention, which is shorter than the peptide consisting of the amino acid sequence of 67LR amino acid numbers 161 to 179 (hereinafter sometimes referred to as “Peptide 161-179”), is less susceptible to protease degradation and is more stable. Let's go. In addition, the portion of 67LR amino acids 173 to 178 (LMWWML) contained in Peptide 161-179 is a binding site with laminin 1 (J. Biol. Chem. 266, 20440-20446 (1991)), and Peptide When 161-179 is administered or used for detection of a target substance, it may bind to laminin 1 in the living body, or the target substance and laminin 1 cannot be distinguished from each other. From this point of view, the peptide of the present invention which is shorter than Peptide 161-179, and more preferably does not contain the portion of amino acids 173 to 178 of 67LR is particularly useful as an anti-prion substance.

Specifically, the anti-prion agent containing the peptide of the present invention can be used for the prevention and / or treatment of prion diseases for the detection of prions in samples such as blood.
When the anti-prion agent of the present invention containing the peptide defined above is used for prevention and / or treatment of prion disease, that is, when the anti-prion agent of the present invention is used as a pharmaceutical composition, its administration route and agent The shape and dose can be appropriately designed by those skilled in the art.

  The administration route and dosage form of the pharmaceutical composition of the present invention containing galloylcatechins can also be appropriately designed by those skilled in the art. This pharmaceutical composition is also excellent in that it can be administered orally and is considered effective. Currently, anti-prion antibodies are promising for the treatment of prion diseases, but in the case of antibodies, the blood-brain barrier prevents them from passing through them. There is no effect if not administered. In contrast, catechin can reach the brain even by oral administration. Anti-prion antibodies have been reported to have serious effects on normal brain (induction of cell death) (Science, 303, 1514-1516 (2004)). No adverse effects on the brain have been reported.

  The pharmaceutical composition of the present invention containing galloylcatechins can be formulated as either a solid or liquid formulation, but in the form of a conjugate with galloylcatechins and the peptides defined above for the sake of stability. It may be included. For livestock such as cattle, galloyl catechins themselves or green tea leaves or tea leaf extracts containing a large amount of galloyl catechins may be mixed with feed. Such feed is also included in the pharmaceutical composition of the present invention. Since the catechin content is higher in the second tea, the third tea, and the autumn / winter tea than the first tea, the second tea, the third tea, and the autumn / winter tea can be used as a raw material of the pharmaceutical composition of the present invention.

  The dosage of the pharmaceutical composition of the present invention containing galloylcatechins can also be appropriately designed by those skilled in the art. With regard to EGCG, it has been demonstrated that it can bind to 67LR on the cell surface at 1 μM, which is the concentration reached in the blood when 500 to 1000 mg is orally ingested (see Non-Patent Document 4). . On the other hand, 500 to 1000 mg of EGCG is also used in commercially available beverages, and it can be said that the safety when ingested continuously has been sufficiently confirmed.

The efficacy of the peptides and galloylcatechins defined above against prion disease is evident from the following points:
(1) The peptide defined above can bind to a prion and can be used as an anti-prion antibody. In addition, galloylcatechins can inhibit prion binding. According to a report by Leucht et al. (EMBO Rep., 4, 290-295 (2003)), when anti-67LR antibodies act on cells infected with pathogenic prions, they prevent and completely eliminate pathogenic prions. It has been demonstrated that anti-67LR antibodies are useful for the treatment of prion diseases.

  (2) It can be confirmed by various methods that the peptide or galloylcatechins defined above inhibit the binding between prion and 67LR. For example, the above-defined peptide or galloylcatechins are allowed to act on a system containing NT2 cells expressing 67LR (see Non-Patent Document 8) and prions, and immunoprecipitated using anti-67LR antibodies or anti-prion antibodies. What is necessary is just to confirm success or failure. Alternatively, using the experimental system described in Non-Patent Document 8, it is confirmed that the peptide or galloylcatechin defined above inhibits the binding between the recombinant prion and 67LR. A more specific approach is shown in Example 11 herein.

  The food or pharmaceutical composition of the present invention has its specific use (indication, symptom that can be improved, etc.) and / or its specific use (eg, administration route, amount, frequency, duration, etc.). Can be displayed on attached instructions, container surfaces, package surfaces, and the like.

The present invention provides a screening method for a candidate galloylcatechin mimetic compound comprising the following steps:
(1) preparing a test compound;
(2) For a test compound, evaluate the binding to the peptide defined above; and (3) If the test compound has binding to the peptide, select the compound.

As used herein, “galloylcatechin mimetic compound” refers to a compound that mimics the physiological activity such as the anticancer activity of catechins or EGCG described above.
Step (1) is a step of preparing a compound for screening. A person skilled in the art can synthesize various compounds by conventional methods, and a commercially available compound library may be used, and the compound may be derived from a natural product. The compound may be subjected to screening as a simple substance, or may be provided as a mixture of a plurality of compounds. The evaluation of the binding property of the test compound to the peptide in step (2) is, for example, by examining whether the test compound binds to the peptide immobilized on the column under specific conditions. For example, when a peptide-immobilized column is used in step (2), step (3) is performed by removing the compound that has passed through the column and eluting the compound captured by the column under appropriate conditions.

  The selected compound (group) can be subjected to further evaluation and examination as a galloylcatechin mimetic compound candidate (group). For example, the first group of compounds selected by the affinity column for screening can be subsequently subjected to a second column to divide into each single component and each component can be identified or determined. Application of such a system based on HPLC as the first column is one of the preferred embodiments of the screening method of the present invention.

  The screening method of the present invention is suitable for a screening method comprehensively and rapidly for candidates of galloylcatechin mimetic compounds, since the peptides defined above are smaller molecules than the 67LR protein, have high binding activity to the target. In the screening method of the present invention, it is preferable to use a peptide consisting of the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 31 or SEQ ID NO: 32 as a peptide. The screening method of the present invention is particularly suitable for selecting EGCG mimetic compound candidates.

The present invention also provides a method for enriching and / or purifying galloylcatechin mimetic candidate compounds using the peptides defined above.
The present invention also provides a method for detecting a disease predisposition that can be treated with galloylcatechins, comprising detecting a mutation in a base sequence encoding a portion consisting of amino acid sequences of 67LR amino acid numbers 161 to 170. . This method makes it possible to diagnose susceptibility to galloylcatechins and / or susceptibility to diseases treatable with galloylcatechins. In the peptide (SEQ ID NO: 15) in which the basic amino acid moiety is substituted with serine in the peptide consisting of the amino acid sequence of 67LR amino acid numbers 161 to 170 (SEQ ID NO: 15), the original neutralizing ability completely disappeared (implementation) Example 5). That is, when it has a 67LR gene mutation that causes a substantial mutation in a basic amino acid in the sequence, it can be said that the sensitivity to galloylcatechins is low. In customizing a food or pharmaceutical composition using galloylcatechins, the sequence information of the catechin binding site is useful for the judgment criteria of the subject's catechin sensitivity. The presence or absence of mutations in the 67LR amino acid numbers 161 to 170 is determined by directly analyzing the gene sequence of the DNA or mRNA from the subject, or the basic amino acid portion genes in 67LR amino acid numbers 161 to 170. This can be done by analyzing the polymorphism.

  In addition, the description of “galloylcatechins” in the present specification also applies to other compounds having a galloyl group, such as strictinin. Strictinin suppresses IL-4 induced εGT expression and IgE production (Biochemical and Biophysical Research Communication 280, 53-60 (2001)). That is, the present invention can also be applied to compounds such as strictinin having a galloyl group. The present invention also provides such modifications.

<Study of inhibitory effect of 67LR partial peptide and recombinant 67LR protein on cell growth inhibitory activity of EGCG>
1) Materials and methods:
1.1) Green tea catechin:
epigallocatechin-3- O- gallate (EGCG, sigma), epicatechin-3- O- gallate (ECG, sigma), epigallocatechin (EGC, sigma), epicatechin (EC, sigma) to 5 mM phosphate buffer ( It was dissolved in PBS), stored frozen at -20 ° C, and diluted appropriately. PBS is 0.1 g of NaCl (Nacalai tesque, Inc.) 8.0 g, 0.2 g of KCl (Nacalai tesque, Inc.), 1.15 g of Na ₂ HPO ₄ (Nacalai tesque, Inc.), KH ₂ PO ₄ (Nacalai tesque, Inc.) 0.2 g was dissolved and prepared.

1.2) Cells and cell culture:
The human hepatocarcinoma cell line HepG2 was passaged and maintained in DMEM medium (COSMO BIO CO., LTD.) Supplemented with 10% FBS (Bio Source International, Camarillo, CA) at 37 ° C under 5% CO ₂ with water vapor saturation. . In DMEM medium, 100 U / mL penicillin (Meiji pharmaceutical Company, Tokyo, Japan), 100 mg / mL streptomycin (Meiji pharmaceutical Company), 23.1 mM NaHCO3 (Wako Pure Chemical), and 25 mM HEPES (Wako Pure Chemical) Was added. Cells were maintained in culture in the logarithmic growth phase.

1.3) Construction of a constant expression system for 67kDa laminin receptor (67LR):
The human 67LR gene was cloned by PCR using the following two primers and a human hepatocyte cDNA library as a template.
h67LR-S: C ggTACC ATg TCC ggA gCC CTT gAT gTC CTg CAA ATg (SEQ ID NO: 1)
h67LR-A: g gCggCCgC TTA AgA CCA gTC AgT ggT TgC TCC TAC CCA (SEQ ID NO: 2)
The obtained PCR amplified fragment was ligated to a pcDNA3.1 (+) (invitrogen) vector to obtain a human 67LR gene expression vector (pcDNA3.1-hLamininR).

pcDNA3.1-hLamininR was introduced into HepG2 cells by electroporation. Details are as described below. 5 × 10 ⁶ cells were collected in a 15 mL centrifuge tube (Nunc, Roskild, Denmark), washed once with phosphate buffer (PBS), and centrifuged at 300 × g for 5 minutes to remove the supernatant. The precipitated cells were resuspended in 300 μL of phosphate buffer (PBS), 10 μg of pcDNA3.1-hLamininR was added and mixed gently, transferred to a 4 mm Gap disposable cuvette, and then left on ice for 10 minutes. The cuvette was set in an Erctro Cell Manipurator ECM395 (A Division of Genetronics, Inc., USA), a voltage of 250 V was applied, and the cuvette was left on ice for 10 minutes. The contents were washed with a medium, suspended in a medium containing 10% FBS, seeded in a 96-well plate (Nunc, Roskild, Denmark) at 100 μL / well, and cultured for 3 days. Thereafter, a medium containing 10% FBS supplemented with neomycin (Invitrogen Corporation) was added at 100 μL / well. Neomycin-resistant clones were obtained by selection in a neomycin-containing medium for about 2 weeks until cells into which the target gene had not been introduced died.

1.4) Synthesis and preparation of 67LR partial peptide:
A partial peptide (amino acid number: 102-295) outside the 67LR cell membrane was synthesized. The name of each partial peptide, amino acid number and amino acid sequence in 67LR protein are shown in the table below.

  Peptide 121-140, Peptide 181-200, Peptide 281-295, Peptide 156-165, and Peptide 171-180 were measured in 50% dimethyl sulfoxide (Nacalai tesque, Inc. Kyoto, Japan) The peptide was dissolved in distilled water to a concentration of 1 mM, stored frozen at -80 ° C, and diluted as appropriate.

1.5) Preparation of recombinant 67LR protein:
The 67LR gene was excised from the human 67LR gene expression vector (pcDNA3.1-hLamininR) by digestion with restriction enzymes KpnI and NotI, and inserted into the KpnI / NotI site of the pET30a vector (pET30a-67LR). The constructed expression vector pET30a-67LR for E. coli was transformed into E. coli BL21 (DE3) strain. Details are as described below.

  1 ng of pET30a-hLamininR was added to the competent E. coli BL21 (DE3) strain and left on ice for 30 minutes. After heat shock at 42 ° C. for 30 seconds, it was cooled back in ice. Then 0.9 mL of SOC medium was added, and the culture was recovered at 37 ° C for 1 hour and centrifuged at 1000 x g for 5 minutes. After removing 0.9 mL of the supernatant, Escherichia coli was suspended in the remaining solution, seeded on a plate containing kanamycin, which is a resistant drug, and cultured for 24 hours, and then kanamycin resistant colonies were obtained to obtain pET30a-67LR-introduced E. coli. The pET30a-hLamininR-introduced Escherichia coli was inoculated into LB medium containing kanamycin and cultured at 37 ° C. for 24 hours. This E. coli culture solution was added to LB medium containing kanamycin and further cultured, and 0.5 mM IPTG was added to enhance gene expression. The Escherichia coli culture was centrifuged to collect the cells as a precipitate. The cooled phosphate buffer was added, and ultrasonic crushing was performed using Handy Sonic (TOMY SEIKO Company, Tokyo, Japan) for 30 seconds. Subsequently, the mixture was centrifuged at 15,000 r.p.m. for 15 minutes, and the supernatant was collected. The recombinant 67LR protein contained in this supernatant was purified using a Hi Trap column (Amersham Biosciences, USA) according to the manual attached to the column.

1.6) Evaluation method:
pcDNA3.1-hLamininR-introduced HepG2 cells are suspended in DMEM medium containing 2% FBS and 5 mg / mL BSA at 1x10 ⁴ cells / mL, seeded in 24-well plates (Nunc, Roskild, Denmark), and left for 24 hours To attach the cells. Then, EGCG and 67LR partial peptide or recombinant 67LR protein were mixed in 2% FBS and 5 mg / mL BSA-containing DMEM medium to a final concentration of 1 μM, respectively, and allowed to stand at room temperature for 15 minutes as cell culture supernatant. Replaced. After culturing at 37 ° C. and 5% CO _{2 for} 5 days, the number of cells was counted with a cell counter (Sysmex). Inhibitory ability (neutralizing ability) of cell growth inhibitory activity is 100 (% )) And the average value (n = 3). Student's t-test was used for statistical processing of experimental results.

2) Result:
The results are shown in FIGS. First, in a peptide group designed to be approximately a certain length (about 20 amino acids) from amino acid number 102 of 67LR, Peptide 161-180 was found to neutralize the cell growth inhibitory effect of EGCG. (Figure 1). In addition to Peptide 161-180, Peptide 161-180 and a peptide group consisting of 10 amino acids in length at least partially overlapping, and Peptide 151-170 were examined. The activity was almost the same as 161-180 (Fig. 2). Furthermore, the effect of Peptide 161-170, Peptide 161-169 lacking one C-terminal amino acid, and Peptide 162-170 lacking one N-terminal amino acid was examined. In both cases, the neutralizing ability of EGCG against the cell growth inhibitory effect disappeared (Figure 3). In addition, the neutralizing ability of Peptide 161-170 exceeded that of recombinant 67LR protein (Fig. 4).

<Confirmation of EGCG binding activity of 67LR partial peptide>
EGCG and a 67LR partial peptide (Peptide 161-180 (SEQ ID NO: 7) or Peptide 161-170 (SEQ ID NO: 15)) were mixed in a 1.5 mL Eppendorf tube to a final concentration of 5 μM, and then mixed at room temperature. Left for a minute. At this time, a 50% ethanol solution containing 1% acetic acid was used as the solvent. Then, it inject | poured into the MS spectrometer (LCQ Advantage, Thermo Finnigan, Australia) using the syringe, and acquired the MS spectrum by the electrospray ionization (ESI) method. The conditions at this time were pH 3.5, capillary temperature 270 ° C., and applied voltage 4.5 kV.

  The results are shown in FIGS. The peak of the complex of Peptide 161-180 or Peptide 161-170 and EGCG was recognized, and it was confirmed that all of these 67LR partial peptides bind to EGCG.

<Measurement of peptide antioxidant activity (TBARS elimination activity)>
The 67LR partial peptide solution was mixed with ₂ mL of dH ₂ O to a final concentration of 1 μM, and 1,1,3,3-tetraethoxypropane (Sigma) was added thereto to a final concentration of 10.5 μM. After adding 500 μL of TBA reagent, the mixture was stirred, and a glass ball was placed on the test tube and incubated at 95 ° C. for 1 hour. The TBA reagent was prepared by mixing and dissolving 10.6 mL of acetic acid (Nacalai tesque, Inc. Kyoto, Japan) and 67 mg of 2-thiobarbituric acid (Sigma) in 9.4 mL of ultrapure water. After the reaction, the reaction mixture was cooled with running water for 5 minutes, added with 2.5 mL of n-butanol (Nacalai tesque, Japan), and stirred for 20 seconds. After centrifugation at 3000 rpm for 10 minutes, the fluorescence intensity of the upper butanol layer was measured using a spectrofluorometer RF-1500 (SHIMAZDU). The measurement conditions were an excitation wavelength of 515 nm and a fluorescence wavelength of 553 nm. Antioxidant activity was expressed as means ± SD (n = 3) by calculating the TBARS decrease amount according to the following formula. In the formula, the standard solution refers to a 10.5 μM solution of 1,1,3,3-tetraethoxypropane (Sigma).

  The amino acid sequences of the peptides subjected to the test and the test results are shown in the table below.

<Measurement of peptide antioxidant activity (DPPH radical scavenging activity)>
The 67LR partial peptide solution was mixed with 30 μL of dH ₂ O to a final concentration of 1 μM, and 90 μL of DPPH (1,1-diphenyl-2-picrylhydrazyl) solution was added thereto. The DPPH solution was prepared by dissolving 1.97 mg of DPPH (Wako Pure Chemicals) in a fine powder state in a mortar in 12.5 mL of ethanol (Nacalai tesque, Inc. Kyoto, Japan). After reacting for 20 minutes after the addition, the absorbance at 520 nm was measured. At this time, a calibration curve was created using Trolox (CALBIOCHEM, US) as a control with reducing power. Antioxidant activity was expressed as average value ± standard error (n = 3) of Trolox equivalent (μmol) of DPPH elimination activity.

  The results are shown in the table below.

<Examination of K-substituted peptides>
Peptides 161-170 were synthesized with peptides (K-substituted peptides) having the sequences shown in the following table in which lysine was substituted.

In addition, Peptide 161-170KS, HS (IPCNNSGASS, SEQ ID NO: 33) in which lysine and histidine were replaced with serine in Peptide 161-170 was synthesized.
The following evaluation was performed about these.

1) Evaluation of inhibitory effect of EGCG on cell growth inhibitory activity:
According to the method described in Example 2, the inhibitory effect of EGCG on cell growth inhibitory activity was evaluated.
2) Evaluation of peptide antioxidant activity (TBARS scavenging activity and DPPH radical scavenging activity):
TBARS scavenging activity and DPPH radical scavenging activity were evaluated for K-substituted peptides according to the methods described in Examples 4 and 5.

3) Result:
FIG. 7 shows the neutralizing activity of the cell growth inhibitory activity of EGCG. The peptide in which K was substituted with R or H also showed neutralizing activity against the cell growth inhibitory effect (A), but the peptide in which K and H were substituted with S each lost neutralizing activity (B).

  The antioxidant activity is shown in the table below.

<Preparation of affinity column using EGCG Binding Peptide 160-170>
The following materials were used.
・ Streptavidin carrier column 1 mL (Hi Trap Streptavidin HP, Amersham)
-Binding buffer: 20 mM sodium phosphate, 0.15 M NaCl, pH 7.5
-Biotinylated Peptide 160-170 (Biot-p160-170): Biotin bonded to the N-terminus of Peptide 160-170. Dissolved in binding buffer, prepared in 2 mM ・ Epicatechin: EC (Sigma): dissolved in 100% Methanol, prepared in 0.5 mM ・ Epigallocatechin-3- O- gallate: EGCG (Sigma): dissolved in 100% Methanol, Prepared to 0.5 mM.

  Immobilization of the biotinylated peptide on the streptavidin carrier column was performed as follows. A streptavidin carrier column (Hi Trap Streptavidin HP, Amersham) was equilibrated with a binding buffer, and 2 μmol (2 mM × 1 mL) Biot-p160-170 was applied. After the peptide was applied, the binding buffer was flowed at 0.5 mL / min, and 1 mL of the eluted fraction from the column was collected. Absorbance at 220 nm, the absorption maximum of Biot-p160-170, was measured, and it was confirmed that the peptide was bound to the column and immobilized (FIG. 8).

  Next, a streptavidin carrier column to which Biot-p160-170 is bound and a column to which Biot-p160-170 is not bound are equilibrated with 100% Methanol, and EC or EGCG dissolved in 100% Methanol is applied to both columns. (The flow rate was 0.5 mL / min). After applying the sample, 1 mL each of the fraction eluted from the column was collected, and the absorbance at 276 nm and 273 nm, the maximum absorption of EC and EGCG, was measured, and the elution of both catechins was monitored. As a result, in the streptavidin carrier column not bound to Biot-p160-170, none of the catechins was bound to the column and collected as a flow-through fraction (FIG. 9).

<Quantification of EGCG using fluorescent-labeled peptide>
FITC-p160-170, in which the N-terminus of Peptide 160-170 is labeled with FITC (fluorescein iso-thiocyanate), was prepared in 1 mL of dH ₂ O to a final concentration of 1 μM. EGCG was mixed with this solution to final concentrations of 0.01, 0.1, 1 and 10 μM and left at room temperature for 15 minutes. Thereafter, the fluorescence intensity at an excitation wavelength of 365 nm and a fluorescence wavelength of 368 nm was measured using a spectrofluorometer RF-1500 (SHIMAZDU). Since the fluorescence intensity of FITC-p160-170 decreases due to the binding of EGCG, the degree of change in FITC-p160-170 was determined as the relative fluorescence intensity by the following formula.

FIG. 10 was obtained by plotting the value of EGCG concentration / FITC-p160-170 concentration against the relative fluorescence intensity.
Further, the following formula was obtained from the plot of FIG.

  The relative fluorescence intensity of FITC-p160-170 obtained by mixing an EGCG-containing sample and a known concentration of FITC-p160-170 is X. From the above formula, Y, that is, the value of EGCG concentration / FITC-p160-170 concentration Get. By multiplying this Y value by the FITC-p160-170 concentration, the amount of EGCG in the EGCG-containing sample can be determined.

<Enhancement of EGCG antioxidant activity by catechin-binding peptide>
Peptide 161-170 solution and EGCG were mixed with 30 μL of dH ₂ O to a final concentration of 1 μM, respectively, and incubated at room temperature for 15 minutes. 90 μL of 1,1-diphenyl-2-picrylhydrazyl solution (Wako Pure Chemical Industries) was added thereto. After reacting for 20 minutes after the addition, the absorbance at 520 nm was measured. At this time, a calibration curve was created using Trolox (CALBIOCHEM, US) as a control with reducing power. The table below shows the results of peptides and EGCG plus peptide when the DPPH radical scavenging activity of EGCG is 1.

<Enhancement of antioxidant activity of EGCG by basic amino acids>
The amino acid solution and EGCG were mixed with 30 μL of dH ₂ O to a final concentration of 1 μM, respectively, and incubated at room temperature for 15 minutes. 90 μL of 1,1-diphenyl-2-picrylhydrazyl solution (Wako Pure Chemical Industries) was added thereto. After reacting for 20 minutes after the addition, the absorbance at 520 nm was measured. At this time, a calibration curve was created using Trolox (CALBIOCHEM, US) as a control with reducing power. The relative values of the activity of EGCG in the presence of peptide and peptide when the DPPH radical scavenging activity of EGCG is set to 1 are shown in the table below.

<Effects of peptides on cell binding properties of EGCG>
In order to directly confirm that the binding of EGCG to cells was inhibited by the peptide, the effect of the 67LR-derived peptide on the binding of EGCG to HepG2 was surfaced. Measurement was performed with a plasmon resonance sensor.

1) Method:
The human cell line HepG2 was immobilized on a gold membrane (Nippon Laser and Electronic Lab.) Using standard immobilization methods such as proteins (which require amino groups). Gold in 10 μM 4,4-Dithio dibutyric acid; DDA (Tokyo Chemical Industry, Tokyo, Japan) ethanol solution (2.38 mg DDA dissolved in 10 mL 99% ethanol and diluted to 1/100 with ethanol) The membrane was immersed (gold side up) and gently stirred at room temperature for 30 minutes. Next, it was washed twice with ethanol without applying water pressure to the gold surface, and a self-assembled film (SA film) was introduced. 25 mg water-soluble carbodiimide; EDC (Wako Pure Chemical Industries) was dissolved in 1 mL ultrapure water, and 15 mg N-hydroxysuccinimide; NHS (Wako Pure Chemical Industries) was dissolved in 9 mL 1,4-Dioxane (Nacalai tesque, Inc.). The respective solutions were mixed, the gold film treated with the SA film was immersed, and gently stirred at room temperature for 10 minutes. To this, 10 mL of ultrapure water was added, and the mixture was further stirred at room temperature for 5 minutes. The gold surface was washed twice with ultrapure water without applying water pressure, dried (air-dried), and mounted on a cartridge. The cells were adjusted to 3 × 10 ⁵ cells / mL (PBS which is a flow buffer), and 20 μL was dropped on the gold membrane, and the cells were fixed for 30 minutes at room temperature. Then, inject EGCG dissolved in PBS 10 μM, or a solution of EGCG and peptide 10 μM mixed and reacted for 15 minutes, and measure the change in surface plasmon resonance angle (Angle) to bind EGCG to cells. The sex was examined. The sample flow rate was 30 μL / min.

2) Result:
The results are shown in FIGS. 11 and 12.
Peptide 151-170, Peptide 161-180, and Peptide 161-170, which neutralize the cell growth inhibitory activity of EGCG, all inhibited the cell binding properties of EGCG. In contrast, Peptide 156-165, Peptide 166-175, Peptide 162-170, and Peptide 161-169, which cannot neutralize the cell growth inhibitory activity of EGCG, did not inhibit EGCG cell binding (FIG. 11). .

  In addition, the amino acid substitutions of Peptide 161-170R and Peptide 161-170H, which neutralize the cell growth inhibitory activity of EGCG, inhibited the cell binding activity of EGCG, whereas Peptide 161-170R failed to neutralize the cell growth inhibitory activity. -170KS and HS did not inhibit EGCG cell binding (FIG. 12).

<Confirmation test of effects as anti-67LR ligand and prion inhibitor>
1) Materials and methods:
1.1) Green tea catechin:
Epigallocatechin-3-O-gallate (EGCG, sigma) is dissolved in phosphate buffer (PBS) to a concentration of 5 mM, stored frozen at −20 ° C., and diluted as appropriate. PBS is 8.0 g of NaCl (Nacalai tesque, Inc.), 0.2 g of KCl (Nacalai tesque, Inc.), 1.15 g of Na ₂ HPO ₄ (Nacalai tesque, Inc.), KH ₂ PO _{4 for} 1 L of ultrapure water. (Nacalai tesque, Inc.) Dissolve 0.2g and adjust.

1.2) Peptides:
67LR derived prion binding Peptide 161-179: IPCNNKGAHSVGLMWWLA
1.3) Cells and cell culture:
The human teratocarcinoma cell line NT2 was subcultured in 10% FBS (Bio Source International, Camarillo, Calif.)-Added DMEM medium (COSMO BIO CO., LTD.) At 37 ° C. under steam-saturated 5% CO ₂ conditions. maintain. In DMEM medium, 100 U / mL penicillin (Meiji pharmaceutical Company, Tokyo, Japan), 100 mg / mL streptomycin (Meiji pharmaceutical Company), 23.1 mM NaHCO ₃ (Wako Pure Chemical), and 25 mM HEPES (Wako Pure Chemical) are added. To do. Cells are maintained in culture in the logarithmic growth phase. In addition, it is recommended that the cells are frozen in large quantities at −80 ° C. at an early stage after the distribution, avoiding passage for a long period of time, and cryopreserved cells are appropriately thawed and used. Use a frozen culture medium prepared by mixing 10% dimethyl sulfoxide (Nacalai tesque, Inc. Kyoto, Japan), 20% FBS, and 70% DMEM medium.

2) Evaluation:
2.1) Examination of the inhibitory action of EGCG (or catechin-binding peptide) on the binding of prion-bound peptide 161-179 and prion:
Sample buffer (0.057M Tris-HCl, pH6.8, 1.8% (w / v) sodium dodecyl sulfate (SDS), 0.65Mβ-merchaptomethanol, 9.1% glycerol, 0.02% bromophenol blue ), And heat-denature at 100 ° C for 5 minutes. This is applied to a 10% polyacrylamide gel and subjected to SDS-polyacrylamide gel electrophoresis (PAGE) at 150V. After SDS-PAGE, the gel is electroblotted at 150V for 90 minutes with ice cooling, and the peptide is transferred to a nitrocellulose membrane. The membrane is blocked by adding 5% Bovine serum albumin (BSA) -TTBS (0.1% Tween 20-containing Tris buffered saline; 20 mM Tris-HCl, pH 7.6) at room temperature for 1 hour.

Prion-expressing NT2 cells were treated with cell lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% (v / v) Triton-X 100, 1 mM EDTA, 50 mM NaF, 30 mM Na ₄ P ₂ O _{7, 1mM Na 3 PO 4,} 1mM Phenylmethysulfonic fluoride, 2μg / mL Aprotinin) cells 1 x 10 ⁷ cells per 1mL was added, shaken for 30 minutes at 4 ° C. to dissolve the cells. Thereafter, the supernatant is collected by centrifugation at 16000 × g.

  The nitrocellulose membrane to which the peptide is transferred and the supernatant containing prion or the supernatant added with 5 μM EGCG (or the supernatant added with 5 μM catechin-binding peptide of the present invention) are reacted at 37 ° C. for 1 hour. After washing the membrane 3 times with TTBS, the membrane is reacted with an anti-prion antibody (MAB1562, CHEMICON International, Inc., USA) diluted 1000 times with BSA-TTBS at 37 ° C. for 1 hour. The membrane is washed 3 times with TTBS and then reacted with a secondary antibody (Mouse IgG-HRP, Santa Cruz Biotechnology) diluted 10,000 times with BSA-TTBS at room temperature for 1 hour. After washing with TTBS three times, luminescence reaction is performed using ECL kit (Enhanced Chemiluminesence; Amersham), and prion protein and peptide on membrane are bound using image analyzer ChemImager 5500 (Alpha Innotech, San Leandro CA.) To detect.

2.2) Examination of the inhibitory action of EGCG on the binding of 67LR and prion (immunoprecipitation method):
NT2 cells are suspended in 10% FBS-DMEM medium at 1 × 10 ⁵ cells / mL, seeded in a 10 mL dish (Nunc, Roskild, Denmark), pre-cultured for 24 hours, and the cells are allowed to adhere. Thereafter, the medium is replaced with a medium supplemented with 5 μM EGCG (or a medium supplemented with 5 μM catechin-binding peptide of the present invention) and incubated for 30 minutes. The cells are collected, washed with PBS, centrifuged, and the supernatant is removed. Then, the cells are suspended in a lysis buffer (200 μL), and lysed by stirring with a rotator at 4 ° C. for 30 minutes. Cell lysis buffer is Tris-HCl (5 mM), NaCl (150 mM), Triton-X100 (1% (v / v)), EDTA (1 mM), NaF (50 mM), Na ₄ P ₂ O ₇ (30 mM), pervanadate (1 mM), PMSF (1 mM), and aprotinin (2 μg / mL) are used to adjust the pH to 7.5, and pervanadate, PMSF, and aprotinin are added immediately before use. The cell lysate is centrifuged at 15,000 xg for 30 minutes to precipitate insoluble material, and the supernatant is used as a sample.

  In parallel with the above procedure, fully suspend protein A sepharose (Pharmacia Biotech Inc.) beads, dispense 20 μL into a 0.6 mL microtube in a 50% slurry, and remove the supernatant after centrifugation at 10,000 xg. Wash three times with 200 μL of cell lysis buffer. The protein A sepharose beads and the cell lysis buffer supplemented with anti-prion antibody (MAB1562, CHEMICON International, Inc., USA) are shaken at 4 ° C for 2 to 4 hours to adsorb the antibodies to the protein A sepharose beads. . Add cell lysis buffer containing anti-67LR antibody (F18, Santa Cruz Biotechnology) to protein G sepharose (Pharmacia Biotech Inc.) beads dispensed and washed as above, and shake at 4 ° C for 2-4 hours. The antibody is then adsorbed to protein G sepharose beads.

  The sample solution obtained above is suspended in addition to the beads adsorbed with the antibody washed 3 times with a cell lysis buffer, and immunoprecipitation is performed by shaking at 4 ° C. for 2 to 4 hours. Thereafter, the supernatant of the added sample solution is removed, and washing is performed 3 times each with 500 μL of cell lysis buffer and PBS. Add 20 μL of SDS Sample buffer (0.057M Tris-HCl, pH6.8, 1.8% (w / v) sodium dodecyl sulfate (SDS), 0.65 M β-merchaptomethanol, 9.1% glycerol, 0.02% bromophenol blue), and vortex Shake for 10 minutes to elute the immunoprecipitate, heat denature at 100 ° C. for 5 minutes, apply to 10% polyacrylamide gel, and perform SDS-polyacrylamide gel electrophoresis (PAGE) at 150V.

  After SDS-PAGE, the gel is electro-blotted at 150V for 90 minutes while ice-cooling to transfer the protein to a nitrocellulose membrane. To this membrane, 5% Bovine serum albumin (BSA) -TTBS (0.1% Tween 20-containing Tris buffered saline; 20 mM Tris-HCl, pH 7.6) is added, and blocking is performed at room temperature for 1 hour. After blocking, anti-67LR antibody (F-18, Santa Cruz Biotechnology) or anti-prion antibody (MAB1562, CHEMICON International, Inc., USA) is diluted 1000 times with 5% BSA-TTBS and reacted overnight at 4 ° C. After washing 3 times with TTBS, anti-67LR secondary antibody (Goat IgG-HRP G2804, Santa Cruz Biotechnology) or anti-prion secondary antibody (Mouse IgG-HRP, Santa Cruz Biotechnology) with 5% BSA-TTBS Dilute 10,000 times and react at room temperature for 1 hour. After washing 3 times with TTBS, color reaction is performed using an ECL kit (Enhanced Chemiluminesence; Amersham), and detection is performed using an image analyzer ChemImager 5500 (Alpha Innotech, San Leandro CA.).

2.3) Examination of the inhibitory action of EGCG (or catechin-binding peptide) on the binding between cell surface 67LR and recombinant prion protein (ELISA method)
NT2 cells are suspended in 10% FBS-DMEM medium at 1 × 10 ⁵ cells / mL, added to a 96-well immunoplate (Nunc, Roskild, Denmark) at 100 μL / wel, and incubated at 37 ° C. for 24 hours. After washing 3 times with TTBS, 3.7% formaldehyde is added at 100 μL / well, and the cells are allowed to stand for 30 minutes at room temperature to fix the cells. After washing 3 times with TTBS, add 300 μL / well of BSA-TTBS and incubate at 37 ° C. for 1 hour to block.

  After washing 3 times with TTBS, a recombinant prion protein (PrP-GST (Molecular Biotechnology)) labeled with glutathione S transferase (GST) diluted with PBS to 10 μg / mL, or PrP− supplemented with 5 μM EGCG GST solution (or PrP-GST solution to which 5 μM catechin-binding peptide of the present invention is added) is added at 100 μL / well and incubated at 37 ° C. for 1 hour. After washing 3 times with TTBS, glutathione S-transferase (GST) -labeled prion (PrP-GST (Molecular Biotechnology)) diluted with PBS to 10 µg / mL is added at 100 µL / well and incubated at 37 ° C for 1 hour To do. After washing 3 times with TTBS, anti-GST antibody (G7781, Sigma) diluted 1000-fold with PBS is added at 100 μL / well and incubated at 37 ° C. for 1 hour. After washing 3 times with TTBS, a secondary antibody (Rabbit IgG-HRP, Santa Cruz Biotechnology) is added at 100 μL / well and incubated at 37 ° C. for 1 hour.

  After washing 3 times with TTBS, the substrate solution is added at 100 μL / well, and the color is developed by incubating at 37 ° C. for 10 minutes. The substrate solution is prepared by mixing 4.5 mL water, 6 mL 0.1 M Citrate buffer, 0.5 mL 6 mg / mL ABTS. When the luminescence is complete, add 1.5% Oxialic acid at 100 μL / well to stop the enzymatic reaction. For color development, the absorbance at 415 nm is measured using a microplate reader.

FIG. 1 is a graph showing the effect of a 67LR extracellular partial peptide on the cell growth inhibitory action of EGCG. In the graph, the white bar indicates a system without EGCG added, and the black bar indicates a system with 1 μM EGCG added (means ± SD (n = 3), p <.0.05 ^* , p <0.01 ^** , p <0.001 ^{**) *} : The same applies to FIGS. Peptide 161-180 was found to have neutralizing activity against the cell growth inhibitory action of EGCG. FIG. 2 is a graph showing the effect of the 67LR extracellular partial peptide on the cell growth inhibitory action of EGCG. Peptide 161-180 and Peptide 151-170 showed almost the same activity as Peptide 161-170. FIG. 3 is a graph showing the effect of Peptide 161-170 and its terminal amino acid lacking peptide on the cell growth inhibitory action of EGCG. When one amino acid was deleted from each of the N and C terminals, the neutralizing activity of Peptide 161-170 on the cell growth inhibitory action of EGCG disappeared. FIG. 4 is a graph showing a comparison of the neutralizing activities of Peptide 161-170 and recombinant 67LR protein on the cell growth inhibitory action of EGCG. Peptide 161-170 exceeded the recombinant 67LR protein in the neutralizing activity of EGCG on the cell growth inhibitory action. FIG. 5 is an MS spectrum showing that the 67LR extracellular partial peptide has binding activity to EGCG. When a mixed solution of EGCG and a 67LR partial peptide was injected into an MS spectrometer, a complex peak of Peptide 161-180 and EGCG was observed. FIG. 6 is an MS spectrum showing that 67LR extracellular partial peptide has binding activity to EGCG. When a mixed solution of EGCG and 67LR partial peptide was injected into an MS spectrometer, a complex peak of Peptide 161-170 and EGCG was observed. FIG. 7 is a graph showing the effect of basic amino acid substitution on the cell growth inhibitory action of EGCG. The peptide in which K was substituted with R or H also showed neutralizing activity against the cell growth inhibitory effect (A), but the peptide in which K and H were substituted with S each lost neutralizing activity (B). FIG. 8 is a graph showing that Biot-p160-170 was immobilized on a streptavidin carrier column. After applying Biot-p160-170 to a streptavidin carrier column, a binding buffer was passed, and the absorbance at 220 nm, the absorption maximum of Biot-p160-170, was measured. FIG. 9 is a graph showing that a streptavidin carrier not bound with Biot-p160-170 does not bind catechins and is recovered as a flow-through fraction. After EC or EGCG was applied to the column, 1 mL of the eluted fraction from the column was collected, and the absorbance at 276 nm and 273 nm, which are the absorption maximums of EC and EGCG, was measured. FIG. 10 is a graph plotting values of EGCG concentration / FITC-p160-170 concentration against relative fluorescence intensity. EGCG is mixed with a solution of FITC-p160-170 (the N-terminal of Peptide 160-170 labeled with FITC) at a predetermined concentration and allowed to stand for a sufficient period of time. Then, fluorescence at an excitation wavelength of 365 nm and a fluorescence wavelength of 368 nm is obtained. The strength was measured. Since the fluorescence intensity of FITC-p160-170 decreases due to the binding of EGCG, the degree of change of this FITC-p160-170 is expressed as the relative fluorescence intensity (formula: relative fluorescence intensity = FITC-p160-170 + EGCG fluorescence intensity). / Fluorescence intensity of FITC-p160-170). From the plot in FIG. 10, the formula: Y = 49.436e ^{? 9.6182X} is obtained (X is the relative fluorescence intensity, Y is the EGCG concentration / FITC-p160-170 concentration), and the amount of EGCG in the EGCG-containing sample can be obtained. . FIG. 11 shows the results of measuring the influence of a 67LR-derived peptide on the EGCG binding property of HepG2 using a surface plasmon resonance sensor. Peptide 151-170, Peptide 161-180, and Peptide 161-170, which neutralize the cell growth inhibitory activity of EGCG, all inhibited the cell binding properties of EGCG. In contrast, Peptide 156-165, Peptide 166-175, Peptide 162-170, and Peptide 161-169, which cannot neutralize the cell growth inhibitory activity of EGCG, did not inhibit the cell binding properties of EGCG. FIG. 12 shows the result of measuring the influence of 67LR-derived peptide on the EGCG binding property of HepG2 using a surface plasmon resonance sensor. Peptide 161-170R and Peptide 161-170H, which neutralize the cell growth inhibitory activity of EGCG, both inhibited the cell binding activity of EGCG, whereas Peptide 161-170KS failed to neutralize the cell growth inhibitory activity HS did not inhibit the cell binding of EGCG.

Claims (15)

The following peptide (a), (b) or (c):
(A) a peptide consisting of the amino acid sequence of SEQ ID NO: 15;
(B) SEQ ID NO: in the amino acid sequence of 15, 1 made of nine amino acids have been pressurized with, and peptides having a binding activity to epigallocatechin gallate (EGCG), or SEQ ID NO: 6 or A peptide consisting of the amino acid sequence of SEQ ID NO: 7 ; or (c) a peptide consisting of the amino acid sequence of SEQ ID NO: 31 or SEQ ID NO: 32 . The peptide according to claim 1, which is a peptide consisting of the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 31 or SEQ ID NO: 32.   The peptide according to claim 1 or 2, which is labeled and / or immobilized. SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 or SEQ ID NO: 32 Lupe peptide such an amino acid sequence. SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 31 or SEQ ID NO: 32 consists of the following amino acid sequence peptides of claim 4.   An antioxidant comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 26.   A method for detecting galloylcatechins, wherein the peptide according to claim 1 is used.   The manufacturing method of galloylcatechin including the process of concentrating and / or refine | purifying galloylcatechin using the peptide of Claim 1.   A method for immobilizing galloylcatechins, wherein the peptide of claim 1 is used.   A conjugate of the peptide of claim 1 and galloylcatechins. A method for promoting the antioxidant activity of galloylcatechins, comprising using a peptide consisting of the amino acid sequence of SEQ ID NO: 15 . An accelerator for the antioxidant activity of galloylcatechins, comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 15 . (1) preparing a test compound;
(2) assessing the binding of the test compound to the peptide of claim 1; and (3) including the step of selecting the compound when the test compound has a binding property to the peptide. Screening method for mimetic compound candidates.   A method for concentrating and / or purifying galloylcatechin mimetic compound candidates using the peptide of claim 1. A method for detecting a disease predisposition that can be treated with galloylcatechins, comprising detecting a mutation in a base sequence encoding a portion consisting of amino acid sequences of amino acid numbers 161 to 170 of a 67 kDa laminin receptor .

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