US 20140349.953A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0349953 A1 Nishida et al. (43) Pub. Date: Nov. 27, 2014

(54) MAILLARD REACTION INHIBITOR (52) U.S. Cl. CPC ...... A6 IK3I/7048 (2013.01); A23L I/3002 (71) Applicant: MORISHITAJINTAN CO.,LTD., (2013.01); A23L 2/52 (2013.01) Osaka (JP) USPC ...... 514/28: 536/18.1 (72) Inventors: Norihisa Nishida, Osaka (JP); Akifumi Nagatomo, Osaka (JP); Hideyuki Ito, Okayama (JP) (57) ABSTRACT

(21) Appl. No.: 14/368,808 A Maillard reaction inhibitor which effectively inhibits the progress of a Maillard reaction in a living body, can be safely (22) PCT Filed: Dec. 27, 2012 applied without adverse side effects, can be manufactured PCT/UP2012/083992 without any complicated process, a skin anti-aging agent, an (86). PCT No.: anti-diabetic complication agent and foods and beverages S371 (c)(1), using the same. The Maillard reaction inhibitor contains 50 to (2), (4) Date: Jun. 26, 2014 90% by mass of as an active ingredient. The Maillard reaction inhibitor has a potent Maillard reaction (30) Foreign Application Priority Data inhibitory activity in a living body and allows for the preven tion and improvement of the various dysfunction of protein in Dec. 27, 2011 (JP) ...... 2011-285694 a living body. Due to this activity, the Maillard reaction Publication Classification inhibitor Suppresses aging and can prevent and/or treat dia betic complications. Furthermore, when the above-described (51) Int. Cl. is mixed in foods and beverages containing collagen, it A6 IK3I/7048 (2006.01) is possible to suppress the Maillard reaction in the foods and A2.3L 2/52 (2006.01) beverages to suppress the deterioration of the foods and bev A2.3L I/30 (2006.01) erages. Patent Application Publication Nov. 27, 2014 US 2014/0349.953 A1

Figure 1

Cor:}pound represented by the formula (VII) (Tetrarier)

Compound represented by the for ruia (WI (Trimer)

---

Eucarbann B D:rner) US 2014/0349.953 A1 Nov. 27, 2014

MALLARD REACTION INHIBITOR 0007 Patent Document 2: Japanese Laid-Open Patent Publication No. 2011-102270 TECHNICAL FIELD 0008 Patent Document 3: Japanese Laid-Open Patent Publication No. 2002-241.299 0001. The present invention relates to Maillard reaction 0009 Patent Document 4: Japanese Laid-Open Patent inhibitors, and more specifically, to Maillard reaction inhibi Publication No. 2005-035911 tors that can be used as a material to be mixed in products Such 0010 Patent Document 5: Japanese Laid-Open Patent as drugs, cosmetics, foods and beverages. Publication No. 2008-214250 0011 Patent Document 6: Japanese Laid-Open Patent BACKGROUND ART Publication No. 2006-256977 0002. A Maillard reaction (glycation reaction) is a reac tion in which various intermediates are formed by nonenzy SUMMARY OF THE INVENTION matic and irreversible reactions of reducing Sugar and protein through a Schiffs base formation and an Amadori rearrange Problem to be Solved by the Invention ment, and then, protein glycation end products (advanced 0012. The present invention was made in order to solve the glycation end products: AGEs) are produced. The Maillard foregoing problems, and it is an object thereof to provide a reaction occurs also in a living body, in which case various Maillard reaction inhibitor which effectively inhibits the biological substances are glycated. The Schiffs base is a progress of a Maillard reaction in a living body (e.g., human reversible compound produced by a reaction of reducing beings, pets, and livestock) and can be safely applied without Sugar Such as and amino groups of protein. Amino any adverse side effects and be manufactured without requir groups of lysine, arginine and an N-terminal amino acid of ing any complicated process, and a skin anti-aging agent, an protein are involved in this reaction. An Amadori rearrange anti-diabetic complication agent, foods and beverages using ment product that is an irreversible compound having a the same. ketoamine structure is produced from a Schiffs base by a rearrangement reaction. The Maillard reaction has three Means for Solving the Problem steps, that is, an initial reaction, intermediate formation and 0013 The present invention provides a Maillard reaction final reaction. inhibitor containing 50 to 90% by mass of polyphenol as an 0003 3-Deoxyglucosone (3DG), glyoxal (GO) and the active ingredient. like are known as such an intermediate. 3DG is an O-dicar 0014. In one embodiment, the polyphenol includes 10 to bonyl compound produced from an Amadori compound and 50% by mass of with respect to a total mass of the is involved in production of AGEs. Although AGEs and the polyphenol. intermediates are metabolized by the involvement of some 0015 Inafurther embodiment, the ellagitannin includes at enzymes and excreted, the activity of these enzymes least one compound selected from the group consisting of decreases with age and consequently AGES accumulate in the , punicalagin, oenotein B, eucarbanin B, punica body. cortein C, pomegraniin A and pomegraniin B. 0004 Carboxymethyllysine (CML), pentosidine, pyrra 0016. In one embodiment, the polyphenol is contained in line, crossline, pyrropyridine, and the like are known as the form of a polyphenol containing plant extract. AGES, and many of these compounds are fluorescent Sub 0017. In one embodiment, the polyphenol containing stances (mainly, the excitation wavelength is 370 nm and the plant extract is an extract derived from at least one plant fluorescent wavelength is 440 nm). Accumulation of AGEs in selected from the group consisting of plants belonging to the tissues and binding of AGES to their receptors cause a dys function of functional protein to develop various symptoms, genera Punica, Quisqualis, Cistus, Terminalia, and Combre which become a major factor of a regressive change associ titlin. ated with age. Production and accumulation of AGEs in the 0018. The present invention also provides a skin anti-ag skin causes the deterioration of the entire skin such as loss of ing agent containing the above Maillard reaction inhibitor. skin firmness and elasticity, wrinkles, looseness of skin, 0019. The present invention also provides an anti-diabetic changes in a skin color, dullness, loss of translucency, and complication agent containing the above Maillard reaction specks on the skin. Moreover, in diabetes patients, AGEs inhibitor. produced by hyperglycemia cause complications such as 0020. The present provides a food or beverage, to which cataracts, arteriosclerosis, renal functional disorder, and the the above Maillard reaction inhibitor is added. like. 0021. The present invention provides an external prepara 0005 Accordingly, these various symptoms can be pre tion, to which the above Maillard reaction inhibitor is added. vented and reduced by inhibiting the Maillard reaction and Suppressing the production of AGES. Recently, various Advantageous Effects of the Invention researches and developments to Suppress the Maillard reac 0022. The Maillard reaction inhibitor of the present inven tion (glycation reaction) have been carried out (Patent Docu tion has a potent Maillard reaction inhibitory activity, effec ments 1 to 6). tively suppresses the production of AGES, and can prevent or improve various symptoms due to AGES. For example, the CITATION LIST Maillard reaction inhibitor can Suppress aging due to the Maillard reaction, and prevent and improve wrinkles, loose Patent Documents ness of skin and loss of the firmness or elasticity of the skin. 0006 Patent Document 1: Japanese Laid-Open Patent Also, the Maillard reaction inhibitor can effectively prevent Publication No. 2005-35911 and treat diabetic complications such as a diabetic neuropa US 2014/0349.953 A1 Nov. 27, 2014 thy, diabetic nephropathy and diabetic retinopathy. Moreover, 0029. Examples of elagitannin include punicalin repre the foods and beverages of the present invention can be easily sented by the following formula (I): taken to suppress the occurrence or progress of the Maillard reaction in a living body because the Maillard reaction inhibi tor is mixed therein. Furthermore, the foods and beverages of Chemical 1 the present invention can also inhibit the occurrence and/or (I) the progress of the Maillard reaction in those foods and bev erages by mixing therein the Maillard reaction inhibitor together with collagen, thereby suppressing the deterioration of those foods and beverages to maintain their quality for a long time.

BRIEF DESCRIPTION OF DRAWINGS 0023 FIG. 1 shows 'H-NMR spectra for identifying a compound isolated in Example 2: (a) 'H-NMR spectrum of pomegraniin B; (b) H-NMR spectrum of pomegraniin A;and (c) 'H-NMR spectrum of eucarbanin B.

DESCRIPTION OF EMBODIMENTS OH 0024 The Maillard reaction inhibitor of the present inven tion contains polyphenol as an active ingredient. 0.025 A Maillard reaction inhibitor is also called an anti glycation agent. The Maillard reaction (glycation reaction) is a reaction in which various intermediates are formed by non 0.030 punicalagin represented by the following formula enzymatic and irreversible reactions of reducing Sugar and (II): protein through a Schiffs base formation and an Amadori rearrangement, and then, protein glycation end products (ad vanced glycation end products: AGES) are produced. The Chemical 2 Maillard reaction inhibitor of the present invention may (II)

inhibit any of the reactions. 0026. In the present invention, the above-described polyphenol is contained at the ratio of 50 to 100% by mass, and preferably 55 to 90% by mass with respect to the total mass of the Maillard reaction inhibitor. If the amount of this polyphenol contained is less than 50% by mass, there is a possibility that the Maillard reaction cannot be sufficiently inhibited in a living body when applied for drugs, cosmetics, foods and beverages described below. 0027. In the present invention, the above-described polyphenol contains elagitannin. The amount of ellagitannin contained is, for example, 10 to 50% by mass, and preferably 10 to 45% by mass, with reference to the total mass of the polyphenol. Alternatively, the amount of ellagitannin con tained is, for example, 5 to 45% by mass, and preferably 10 to 45% by mass, with reference to the total mass of the Maillard reaction inhibitor of the present invention. When elagitannin is used in Such a range of the amount, the Maillard reaction inhibitor of the present invention can effectively inhibit the Maillard reaction in a living body and be efficiently manu factured industrially. 0028 Ellagitannin is a water-soluble compound that, for example, is derived from plants and can react with protein, alkaloids and metal ions to strongly bind thereto and form hardly soluble salts. US 2014/0349.953 A1 Nov. 27, 2014

0031 oenotein B represented by the following formula 0033 punicacortein C represented by the following for (III): mula (V):

Chemical 3 Chemical 5

(III) (V)

0032 eucarbanin B represented by the following formula (IV):

Chemical 4 (IV) OH HO O OH HO O OH O HO O O OH 5 O O OH HO O O OH OH HO O HO OH HO O HO O1O OH O O HO OH OH OH OH HO OH O OH US 2014/0349.953 A1 Nov. 27, 2014

0034 pomegraniin A represented by the following for mula (VI):

Chemical 6 (VI) OH HO O OH HO O OH O HO OO OH O OH HO O O

O HO OH O OH HO O O OH HO As. OH 6 O O OH O O OH OH HO O

HO OH HO C O O HO OO OH O O O OH HO OH OH OH HO OH O OH

0035 pomegraniin B represented by the following for mula (VII):

Chemical 7 (VII)

HO O OH HO O O OH HO O O OH O HO O

HO OH US 2014/0349.953 A1 Nov. 27, 2014

-continued OH OH

OH O O HO O OH HO O OH HO O O O OH O OH O HO O O O OH OH HO

HO OH HO HO

O HO C O

OH

HO OH

0036) and a combination thereof, but are not particularly 0043. The plants belonging to the genus Terminalia limited thereto. belong to Combretaceae as Rangoon creeper. Terminalia 0037. In the present invention, ellagitannin may be a single arborea, Terminalia calamansanai, Terminalia catappa, Ter compound itself selected from punicalin, punicalagin, minalia chebula, Terminalia horrida and Terminalia triflora oenotein B, eucarbanin B, punicacortein C, pomegraniin A or are preferably used. pomegraniin B as above or a mixture thereof, or a polymer or a copolymer including at least one of punicalin, punicalagin, 0044) The plants belonging to the genus Combretum also oenotein B, eucarbanin B, punicacortein C, pomegraniin A or belong to Combretaceae. is prefer pomegraniin B as a basic structure. Alternatively, ellagitannin ably used. may be a mixture of the single compound and the polymer 0045. The plants (whole plant or entire plant) or a portion and/or the copolymer. There is no particular limitation on thereof, processed products thereof (e.g., dried plants, cut degree of polymerization of ellagitannin in the form of a plants, chopped plants, or dry powder obtained by pulverizing polymer or a copolymer, and the degree thereof is 2 to 10. them, or products obtained by shaping the pulverized dried 0038. In the present invention, the above-described plants) or crude extracts thereof can be used for the polyphe polyphenol is preferably contained in form of a polyphenol nol containing plant extract. Herein, when merely referring to containing plant extract. “plant, processed products of plants (e.g., dried plants, cut 0039 Examples of plants providing the polyphenol con plants, chopped plants, or dry powder obtained by pulverizing taining plant extract include, but are not particularly limited them, or products obtained by shaping the pulverized dried to, plants belonging to the genera Punica, Quisqualis, Cistus, plants) are also included. Examples of a portion of a plant Terminalia, and Combretum. The polyphenol containing include, but are not necessarily limited to, parts of a plant Such plant extract may be a single extract obtained from the plants as fruit, fruit skin, flower, stem, leaf trunk, seed and root. or a combination thereof. Herein, the “extract' includes liquid extract obtained by 0040. The plants belonging to the genus Punica belong to being extracted from the above-described plants or processed Punicaceae. Pomegranate (Punica granatum) is preferably products thereof with a solvent, a diluted solution or a con used. Examples of parts thereof used for extraction include centrated solution thereof, or dried products thereof or puri fruit, trunk bark, branch bark and root bark, and fruit is fied products thereof. preferable. 0046 Examples of a solvent used for the extraction of the 0041. The plants belonging to the genus Quisqualis above-described plants include water, lower alcohol such as belong to Combretaceae. Rangoon creeper (Ouisqualis ethanol and methanol, lower ester such as ethyl acetate and indica) is preferably used. Mature fruit of this plant are called methyl acetate, acetone and a mixture of these organic Solvent “Shikun-shi' and have been used for medicine over a long and water (hydrous organic solvent). From the viewpoint of period. They are particularly well-known as anthelmintics. further enhancing safety of ingestion by a living body, it is Examples of parts thereofused for extraction include fruit and preferable to use water alone, ethanol alone or a mixture of seed. ethanol and water (so-called hydrous ethanol or aqueous 0042. The plants belonging to the genus Cistus belong to solution of ethanol). Furthermore, for example, a 20% (v/v) Cistaceae. Twenty species belong to the genus Cistus. Cistus or more, preferably 30 to 90% (v/v), aqueous solution of ladaniferus and Cistus populifolius are preferably used. ethanol can be used to provide an extract that abundantly US 2014/0349.953 A1 Nov. 27, 2014

contains elagitannin described above, or effectively provides 0050. The extract obtained in this manner is used as it is or the inhibition of the Maillard reaction. is concentrated to be used in liquid form, a concentrate, a 0047. There is no particular limitation on extraction con paste or a dried product obtained by further drying them. ditions (e.g., amount of a solvent, temperature and time). For Drying is performed by a method commonly used by a person example, the amount of an extraction solvent is preferably 5 skilled in the art, Such as spray drying, lyophilization, vacuum to 50-times the volume per dry mass with respect to pulver drying and fluidized drying. ized plants, and more preferably 10 to 30-times the volume 0051. It should be noted that, in the present invention, the per dry mass. Although varying with the type of a solvent “extract also includes a water extract or a solvent extract used, for example, a temperature from a room temperature to obtained by the above-described procedure, and a supercriti a temperature below the boiling point of the solvent used can cal carbon dioxide extract, solid contents obtained by remov be adopted as an extraction temperature. An extraction time ing the solvent from these obtained extracts by a method can vary in accordance with the type, amount, and extraction known in the art Such as evaporation to dryness under reduced temperature of the solvent used, and a person skilled in the art pressure or spray drying, and fractions obtained by further can determine a suitable extraction time. When the extraction purifying them. is performed at room temperature, the extraction time may be, 0.052 The Maillard reaction inhibitor of the present inven for example, 1 to 48 hours, and preferably 6 to 24 hours, and tion may contain other additives than the above-described when the extraction is performed near the boiling point of the polyphenol or polyphenol containing plant extract. Examples Solvent, the extraction time may be, for example, approxi of the other additives include cellulose, (cyclo)dextrin, and a mately 1 to 60 minutes. Furthermore, (a) the extraction may combination thereof. There is no particular limitation on the be a single extraction using one extraction solvent; (b) the amount of the other additives contained in the present inven extraction may be a stepwise extraction using different sol tion, but any amount can be set by a person skilled in the art vents in which an extraction is performed with one solvent, within the range where the other additives do not inhibit or and another extraction is Subsequently performed on the reduce the above-described Maillard reaction inhibitory obtained extract with a different solvent; or (c) an extract effect exhibited by the above-described polyphenol or obtained with one solvent may be combined with an extract polyphenol containing plant extract. obtained with a different solvent. By way of a specific 0053. The Maillard reaction inhibitor of the present inven example, the extraction operation (b) can be performed by tion is useful as a material constituting, for example, drugs, extracting the above mentioned plant with approximately cosmetics or foods and beverages. 20-times the volume of hot water with respect to the dry mass 0054. One embodiment of the Maillard reaction inhibitor of the plant for about 10 minutes, adding the same amount of of the present invention includes anti-aging for the skin of a ethanol thereto and mixing them, and filtrating the mixture to living body due to the Maillard reaction. For example, the obtain the filtrate as the extract. It should be noted that such an Maillard reaction inhibitor of the present invention can be extraction can be performed under any condition Such as still used to prevent or improve aging of the skin of a living body. standing, shaking, stirring or refluxing. Alternatively, the Here, aging due to the Maillard reaction means regressive extraction is performed by homogenization after the addition changes resulting from the dysfunction of functional protein of the solvent. due to an uncontrollable reaction of AGES produced by the 0048. In the present invention, a preferable purification Maillard reaction with biological substances. For example, procedure and condition is made by combining, for example, the accumulation of AGES in the skin reduces skin translu silica gel column chromatography, reversed phase ODS col cency and yellows the skin. Although collagen protein plays umn chromatography, preparative HPLC and the like, using a roll of maintaining the elasticity of the skin together with an eluent (e.g., water, organic solvents such as hexane, ethyl elastic fibers, when collagen protein bind to AGEs to form acetate, chloroform, methanol and n-butanol, or a mixture crosslinks therebetween, the movability of collagen is lost thereof) under a preferable condition, but are not particularly and this results in hardening of the skin and wrinkles. The limited thereto. Maillard reaction inhibitor of the present invention can Sup 0049. Alternatively, the extract of the above-described press the production of AGES to Suppress Such a dysfunction plants may be subjected to purification as set forth below in of protein in a living body. order to enhance the Maillard reaction inhibitory activity. For 0055. That is, the Maillard reaction inhibitor of the present example, the above-described filtrate obtained by the filtra invention can be used as an active ingredient providing a skin tion following the extraction is concentrated by a means com anti-aging agent. The term "skin anti-aging agent' includes a monly used by a person skilled in the art, and then Subjected composition capable of exhibiting an anti-aging action on the to a column chromatography using preferably a 40 to 90% skin of a living body. Such as drugs (including quasi-drugs) (v/v), more preferably 50 to 80% (v/v), aqueous solution of and cosmetics. C-C alcohol (preferably an aqueous solution of ethanol). An 0056. When the Maillard reaction inhibitor of the present absorbent useful for this column chromatography is prefer invention is used as a skin anti-aging agent, there is no limi ably an aromatic absorbent, and a specific example thereof is tation on routes or methods for administration and ingestion a styrene-divinylbenzene based absorbent. Examples of the thereof. styrene-divinylbenzene based absorbent include Amberlite 0057 For example, when the Maillard reaction inhibitor XAD series (manufactured by Organo Corporation). It should of the present invention is used as a drug, transcutaneous be noted that, in the present invention, before the purification administration, Subcutaneous administration, transvenous using the aqueous solution of C-C alcohol as mentioned administration and oral administration are exemplified. There above, a preliminary purification may be performed on the is no limitation on a dosage form or physical form. Solid, above-described column using water (e.g., distilled water) in semi-solid and liquid are exemplified, and an injection, an order to further enhance the Maillard reaction inhibitory external preparation and an oral preparation are exemplified. activity. A liquid medicine and a dry preparation for being dissolved in US 2014/0349.953 A1 Nov. 27, 2014 use are exemplified as an injection. A patch, an ointment, a agent, a Suspending agent and an emulsion. The foods and plaster, a lotion, a milky lotion, a liquid medicine and Sus beverages may contain other food materials, other active pending agent are exemplified as an external preparation. A ingredients, and/or additives that can be generally used in the powder medicine, a granular medicine, a tablet, a capsule and art (e.g., a vehicle Such as dextrin, starch, Sugars and calcium a liquid medicine (including a suspending agent, an emulsion, phosphate, a solvent, a flavor and perfumed oil) other than the and the like) are exemplified as an oral preparation. An exter above-described Maillard reaction inhibitor. nal preparation is preferably used for Maillard reaction inhi 0063. In the present invention, processed foods, noodles, bition in skin tissue. confectionery, drink, seasonings, powdered milk, health 0.058 Drugs may contain an optional ingredient (e.g., a foods, Supplements and the like are exemplified as the foods binding agent, a disintegrating agent, a lubricant, a vehicle, a and beverages to which the above-described Maillard reac coloring agent, a flavoring agent, a Surfactant, a preservative, tion inhibitoris added. There is no limitation on a dosage form an antioxidant, an ultraviolet absorbing agent, a moisturizing or physical form of the composition for addition to a food or agent and a pH adjusting agent) other than the Maillard reac a beverage as long as it is in the form that is easily added. Such tion inhibitor of the present invention as mentioned above. as powdery form or liquid form. Although the compositions Examples of Such an optional ingredient include cellulose, including a composition, Such as a rice seasoning or season (cyclo)dextrin, powdered acacia, methylcellulose, crystalline ing, having Such a form and amount that can be added to a cellulose, ethylcellulose, polyvinylpyrrolidone, polyethylene food or a beverage and mixed therewith by a person who eats glycol, talc, silicon dioxide (e.g., light anhydrous silicic acid or drinks and a composition having a form and amount to be or hydrous silicon dioxide), magnesium Stearate, Stearic acid, added on processing a food or a beverage varies the form, Stearyl alcohol, starches, hydroxypropyl Starch, Sodium car amount, and degree of processing of the composition in boxymethyl starch, powdered agar, carboxymethylcellulose accordance with its addition method or purpose, the compo (calcium), low Substituted hydroxypropylcellulose, ester oil, sition of the present invention includes all of them. wax, higher fatty acid, higher alcohol, anionic Surfactant, cationic Surfactant, nonionic Surfactant, amphoteric Surfac 0064. Furthermore, by adding the above-described Mail tant and polyalcohol. In this case, any amount can be set for lard reaction inhibitor, that is, an ingredient having an inhibi the contained ingredient by a person skilled in the art within tory activity on the Maillard reaction in a living body, the the range where the ingredient does not inhibit or reduce the amount of the ingredient contained in the foods and beverages above-described the Maillard reaction inhibitory effect of the of the present invention can be increased compared with present invention. conventional food materials. Therefore, it is possible to 0059. When used for cosmetic, cosmetics may contain an ingest, at one time, the inhibition active ingredient with the optional ingredient (e.g., an oily ingredient such as ester oil, unexperienced amount in conventional food materials. wax, higher fatty acid and higher alcohol; a surfactant Such as 0065. The present invention also provides foods and bev an anionic Surfactant, cationic Surfactant, nonionic Surfactant erages containing collagen and the above-described ellagi and amphoteric Surfactant; a moisturizing agent such as poly tannin. The present invention further provides a method for alcohol; a base ingredient Such as water, lower alcohol and inhibiting the Maillard reaction in foods and beverages by silicone; a preservative; an antioxidant; an ultraviolet absorb mixing the above-described elagitannin in foods and bever ing agent; a flavor, and a pH adjusting agent) other than the ages containing collagen. The elagitannin is preferably Maillard reaction inhibitor of the present invention as men hydrolyzed tannin. There is no particular limitation on the tioned above. In this case, any amount can be set for the type and molecular weight of collagen. The foods and bever contained ingredient by a person skilled in the art within the ages may contain Sugars that can be generally used in a food range where the ingredient does not inhibit or reduce the field. When the ellagitannin is mixed in the foods and bever above-described the Maillard reaction inhibitory effect of the ages containing collagen, the Maillard reaction of Sugars in present invention. The "cosmetics” include base cosmetics the foods and beverages with collagen is inhibited, so that it is Such as a face lotion and a milky lotion; an application for the possible to suppress the deterioration of the foods and bever entire face Such as a foundation and face powder, and an ages and to maintain their quality. Furthermore, after the application for a portion of the face Such as an eye-shadow, an foods and beverages are ingested, it is possible to Suppress the eyeliner and a cheek rouge. Maillard reaction in a living body. For example, 0.0001 to 100 0060. The Maillard reaction inhibitor of the present inven mg of punicalin and/or punicalagin, preferably 0.001 to 1 mg, tion can be also used as anti-diabetic complication agent. In is mixed with respect to 1 g of collagen contained in foods and diabetes patients, since the concentration of glucose in blood beverages. is high, protein in a living body is easily glycated to cause its 0066. The present invention also provides an external dysfunction. The Maillard reaction inhibitor of the present preparation containing collagen and the above-described invention can Suppress the glycation of protein in a living ellagitannin. When used for an external preparation, the exter body to prevent or treat diabetic complications. Examples of nal preparation may contain an optional ingredient (e.g., an the diabetic complications include diabetic retinopathy, dia oily ingredient such as ester oil, wax, higher fatty acid and betic nephropathy, diabetic neuropathy, cerebrovascular dis higher alcohol; a Surfactant such as an anionic Surfactant, order, ischemic heart disease, diabetic gangrene, hyperlipi cationic Surfactant, nonionic Surfactant and amphoteric Sur demia, chronic infection, cholelithiasis and cataracts. factant; a moisturizing agent such as polyalcohol; a base 0061 The present invention also provides foods and bev ingredient such as water, lower alcohol and silicone; a pre erages to which the above-described Maillard reaction inhibi servative; an antioxidant; an ultraviolet absorbing agent; a tor is added. flavor, and a pH adjusting agent) other than the above-de 0062. The foods and beverages include feed and pet food, scribed Maillard reaction inhibitor of the present invention. In and examples thereof include foods and beverages formu this case, any amount can be set for the contained ingredient lated into a powder, a granule, a tablet, a capsule, a liquid by a person skilled in the art within the range where the US 2014/0349.953 A1 Nov. 27, 2014

ingredient does not inhibit or improve the above-described (0070. Next, the Maillard reaction inhibition ratio (%) with Maillard reaction inhibitory effect of the present invention. respect to the above-described prepared solutions (concentra EXAMPLES tion (ug/mL)) of the samples was calculated from the obtained quantitative value using the following equation. 0067 Next, the present invention will be further described by way of examples, but is not limited to the following Equation: Inhibition ratio (%)=100-(a-b)f(c-d)x examples. 100 Formula 1 Example 1 0071 where a represents a relative value (-) of the fluo rescent products of the Maillard reaction with the reaction Measurement of the Maillard Reaction Inhibitory Solution A calculated in terms of quinine Sulfate, Activity 0068 A pomegranate extract (containing 50% by mass of 0072 b represents a relative value (-) of the fluorescent polyphenol; manufactured by Morishita Jintan Co., Ltd.), products of the Maillard reaction in terms of quinine sulfate punicalin, punicalagin, oenotein B, eucarbanin B, punica when the reaction solution B is used, cortein C, pomegraniin A and pomegraniin B were used as samples to be examined. Tannic acid and dihy 0073 c represents a relative value (-) of the fluorescent drate were used for comparison. Aminoguanidine was used as products of the Maillard reaction in terms of quinine sulfate a positive control. These samples were diluted with water to when the reaction solution C is used, and prepare solutions at concentrations of 0.01 lug/mL, 0.03 ug/mL, 0.1 g/mL, 0.3 ug/mL, 1 g/mL, 10 g/mL, 100 0074 d represents a relative value (-) of the fluorescent ug/mL, 300 g/mL and 1,000 ug/mL, and the Maillard reac products of the Maillard reaction in terms of quinine sulfate tion inhibitory activity of the prepared solutions was mea when the reaction solution D is used. sured. The inhibitory activity was measured as follows. 0069. Four reaction solutions A to D shown in Table 1 (0075. Thereafter, 50% inhibition ratio (ICs) of each below were prepared using the above-described prepared sample was calculated as follows. That is, the inhibition ratio solutions of the samples, and were incubated at 60° C. for 40 was plotted with respect to the sample concentration, 4-pa hours to cause the Maillard reaction. Thereafter, 400 uL of the rameter plot was performed, and the concentration showing reaction solutions A to D was diluted with 2,400 uL of dis tilled water and diluted samples were obtained. The fluores an inhibition ratio of 50% was taken as ICs. cence intensity (the excitation light: 370 nmi; the measure 0076 Table 2 shows the Maillard reaction inhibition ratios ment light: 440 nm) of the diluted samples was measured and was expressed as a relative value with respect to the fluores (%) with respect to the prepared solutions. Furthermore, cence intensity of 0.1 g/mL Solution of quinine Sulfate that Table 3 shows ICs values calculated from the Maillard reac was taken as 100 to calculate the amount of fluorescent prod tion inhibition ratios (%) with respect to the prepared solu ucts of the Maillard reaction (quantitative value). tions shown in Table 2.

TABLE 1.

Sample (Concentration in Reaction Reaction Reaction Reaction Reation Solution) Solution A Solution B Solution C Solution D

Human Serum 0.8 mg/mL 0.8 mg/mL 0.8 mg/mL 0.8 mg/mL. Albumin Solution Glucose Solution 200 mmol/L 200 mmol/L Phosphate Buffer 50 mmol/L 50 mmol/L 50 mmol/L 50 mmol/L Distilled Water 100 L 200 IL 200 IL 300 IL Sample to 100 L 100 IL be Examined

Total Amount 1000 L 1000 IL 1000 IL 1000 IL US 2014/0349.953 A1 Nov. 27, 2014

TABLE 2

Maillard reaction inhibition ratio (%

Conc. (mg/mL) O.O1 O.O3 O1 O.3 1 10 1OO 1OOO Pomegranate extract 8.4 40.7 70.4 8S.S 76.6 Punicalin 7.6 6.5 32.1 69.6 73.1 84.9 - Punicalagin 4.3 10.9 36.2 71.4 73.5 86.1 - Oenotein B 15.5 - – 69.2 64.6 — Eucarbanin B – 21.2 – 72.9 79.4 — Punicacortein C - 39.6 76.8 78.2 - Pomegraniin A — 46.6 – 76.7 95.4 — Pomegraniin B 30.4 – 40.3 – 74.3 82.5 — Aminoguanidine — 40.2 Tannic acid 12.7 - 48.7 - 65.1 68.8 - Ellagic acid dihydrate 1.9 2.2 12.O 44.8 68.3 784 -

—: not measured

TABLE 3 as a lyophilization aid was added to the obtained concentrate of the ethanol-water fractions, and the mixture was lyo ICso philized. In this manner, a sample S constituted of powder (mg/mL) derived from pomegranate powder was prepared. Pomegranate extract O.S6 0079 Next, the following measurements were performed Punicalin O16 on the sample S obtained as described above. Punicalagin O.15 0080. Total Amount of Polyphenol Oenotein B O43 Eucarbanin B O43 I0081. The amount of the contained polyphenol was mea Punicacortein C O16 Sured as ethyl gallate equivalence according to Folin-Denis Pomegraniin A O.13 method (“Explanation on Analytic Manual of Food Recorded Pomegraniin B O.21 Aminoguanidine 273.31 in 5th Revised Standard Table of Food Composition in Tannic acid O.31 Japan', p. 254, 2001, Chuohoki Publishing Co., Ltd.). That is, Ellagic acid dihydrate O.34 a sample solution was prepared by dissolving the sample in distilled water so as to have a concentration of 15 mg/mL. One milliliter of this sample solution and 1 mL of a Folin 0077. As shown in Tables 2 and 3, it is found that each of reagent (obtained by adding 180 mL of distilled water to 25g the pomegranate extract, punicalin, punicalagin, oenotein B, of Sodium tungstate, 5 g of phosphomolybdic acid and 12.5 eucarbanin B, punicacortein C, pomegraniin A and pomeg mL of phosphoric acid, boiling the mixture under reflux for 2 raniin Bhad a superior Maillard reaction inhibitory activity to hours, and then making the total volume 1 L with distilled aminoguanidine serving as a positive control. Furthermore, as water) were mixed and were allowed to stand for 5 minutes. is clear from Table 2, it is found that punicalin, punicalagin, One milliliter of 10% aqueous solution of sodium carbonate oenotein B and pomegraniin B were particularly Superior in was further added thereto, the solution was allowed to stand Maillard reaction inhibition ratio (%) to elagic acid dihydrate for 1 hour, and then, the absorbance was measured for the and aminoguanidine serving as a positive control even when solution at 700 nm. The absorbance measured in the same prepared at lower concentrations. In addition, as is clear from manner as described above, except that distilled water alone Tables 2 and 3, it is found that all ICs values of punicalin, was used instead of the sample solution, was used as a control. punicalagin, punicacortein C, pomegraniin A and pomegra Aqueous solutions of ethyl gallate at various concentrations niin B were particularly low compared with those of tannic were prepared and were measured in the same manner as acid, ellagic acid dihydrate and aminoguanidine serving as a described above to make a calibration curve. Then, the positive control, and each of them has a superior Maillard amount of polyphenol contained in the sample S obtained as reaction inhibitory activity. described above was measured. Example 2 I0082. The total amount of polyphenol contained in the sample S was 72.1+0.6% with respect to the mass of the sample S. Extraction of Ellagitannin from Pomegranate Powder I0083. Amount of Ellagitannin 0078. Seven hundred milliliters of water was added to 300 I0084. The amount of elagitannin contained in the above g of commercially available dry pomegranate powder (made described sample S was determined with HPLC (model num in China). The mixture was allowed to stand with stirring at ber: Inertsil ODS-3; manufactured by GL Sciences Inc.) 50° C. for 24 hours, was allowed to cool, and was centrifuged according to the following condition described in literature (J. to provide 900 ml of liquid extract. The liquid extract was Agric. Food Chem., 2009, 57(16), p. 7395). injected to a column packed with 100 g of Amberlite XAD4 I0085

0090. Injection amount: 25 LL using a collagen gel contraction activity of human dermal 0091 Mobile phase condition: a linear gradient with 0.5% fibroblasts as an index. That is, collagen gel was prepared in phosphoric acid (A) and acetonitrile (B) under the following a 12-well cell culture plate using a collagen gel culture kit conditions: (Cellmatrix, manufactured by Nitta Gelatin Inc.). 0104. The samples S obtained in Example 2 at various concentrations were prepared using a 10 mM solution of A. B glucose-6-phosphate, were added on the collagen gel, and O min. 95% 59% were then incubated at 37° C. for 10 days so as to cause the 10 min. 85% 15% glycation reaction. Unreacted glucose-6-phosphate was 30 min. 75% 25% washed away, and 1x10 cells/mL of fibroblasts were seeded 35 min. 95% 5%. on the collagen gel and cultured in DMEM medium contain ing 0.25% FBS. After 3 hours, the collagen gel was peeled off 0092 Table 4 shows the obtained results. the wall of the well and collagen was contracted. After 48 hours, the medium was Sucked and removed, the diameter of TABLE 4 the collagen gel was measured, and the area of the collagen gel was calculated. Compound Elution time?min. 0105. As Control 1, the area of the collagen gel was cal Punicalin 7.3 culated in the same manner as described above, except that Punicalagin 11.2, 12.9 the above-described solution of glucose-6-phosphate was not Oenotein B 12.1 added and the glycation reaction was not caused. In addition, Eucarbanin B 13.1 Punicacortein C 12.5 as Control 2, the area of the collagen gel was calculated in the Pomegraniin A 12.3 same manner as described above, except that the sample S Pomegraniin B 13.3 obtained in the above-described Example 2 was not added. Ellagic acid dihydrate 24.3 The area calculated from Control 1 was denoted as A, the area calculated from Control 2 was denoted as B, the area calcu 0093. As shown in Table 4, it is found that the sample S lated from the sample was denoted as C, and the inhibition obtained in this example contained polyphenol, specifically ratio was calculated from the following equation. certain amounts of various . Inhibition ratio (%)=(C-A) (B-A)x100 Formula 2 0094) Moreover, the above-described sample S was sub jected to the separation according to the following conditions 01.06 Table 5 shows the obtained results. to isolate oenotein B, eucarbanin B, punicacortein C, pome graniin A and pomegraniin B. Of these compounds, pomeg TABLE 5 raniin A, pomegraniin B and eucarbanin B were identified as Test Concentration Glycation Area of Collagen Inhibition these compounds by 'H-NMR spectrum measurement (600 Material (mg/mL) reaction gel (cm) rate (%) MHz, acetone-de-DO) as shown in FIG. 1. 'H-NMR data Control 1 O.O absence 2.47 O.12 (600 MHz, acetone-de-DO) of pomegraniins A and B are Control 2 O.O presence 3.25. O.15 shown below: 6.97-7.05 (galloyl-H), 6.11-6.67 (HHDP-H, O.O1 presence 3.22 O.22 3.8 valoneoyl-H), 5.55-5.85 (glucose H-3), 5.49-5.61 (glucose O.O3 presence 3.14 O.19 14.1 Sample S of O.1 presence 2.90 O.25 44.9 H-1 (C.-anomer), H-3), 5.24-5.26 (glucose H-6), 5.03-5.22 Example 2 O.3 presence 2.74 - 0.24 65.4 (glucose H-1 (B-anomer), H-2, 4, 6), 4.61-4.63 (glucose H-5 1.O presence 2.70 - O.21 70.5 (3-anomer)), 4.30-4.32 (glucose H-1 (B-anomer)), 4.08-4.26 3.0 presence 2.65 0.18 76.9 (glucose H-5 (C-anomer)), 3.66-3.93 (glucose H-6). In addi tion, results of electrospray mass spectrometry (ESI-MS) of —: not calculated pomegraniins A and B are also shown below: 0107 As shown in Table 5, the higher the added concen 0095 Pomegraniin A (trimer) ESI-MS: m/Z 2353 (M-H), tration of the sample S obtained in Example 2 was, the smaller 1176 (M-2H)? the area of the collagen gel was and the higher contraction 0096 Pomegraniin B (tetramer) ESI-MS: m/z. 3137 activity was exhibited. Therefore, the sample S exhibited a (M-H), 1568 (M-2H). Superior anti-aging action on the skin. 0097

Example 2 was mixed at a ratio of 1%, and both groups were Example 5 housed for 12 weeks. The normal control mice were fed with the normal diet and were housed for 12 weeks in the same Preparation of a Beverage manner. The mice freely drank tap water as drinking water. O112 A beverage was prepared based on the following 0109. After the administration for 12 weeks was finished, formula. blood of all the mice was collected from the abdominal aorta under anesthesia, the mice were put to death by exsan guination, and the thoracic aorta was extracted. In addition, Ingredients Compounding ratio (Mass ratio) the eyeball was extracted and the crystalline lens was col Glycerin 1O.O Sample 2 obtained in Example 2 1.O lected. The blood sample was centrifuged to collect blood Cellulose O.1 plasma, and the CML concentration was measured by Citric acid O.3 ELISA. After fat was removed, the extracted aorta was lyo Flavor O.1 philized and was pulverized finely. A portion thereof was Purified water Remaining quantity treated with a protease and was centrifuged to collect the supernatant. This supernatant was measured at 370 nm of the 0113. The above-described ingredients were mixed excitation wavelength and 430 nm of the fluorescent wave together and were stirred to produce a beverage. length, and the amount of AGE was calculated. The crystal line lens was homogenized and was centrifuged, and the Example 6 amount of AGE contained in the Supernatant was measured by ELISA. Preparation of Face Lotion 0110 Tables 6 to 8 show the obtained results. 0114. The following ingredients were uniformly mixed at the following ratio to provide a face lotion. TABLE 6 CML in blood Ingredients Compounding ratio (Mass ratio) Absorbance (450 nm) Absorbance(-) SE Glycerin 1O.O 1,3-butylene glycol 6.O TSNO O.S2 O.14 Sample 2 obtained in Example 2 1.O TSOD 1.13 O.21 Citric acid O.1 TSOD Sodium citrate O.3 (After ingested a sample O.84 O.11 Polyoxyethylene 1.O S of Example 2) Ethyl alcohol 8.0 Paraben O.1 Flavor O.1 Purified water Remaining quantity TABLE 7

AGE in Aorta INDUSTRIAL APPLICABILITY Fluoresence Fluoresence(-) SE 0115 The Maillard reaction inhibitor of the present inven NO 7543 6SO tion has a potent Maillard reaction inhibitory activity to effi TSOD 962O 745 TSOD ciently inhibit the Maillard reaction in a living body and allow (After ingested a sample 846O 469 for the prevention and improvement of the various dysfunc S of Example 2) tion of protein in a living body. Due to this activity, the Maillard reaction inhibitor of the present invention Sup presses aging and is useful for the prevention and treatment of diabetic complications. Furthermore, when the above-de TABLE 8 scribed tannin is mixed in foods and beverages containing AGE in Crystalline collagen, it is possible to Suppress the Maillard reaction in the foods and beverages to suppress the deterioration of the foods Absorbance (450 nm) Absorbance(-) SE and beverages, and to also suppress the Maillard reaction in a TSNO O.65 O.18 living body. The Maillard reaction inhibitor of the present TSOD 1.27 O.32 invention is useful in various fields Such as drugs, cosmetics TSOD and food. (After ingested a sample 1.04 O.36 S of Example 2) 1. A Maillard reaction inhibitor containing 50 to 90% by mass of polyphenol as an active ingredient. 2. The Maillard reaction inhibitor according to claim 1, 0111. As shown in Tables 6 to 8, in the TSOD mice ingest wherein the polyphenol includes 10 to 50% by mass of ella ing the sample S obtained in Example 2, the formation of gitannin with respect to a total mass of the polyphenol. AGE in the blood, aorta and crystalline lens was suppressed 3. The Maillard reaction inhibitor according to claim 2, compared with the case of TSOD mice ingesting only the wherein the ellagitannin comprises at least one compound normal diet. Therefore, it is possible that the sample S selected from the group consisting of punicalin, punicalagin, obtained in Example 2 is useful for the prevention or delay of oenotein B, eucarbanin B, punicacortein C, a compound rep development of the diabetic complications. resented by the following formula (VI): US 2014/0349.953 A1 Nov. 27, 2014

Chemical 6 (VI)

HO O OH HO Oray. O OH O OH HO O O

O HO OH C OH HO O OH HO As.O OH 6 O O OH O O OH HO

O O O O O OH O O O OH OH OH OH, HO OH O OH

and a compound represented by the following formula (VII): US 2014/0349.953 A1 Nov. 27, 2014 13

(IIA) ‘HO HO HO

HO HO HO

OH HO HO HO OH OH OH

HO HO HO OH OH OH

HO HO HO OH OH OH

HO

OH

OH OH OH OH US 2014/0349.953 A1 Nov. 27, 2014

4. The Maillard reaction inhibitor according to claim 1, 10. The Maillard reaction inhibitor according to claim 2, wherein the polyphenol is contained in the form of a polyphe wherein the polyphenol is contained in the form of a polyphe nol containing plant extract. nol containing plant extract. 5. The Maillard reaction inhibitor according to claim 4, 11. The Maillard reaction inhibitor according to claim 10, wherein the polyphenol containing plant extract is an extract wherein the polyphenol containing plant extract is an extract derived from at least one plant selected from the group con derived from at least one plant selected from the group con sisting of plants belonging to the genera Punica, Quisqualis, sisting of plants belonging to the genera Punica, Quisqualis, Cistus, Terminalia, and Combretum. Cistus, Terminalia, and Combretum. 12. The Maillard reaction inhibitor according to claim 3, 6. A skin anti-aging agent containing the Maillard reaction wherein the polyphenol is contained in the form of a polyphe inhibitor according to claim 1. nol containing plant extract. 7. An anti-diabetic complication agent containing the 13. The Maillard reaction inhibitor according to claim 12, Maillard reaction inhibitor claim 1. wherein the polyphenol containing plant extract is an extract 8. A food or beverage, to which the Maillard reaction derived from at least one plant selected from the group con inhibitor according to claim 1 is added. sisting of plants belonging to the genera Punica, Quisqualis, 9. An external preparation, to which the Maillard reaction Cistus, Terminalia, and Combretum. inhibitor according to claim 1 is added. k k k k k