JP2009096731A - Carboxymethyl arginine production inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a carboxymethyl arginine production inhibitor that controls production of carboxymethyl arginine being a Maillard reaction advanced glycation end product (AGEs) of collagen and functional disorder of in vivo protein. <P>SOLUTION: The carboxymethyl arginine production inhibitor comprises rutin, quercetin, astragalin, quercetin 3-O-sambubioside, luteolin-7-O-glucoside, pentaacetate quercetin or the like as an active ingredient. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a carboxymethylarginine production inhibitor.

  Proteins in the body are saccharified by reacting with reducing sugars non-enzymatically. This reaction is generally called the Maillard reaction, and is composed of an early stage reaction and a late stage reaction. The early stage of the Maillard reaction is that the amino acid side chain amino group or N-terminal amino group of the protein reacts with the carbonyl group of the sugar to produce an Amadori rearrangement compound via a Schiff base. As products of this reaction, hemoglobin A1C and glycated albumin are known, and these products are widely used as clinical markers of diabetes. Basically, all proteins in the living body are considered to be glycated at present, and many protein dysfunctions as a result have been reported.

  The latter stage of the Maillard reaction is a Maillard reaction characterized by fluorescence, browning change or intramolecular / intermolecular cross-link formation through complex irreversible reactions such as dehydration, oxidation, condensation, etc. To yield the final product. The final product in the late stage is called AGEs (Advanced Glycation End products). It is known that late products of the Maillard reaction (AGEs) accumulate in biological proteins with aging, for example, collagen subjected to AGE induces apoptosis in fibroblasts. Carboxymethylarginine (CMA) is known as one of the AGE structures that specifically accumulate in collagen. Patent Document 1 describes an antibody against a peptide or protein having an N-carboxymethylarginine or N-carboxymethylarginine residue, and an immunoreagent containing the antibody, and the antibody is used for CMA detection or glycation. It is described that it is useful for research.

JP 2002-243732 A

  As described above, carboxymethylarginine (CMA) is a type of AGE structure that specifically accumulates in collagen. If a substance that suppresses the production of CMA is found, it may be possible that the substance can suppress the denaturation of biological proteins such as skin and blood vessels by suppressing the AGE conversion of collagen. That is, the present invention has identified a substance having an activity capable of suppressing the production of carboxymethylarginine and provided a carboxymethylarginine production inhibitor.

  The present inventors have intensively studied to solve the above-mentioned problems.First, an antibody specific to carboxymethylarginine (CMA), which is one of AGE structures that specifically accumulate in collagen, is prepared, and CMA Compounds that inhibit production were searched. As a result, it was clarified that the compound represented by the formula (1), formula (2) or formula (3) described in the present specification remarkably suppresses the production of CMA. Furthermore, it was also confirmed that these compounds represented by the formulas (1) to (3) did not show toxicity to fibroblasts. The present invention has been completed based on these findings.

That is, according to the present invention, the following inventions are provided.
(1) A carboxymethylarginine production inhibitor comprising as an active ingredient a compound represented by the following formula (1), formula (2) or formula (3), or a salt thereof, or a hydrate or solvate thereof .
(Wherein R ₁ represents a hydrogen atom, —COCH ₃ , or a sugar residue, R ₂ represents a hydrogen atom or —COCH ₃ , and R ₃ represents a hydrogen atom, —OH, —OCOCH ₃ , or — O ₄ represents an O-sugar, R ₄ represents a hydrogen atom, —OH, or —OCOCH ₃ , R ₅ represents —OH, or —OCOCH ₃ , Xyl represents a β-D-xylopyranosyl group, glcA represents β -D-glucuronopyranosyl group, rha represents α-L-rhamnopyranoside group.

(2) The compound represented by the formula (1) is rutin, quercetin, astragalin, quercetin 3-O-sambubioside, luteolin-7-O- The carboxymethylarginine production inhibitor according to (1), which is either glucoside (Luteolin-7-O-glucoside) or pentaacetate quercetin.

(3) The carboxymethylarginine production inhibitor according to (1) or (2), which is used for inhibiting the late product (AGE) of collagen from the Maillard reaction.

(4) The carboxymethylarginine production inhibitor according to any one of (1) to (3), which is used as a skin external preparation.

  The present invention provides a novel carboxymethylarginine production inhibitor. Moreover, it has been confirmed that the carboxymethylarginine production inhibitor of the present invention does not show toxicity to fibroblasts. The carboxymethylarginine production inhibitor of the present invention can suppress denaturation of biological proteins such as skin and blood vessels by suppressing collagen AGE formation.

Hereinafter, the present invention will be described in detail.
Attempts have been made to suppress the generation of AGE accumulated in the skin and suppress the aging of the skin, but the AGE is quantified by measuring fluorescence, and the AGE structure is not specified. The present inventor paid attention to carboxymethylarginine (CMA), and since this structure is unstable to acid hydrolysis, it is difficult to measure by instrumental analysis by a normal method. An antibody was prepared and a measurement system for CMA was established. As a result, it became possible to search for compounds showing CMA production inhibitory action. Moreover, although the compound which suppresses the production | generation of carboxymethyl lysine (CML) is reported, this inventor also confirmed that carboxymethyl arginine (CMA) is an AGE structure which produces | generates significantly in collagen.

(1) Method for Producing Monoclonal Anti-CMA Antibody Carboxymethylarginine can be used as an antigen for producing a monoclonal anti-CMA antibody. Carboxymethylarginine reacts arginine and glyoxal in NaOH solution, neutralized with HCl, applied to an ion exchange column, and each fraction was developed with acetonitrile using thin layer chromatography, and ninhydrin positive And can be obtained by collecting a fraction containing a compound different from arginine.

(2) Preparation of monoclonal antibody A monoclonal antibody can be prepared by a conventional method. In order to produce a monoclonal antibody, first, CMA conjugated to a protein such as hemocyanin protein or human serum albumin is used as an antigen to a mammal such as a rat, mouse, rabbit or the like. The dose of the antigen per animal is 0.1 to 100 mg when no adjuvant is used, and 1 to 100 μg when an adjuvant is used. Examples of adjuvants include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminum hydroxide adjuvant. Immunization is performed mainly by injecting intravenously, subcutaneously or intraperitoneally. The immunization interval is not particularly limited, and immunization is performed 1 to 10 times, preferably 2 to 5 times at intervals of several days to several weeks, preferably 2 to 5 weeks. Then, antibody-producing cells are collected 1 to 60 days after the last immunization day, preferably 1 to 14 days later. Examples of antibody-producing cells include spleen cells, lymph node cells, peripheral blood cells, etc., but spleen cells or local lymph node cells are preferred.

  In order to obtain a cell fusion hybridoma, cell fusion between antibody-producing cells and myeloma cells is performed. As myeloma cells to be fused with antibody-producing cells, generally available cell lines of animals such as mice can be used. The cell line to be used has drug selectivity and cannot survive in a HAT selection medium (including hypoxanthine, aminopterin, and thymidine) in an unfused state, but can survive only in a state fused with antibody-producing cells. Those having the following are preferred. Examples of myeloma cells include mouse myeloma cell lines such as P3X63-Ag.8.U1 (P3U1) and NS-I.

Next, the myeloma cell and the antibody-producing cell are fused. Cell fusion is performed in animal cell culture media such as serum-free DMEM and RPMI-1640 media, and 1 × 10 ⁶ to 1 × 10 ⁷ antibody-producing cells and 2 × 10 ⁵ to 2 × 10 ^6. 1 / ml myeloma cells are mixed (a cell ratio of antibody-producing cells to myeloma cells is preferably 5: 1), and a fusion reaction is performed in the presence of a cell fusion promoter. As the cell fusion promoter, polyethylene glycol having an average molecular weight of 1000 to 6000 daltons or the like can be used. Alternatively, antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device utilizing electrical stimulation (for example, electroporation).

The target hybridoma is selected from the cells after cell fusion treatment. As a method, the cell suspension is appropriately diluted with, for example, fetal bovine serum-containing RPMI-1640 medium, then plated on a microtiter plate at about 3 × 10 ⁵ cells / well, a selective medium is added to each well, and thereafter appropriate. The culture is carried out with the selective medium changed. As a result, cells that grow from about 14 days after the start of culture in the selective medium can be obtained as hybridomas.

  Next, it is screened whether the target antibody exists in the culture supernatant of the hybridoma which has proliferated. Hybridoma screening is not particularly limited, and may be performed according to ordinary methods. For example, a part of the culture supernatant contained in a well grown as a hybridoma can be collected and screened by a labeled immunoassay such as an enzyme immunoassay or a radioimmunoassay. Cloning of the fused cells is performed by limiting dilution or the like, and finally a hybridoma that is a monoclonal antibody-producing cell is established.

As a method for collecting the monoclonal antibody from the established hybridoma, a normal cell culture method or ascites formation method can be employed. In the cell culture method, the hybridoma is cultured in an animal cell culture medium such as RPMI-1640 medium containing 10% fetal bovine serum, MEM medium, or serum-free medium under normal culture conditions (eg, 37 ° C., 5% CO ₂ concentration) Cultivate for 7 to 14 days and obtain antibody from the culture supernatant.

In the case of the ascites formation method, about 1 × 10 ⁷ hybridomas are administered into the abdominal cavity of a mammal derived from a myeloma cell and the same strain, and the hybridomas are proliferated in large quantities. And ascitic fluid is collected after 1-2 weeks. When antibody purification is required in the antibody collection method, known methods such as ammonium sulfate salting-out method, ion exchange chromatography, gel filtration, affinity chromatography are appropriately selected, or a combination thereof is used. Can be purified.

(3) ELISA method As a suitable example of the labeled immunoassay, an ELISA method which is an immunoassay using an enzyme as a label can be mentioned. In the ELISA method, for example, a specimen or a diluted solution thereof is placed in a 96-well plate and left overnight at 4 ° C. to room temperature or at 37 ° C. for about 1 to 3 hours to adsorb carboxymethylarginine to be detected. Solidify. Next, the antibody of the present invention is reacted, and then an anti-immunoglobulin antibody (secondary antibody) conjugated with an enzyme in advance is reacted. Finally, an appropriate chromogenic substrate that reacts with the enzyme (for example, p-nitrophenyl phosphate if the enzyme is phosphatase) is added, and the antibody is detected by this color development.

(4) Carboxymethylarginine production inhibition (inhibition) test Collagen or gelatin was incubated in a test tube at 37 ° C. for 1 week in the presence of glucose or ribose and a candidate compound for carboxymethylarginine production inhibitor and dialyzed against PBS. Thereafter, the production inhibitory effect can be examined by ELISA using a monoclonal antibody against carboxymethylarginine.

In the present invention, the compound represented by the following formula (1), formula (2) or formula (3) was identified as a compound having a carboxymethylarginine production inhibitory action by the above test.
(Wherein R ₁ represents a hydrogen atom, —COCH ₃ , or a sugar residue, R ₂ represents a hydrogen atom or —COCH ₃ , and R ₃ represents a hydrogen atom, —OH, —OCOCH ₃ , or — O ₄ represents an O-sugar, R ₄ represents a hydrogen atom, —OH, or —OCOCH ₃ , R ₅ represents —OH, or —OCOCH ₃ , Xyl represents a β-D-xylopyranosyl group, glcA represents β -D-glucuronopyranosyl group, rha represents α-L-rhamnopyranoside group.

The type of sugar in the sugar residue represented by R ₁ and the —O-sugar represented by R ₃ is not particularly limited, and examples thereof include glucose, galactose, rhamnose, glucose-glucose, and glucose-rhamnose.

  As an active ingredient of the carboxymethylarginine production inhibitor of the present invention, a physiologically acceptable salt is used in addition to a free form compound represented by formula (1), formula (2) or formula (3). Also good. Physiologically acceptable salts include salts with alkali metals such as sodium and potassium; salts with alkaline earth metals such as magnesium; ammonia, ethanolamine, 2-amino-2-methyl-1-propanol and the like Examples include salts with amines. In addition, the type of salt is not particularly limited as long as it is physiologically acceptable.

  The compounds of the formulas (1) to (3) used in the present invention are known compounds, or can be prepared and obtained by the method described in Example 1 in the present specification or a method analogous thereto. In order to extract and separate a compound from a crude drug / natural product, first, components contained in the crude drug / natural product are eluted in methanol or water. Thereafter, the obtained methanol extract and water extract are gradually separated by various chromatography and finally separated into a single compound. Some compounds are derivatized by organic synthesis techniques. The isolated compound can be subjected to structural analysis using nuclear magnetic resonance (NMR) spectroscopy or mass (MS) analysis to determine the chemical structure.

The compounds used in the present invention are also described in the following documents.
Rutin and Quercetin;
Li, Xiangjun; Zhang, Yuping; Yuan, Zhuobin. Chromatographia (2002), 55 (3/4), 243-246.

Astragalin;
Takamura, Chika; Hirata, Tetsuya; Yamaguchi, Yasuyo; Ono, Masateru; Miyashita, Hiroyuki; Ikeda, Tsuyoshi; Nohara, Toshihiro. Journal of Natural Medicines (2007), 61 (2), 220-221.

Quercetin 3-O-sambubioside;
Webby, Rosemary F. DSIR, Petone, NZ Phytochemistry (1991), 30 (7), 2443-4.

Luteolin-7-O-glucoside;
Hatam, Natiq AR; Seifert, K. Planta Medica (1994), 60 (6), 600.

  The dosage when the carboxymethylarginine production inhibitor of the present invention is administered to a subject can be appropriately set according to the age, weight, symptom, etc. of the subject. As an active ingredient, 0.1 to 1000 mg / kg body weight, particularly 0.1 to 500 mg / kg body weight can be administered in 1 to several divided doses.

  The administration route of the carboxymethylarginine production inhibitor of the present invention is not particularly limited. For example, oral administration or parenteral administration (applied to the skin, or intravenous injection, subcutaneous injection, intradermal injection, intraperitoneal injection, intramuscular injection) Administration).

  In addition to the compound represented by the formula (1), formula (2) or formula (3) contained as an active ingredient, the carboxymethylarginine production inhibitor of the present invention contains additives usually used in pharmaceutical compositions. Can be contained. Such optional additives include excipients, binders, disintegrants, emulsifiers, solubilizers, dispersants, lubricants, coating agents, colorants, stabilizers, isotonic agents, and the like. However, it is not limited to these. As an acupuncture agent, saccharides such as lactose, sucrose, glucose etc., inorganic substances such as starch, calcium carbonate, calcium sulfate, crystalline cellulose, distilled water, purified water, sesame oil, soybean oil, corn oil, olive oil, cottonseed oil, etc. What is being done can be illustrated. The carboxymethylarginine production inhibitor of the present invention can be formulated by a conventional method using these additives. In addition, the carboxymethylarginine production inhibitor of the present invention can be used in combination with other pharmaceuticals or used in combination.

  For example, the carboxymethylarginine production inhibitor of the present invention can be used as a skin external preparation. Examples of the external preparation for skin include, but are not limited to, lotions, creams, ointments, emulsions, packs and the like. In addition, for topical skin preparations, polyhydric alcohols, moisturizers, thickeners, hydrocarbons, esters, alcohols, higher fatty acids, surfactants, powder components, colorants, fragrances, antioxidants, ultraviolet rays are optionally used One or more components selected from an absorbent, an anti-inflammatory agent and the like can be appropriately blended.

  The carboxymethylarginine production inhibitor of the present invention can also be produced as a food form. Examples of the food form include beverages, solid or semi-solid foods, and the like, and can also be used as food for specified health use.

  The following examples further illustrate the present invention. However, the scope of the present invention is not limited by these examples.

Example 1: Preparation of various inhibitors (1) Extraction of 3-O-β-D-xylopyranosyl- (1 → 2) -β-D-glucuronopyranosyl complogenin 22-O-α-L-rhamnopyranoside (Complogenin glycoside) Separation method Put 1.5 kg of the root of the herbal medicine "Gueldenstaedtia multiflora BGE." Add methanol, and then heat in a water bath at 80-100 ° C. for 5 hours to elute the components contained in the paddle in methanol. The methanol eluate was placed in an eggplant-shaped flask, and the flask was attached to an evaporator and methanol was removed to obtain 111 g of a methanol extract. This methanol extract was applied to Diaion HP-20 column chromatography (70 mm × 500 mm glass open column) and eluted successively with water (6 L), methanol (6 L) and acetone (3 L). Next, the methanol elution fraction (10 g out of 43 g) was subjected to MCI gel column chromatography (40 mm x 600 mm glass open column), and 40% methanol / water (4 L), 50% methanol / water (2 L). ), 60% methanol / water (2 L), 70% methanol / water (2 L), 80% methanol / water (2 L), and 6 fractions (60% methanol / water) Fr. 1 to Fr. 6) were fractionated. Further, the Fr. 4 (468.7 mg) was subjected to Chromatrex ODS column chromatography (20 mm × 400 mm glass open column), and 8 fractions (Fr. I to Fr.) were added using a solvent system 60% methanol / water (1 L). VIII) Fractionation, apply Fr. II (145.4 mg) to silica gel chromatography (15 mm x 170 mm glass open column), and elute with solvent system (chloroform: methanol: water = 7: 3: 0.5, 0.3 L) Thus, 3-O-β-D-xylopyranosyl- (1 → 2) -β-D-glucuronopyranosyl complogenin 22-O-α-L-rhamnopyranoside (9.5 mg) was isolated.

(2) Extraction and separation of Astragalin and Quercetin 3-O-sambubioside Put 577.6 g of Eucommia ulmoides Oliv. Green leaves into a 2 L container and extract with water (65 ° C) for 5 hours. The liquid was filtered to obtain a filtrate (1.5 L). The filtrate was then subjected to Diaion HP-20 column chromatography (70 mm x 500 mm glass open column), water (6 L), 30% methanol / water (10 L), 50% methanol / water (6 L ), 80% methanol / water (6 L), and methanol (6 L). The 50% methanol / water elution fraction (9.0 g) was subjected to Shephadex LH-20 column chromatography (35 mm x 700 mm glass open column), and the solvent system 50% methanol / water (2 L) and methanol (2 L ) Were fractionated into 8 fractions (Fr. 1 to Fr. 8). The Fr. 6 was astragalin (55 mg). Furthermore, Fr. 4 (441 mg) was applied to Chromatrex ODS column chromatography (30 mm x 300 mm glass open column) and eluted with 30% methanol / water (1 L) solvent system to obtain quercetin 3-O-sambubioside ( 155 mg) was isolated.

(3) Extraction and separation of Luteolin-7-O-glucoside Place 310 g of leaves of Catalpa bignonioides Walt. Then, heat is applied in a water bath at 80 to 100 ° C. for 5 hours to elute the components contained in the red bean in methanol. The methanol eluate was placed in an eggplant-shaped flask, and the flask was attached to an evaporator and methanol was removed to obtain 50.8 g of a methanol extract. This methanol extract was applied to Diaion HP-20 column chromatography (50 mm × 300 mm glass open column) and eluted successively with water (2 L), methanol (1.8 L) and acetone (1 L). Next, the methanol elution fraction (19.5 g) was applied to Toyo peal HW-40C column chromatography (40 mm x 500 mm glass open column), and 6 fractions (Fr. 1 to Fr. Fractionated into 6). Further, the Fr. 4 (1.73 g) was subjected to Sephadex LH-20 column chromatography (25 mm × 700 mm glass open column), and 7 fractions (Fr. I to Fr. VII) were added using a solvent system, methanol (1 L). Fr. IIL (306 mg) is subjected to silica gel chromatography (25 mm x 300 mm glass open column) and eluted with a solvent system (chloroform: methanol: water = 9: 1: 0.1, 1 L). Luteolin-7-O-glucoside was isolated.

(4) Preparation of Penta acetate quercetin
Quercetin (300 mg) is weighed into a 100 ml cocoon flask, pyridine (2 ml) and acetic anhydride (2 ml) are added, and the mixture is stirred at room temperature for 2 hours. Ice is added to the reaction solution, and the deposited precipitate is collected by suction filtration with a Buchner funnel (diameter 3 cm). Ethanol (5 ml) was added to the resulting precipitate (400 mg), heated and dissolved, and left at room temperature overnight to obtain Penta acetate quercetin (280 mg) as needle crystals.

(5) (+)-Catechin, Rutin, Quercetin purchased commercially available products (Wako Pure Chemical Industries) and used in the following.

(6) About the substance for comparison The substance for comparison is described in the following documents.
Kakkalide;
Yamazaki, Takashi; Nakajima, Yoshijiro; Niiho, Yujiro; Hosono, Tsuyoshi; Kurashige, Tatsuo; Kinjo, Junei; Nohara, Toshihiro. Journal of Pharmacy and Pharmacology (1997), 49 (8), 831-833.
Medicarpin;
Soby, Scott; Caldera, Sriyani; Bates, Robert; VanEtten, Hans. Phytochemistry (1996), 41 (3), 759-65.
Medicarpin-β-glucoside;
Al-Khalil, Suleiman; Masalmeh, Amneh; Abdalla, Shtaywy; Tosa, Hideki; Iinuma, Munekazu. Journal of Natural Products (1995), 58 (5), 760-3.

Esculeoside A and Esculeogein A;
Fujiwara, Yukio; Takaki, Ayumi; Uehara, Yukie; Ikeda, Tsuyoshi; Okawa, Masafumi; Yamauchi, Ken; Ono, Masateru; Yoshimitsu, Hitoshi; Nohara, Toshihiro.Tetrahedron (2004), 60 (22), 4915-4920.
Tomatine;
Ikeda, Tsuyoshi; Yamauchi, Ken; Nakano, Daisuke; Nakanishi, Kenji; Miyashita, Hiroyuki; Ito, Shin-ichi; Nohara, Toshihiro. Tetrahedron Letters (2006), 47 (26), 4355-4359.
Desgalactotigonin;
Ikeda, Tsuyoshi; Tsumagari, Hidetsugu; Honbu, Takehiko; Nohara, Toshihiro. Biological & Pharmaceutical Bulletin (2003), 26 (8), 1198-1201.

Neoaspidistrin;
Chen, Changxiang; Zhou, Jun. Yunnan Zhiwu Yanjiu (1994), 16 (4), 397-400.
Timosaponin AIII;
Meng, Zhi-Yun; Zhang, Jian-Ying; Xu, Sui-Xu; Sagahara, Kazunori.Plana Medica (1999), 65 (7), 661-663.

Dioscin;
Ono, Masateru; Nishimura, Kazuya; Suzuki, Keita; Fukushima, Takeshi; Igoshi, Keiji; Yoshimitsu, Hitoshi; Ikeda, Tsuyoshi; Nohara, Toshihiro. Chemical & Pharmaceutical Bulletin (2006), 54 (2), 230-233.
Isotubocaposide B and Isotubocaposigenin;
Kiyota, Naoko; Shingu, Kazushi; Yamaguchi, Koki; Yoshitake, Yasuyuki; Harano, Kazunobu; Yoshimitsu, Hitoshi; Ikeda, Tsuyoshi; Nohara, Toshihiro. Chemical & Pharmaceutical Bulletin (2007), 55 (1), 34-36.
Diosgenin;
Noguchi, Eishin; Fujiwara, Yukio; Matsushita, Sayaka; Ikeda, Tsuyoshi; Ono, Masateru; Nohara, Toshihiro. Chemical & Pharmaceutical Bulletin (2006), 54 (9), 1312-1314.
Cilistol A
Zhu, X.-H .; Takagi, M .; Ikeda, T .; Midzuki, K .; Nohara, T. Phytochemistry (2001), 56 (7), 741-745.

Example 2: Production of monoclonal antibody against CMA or CMA-modified protein (1) Production method of carboxymethylarginine (CMA)
0.1 M arginine and 0.1 M glyoxal were reacted in 25 mL of 1 N NaOH solution at 37 ° C. for 24 hours, and neutralized with 1H HCl. After applying to 10 mL of ion exchange column Dowex 50, 1 mL was collected, and each fraction was developed with 80% acetonitrile using thin layer chromatography, and fractions containing compounds that were ninhydrin positive and different from arginine were collected. It was collected. Then, it confirmed that it was a single compound with the amino acid analyzer. Thereafter, structural analysis was performed by NMR, and it was confirmed that it was the same as CMA (Iijima K et al., Biochem. J. 347, 23-27, 2000).

(2) Production of monoclonal antibodies against CMA or CMA-modified proteins
(i) Collection of antibody producing cells 1 mg CMA 1 mg in 10 mg carbodiimid and 4 mg hemocyanin protein (KLH: keyhole limpet hemocyanin) or human serum albumin (HSA) and 1 ml phosphate buffer After reacting for a while, CMA-KLH and CMA-HSA, which are CMA adducts, were prepared. Each mouse was then immunized with 0.1 mg of CMA-KLH with Freund's complete adjuvant (FCA), and after 2 weeks and 4 weeks, the same amount of CMA-KLH was boosted intradermally with Freund's incomplete adjuvant (FIA). Went. Two weeks after the third immunization, blood was collected from the tail vein and the antibody titer was evaluated by ELISA using CMA-HSA as an antigen. Antisera diluted 5000 times and CMA-HSA reacted significantly. After confirming that the spleen was removed from the animal, antibody-producing cells were collected.

(ii) Cell fusion In order to obtain a hybridoma, cell fusion between antibody-producing cells and myeloma cells (P3U1) is performed. Cell fusion was performed by averaging 1 × 10 ⁶ to 1 × 10 ⁷ antibody-producing cells and 2 × 10 ⁵ to 2 × 10 ⁶ cells / ml myeloma cells in RPMI-1640 medium without serum. Mixing was performed in the presence of polyethylene glycol having a molecular weight of 1000 to 6000 daltons. Then, after culturing for 10 days in the presence of HAT medium, 0.05 ml of the culture supernatant was collected and used as the primary antibody of ELISA. In ELISA, 0.05 μl of 10 μg / ml CMA-KLH or CML-HSA was immobilized on each well. The primary antibody bound to the antibody is detected with an anti-mouse IgG antibody labeled with HRP, and then 1,2-phenylenediamine The color was developed with dihydrochloride, and the absorbance at 492 nm was measured with an ELISA reader. Cloning of the fused cells was performed by limiting dilution or the like, and finally hybridomas that were CMA-KLH positive and CML-HSA negative monoclonal antibody producing cells were established. Thereafter, a similar AGE ^{structures, CML, N e - (carboxyethyl} ) lysine (CEL), S- (carboxymethyl) cysteine (CMC), a strain that does not exhibit arginine crossreactive unmodified selected by ELISA.

(iii) Collection of Monoclonal Antibody About 1 × 10 ⁷ hybridomas obtained were intraperitoneally administered to Balb / c mice pre-administered with 0.5 ml of pristane, and ascites was collected approximately 10 days later. Immunoglobulin G was purified from the obtained ascites using protein G affinity chromatography. The antibody (3F5) produced by this hybridoma was IgG1 as a result of isotyping.

(Iv) Reactivity of monoclonal anti-CMA antibody The reactivity of the monoclonal antibody (hereinafter referred to as monoclonal antibody 3F5) produced by the hybridoma 3F5 obtained above was examined. Specifically, after 0.1 ml of CMA-HSA (0.1 μg / ml) was immobilized on an immunoplate, 0.05 ml of CMA, CML, CEL, CMC, arginine and 0.1 μg / ml were serially diluted from 1 mM. An equal amount of 3F5 was mixed, and 0.1 ml of the mixture was added to each well of the immunoplate. Thereafter, antibody 3F5 bound to the immunoplate was detected with an HRP-labeled anti-mouse IgG antibody.

The reactivity of monoclonal antibody 3F5 with CMA is shown in FIG. As shown in FIG. 1, the monoclonal antibody 3F5 of the present invention is its similar structure ^{N e - (carboxymethyl) lysine (} CML), did not show CEL, CMC, and arginine and cross-reactivity. Normally, it shows a cross-reactivity with a similar AGE structure, but the antibody of the present invention has a very high specificity, so that CMA can be specifically detected.

Example 3: Measurement of carboxymethylarginine in a reaction solution of ribose and gelatin
Incubate 5 mg / ml ribose and 2 mg / ml gelatin in the presence of 0.1 mM each in 0.1 M phosphate buffer (pH 7.4) at 37 ° C. for 1 week, and then dialyze against PBS for 24 hours. went. After immobilizing 10 μg / ml and 0.1 ml of each sample on an immunoplate, the plate was blocked with a 0.5% gelatin solution solubilized with PBS. Thereafter, 0.1 ml of monoclonal antibody 3F5 (1 μg / ml) against carboxymethylarginine was added to each well and incubated for 1 hour. After washing, HRP-labeled anti-mouse IgG antibody was added to each well and incubated for 1 hour. After washing, color was developed with 1,2-phenylenediamine dihydrochloride for 5 minutes and the absorbance was measured at 492 nm.

  The measurement result of CMA production | generation inhibition rate is shown in FIG.2 and FIG.3. As a result of measuring the production rate of CMA produced from the gelatin-ribose system using monoclonal antibody 3F5, which is a CMA-specific antibody, the compound having the structure shown in FIG. 4 showed a high CMA production inhibitory effect.

FIG. 1 shows the reactivity of monoclonal antibody 3F5 with CMA. FIG. 2 shows the measurement results of the CMA production inhibition rate. FIG. 3 shows the measurement results of the CMA production inhibition rate. FIG. 4 shows a compound having a CMA production inhibitory effect.

Claims (4)

A carboxymethylarginine production inhibitor comprising a compound represented by the following formula (1), formula (2) or formula (3), or a salt thereof, or a hydrate or solvate thereof as an active ingredient.
(Wherein R ₁ represents a hydrogen atom, —COCH ₃ , or a sugar residue, R ₂ represents a hydrogen atom or —COCH ₃ , and R ₃ represents a hydrogen atom, —OH, —OCOCH ₃ , or — O ₄ represents an O-sugar, R ₄ represents a hydrogen atom, —OH, or —OCOCH ₃ , R ₅ represents —OH, or —OCOCH ₃ , Xyl represents a β-D-xylopyranosyl group, glcA represents β -D-glucuronopyranosyl group, rha represents α-L-rhamnopyranoside group. The compound represented by the formula (1) is rutin, quercetin, astragalin, quercetin 3-O-sambubioside, luteolin-7-O-glucoside (Luteolin). The carboxymethylarginine production inhibitor according to claim 1, which is any one of -7-O-glucoside and Penta acetate quercetin. The carboxymethylarginine production inhibitor according to claim 1 or 2, which is used for inhibiting the late product (AGE) of collagen by Maillard reaction. The carboxymethylarginine production inhibitor according to any one of claims 1 to 3, which is used as a skin external preparation.