JP6673864B2 - Advanced saccharification product formation inhibitor - Google Patents

Description

  The present invention relates to a novel terminal glycation end product production inhibitor, and more specifically, to a terminal glycation end production inhibitor which is safe and has high terminal glycation end production inhibition activity.

  Advanced Glycation Endproducts (AGEs) are also called glycated proteins, Maillard reaction products, etc., and are protein derivatives having various structures that are generated by a non-enzymatic reaction between a reducing sugar such as glucose and an amino group of the protein. It is. AGEs may be disrupted by the glycational modification of extracellular matrix proteins, membrane proteins and intracellular proteins, resulting in disruption of the function of these proteins and of their dependent cellular functions, or as a result of cellular responses triggered by AGEs ligand receptors. Involved in the onset and exacerbation of the lesions.

  For example, when AGEs are recognized by RAGE, which is one of the AGEs receptors, the production of intracellular oxidative stress substances by intracellular NADPH oxidase is enhanced, and this changes gene expression in epithelial cells. It is considered that vascular disorders occur (see Non-Patent Document 1).

  In recent years, in addition to diabetic vascular disorders, AGEs have recently been used in cardiovascular disorders such as myocardial infarction and arteriosclerosis (see Non-Patent Document 2), and neurodegeneration such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis Diseases (see Non-Patent Document 3), brain disorders and liver disorders due to alcohol dependence (see Non-Patent Document 4), diabetic complications such as diabetic nephropathy, diabetic retinopathy, and diabetic neuropathy (Non-patent Document 5) Bone metabolism disorders such as osteoporosis (see Non-Patent Document 6), aging (see Non-Patent Document 7), insulin resistance (see Non-Patent Document 8), tumor growth and metastasis (see Non-Patent Document 9) It has been suggested that they are also involved in such activities.

As a drug for treating or preventing a disease related to AGEs by inhibiting the formation of AGEs in a living body, for example, in Patent Document 1, a cultured cell extract of a purple genus plant or a processed product thereof, or caffeic acid A Maillard reaction inhibitor containing a polymer as an active ingredient is disclosed. In Patent Document 2, a Maillard reaction inhibitor, AGEs product inhibiting composition containing an acceptable salt vitamin B ₆ or a pharmaceutically is disclosed. Patent Document 3 discloses an agent for improving carbonyl stress in the peritoneal cavity in peritoneal dialysis, which comprises a carbonyl compound trapping agent as an active ingredient. Patent Document 4 discloses an activity of inhibiting one or more reactions related to the production of advanced glycation end products from proteins and sugars, which are isolated from hot water extracts of one or both of pericarp and fruit of a plant belonging to the family Lamiaceae. A compound having one or more compounds having the following formulas as an active ingredient, wherein the one or more compounds preferably have a molecular weight measured by gel filtration chromatography in the range of 70 to 130 or 290 to 380. An advanced glycation end product formation inhibitor comprising one or more of the above is disclosed.

  In addition, for the treatment and prevention of diseases related to AGEs, a substance having a combination of a plurality of actions such as chelating ability and antioxidant action is considered to be promising (see Non-Patent Document 10).

JP 2008-214250 A JP-A-10-324629 JP 2006-305345 A JP 2015-209420 A

Marie-Paule Wautier et al., "Activation of NADPH oxidase by AGE links oxidant stress to altered gene expression via RAGE", Amarican Journal of Physiology-Endocrinology and Metabolism, (USA), American Physiological Society, May 2001 Mon, Volume 280, E686-E694 Yoshihide Jinnouchi et al., "Glycolaldehyde-modified low density lipoprotein leads macrophages to foam cells via the macrophage scavenger receptor", The Journal of Biochemistry, The Japanese Biochemical Society, 1998, 123, No. 6, p. 1208-1217 Nobuyuki Sasaki et al., "Advanced Glycation End Products in Alzheimer's Disease and Other Neurodegenerative Diseases", The American Journal of Pathology, (USA), American Society for Investigative Pathology, October 1998, Volume 153, No. 4, p. 1149-1155 Keiko Iwamoto et al., "Advanced glycation end products enhance the proliferation and activation of hepatic stellate cells", Journal of Gastroenterology, Springer Japan, April 2008, Vol. 43, No. 4, p. 298-304 CW Yang et al., Advanced glycation end products up-regulate gene expression found in diabetic glomerular disease, Proceedings of the National Academy of Sciences of the United States of America, (USA), National Academy of Sciences of the United States of America), September 27, 1994, Vol. 91, No. 20, p. 9436-9440 James J. Tomasek et al., "Diabetic and age-related enhancement of collagen-linked fluorescence in cortical bones of rats", Life Sciences, (Netherlands), Elsevier B.V., 1994, Vol. 55, p. 855-861 Melpomeni Peppa et al., "Aging and glycoxidant stress", Hormones, (Greece), Hellenic Endocrine Society, 2008, Vol. 7, No. 3, p. 123-132 Masaya Mizuta, “Insulin Insufficiency and Oxidative Stress”, Pharmacological Journal of Japan, The Pharmacological Society of Japan, Vol. 125, No. 3, p. 125-128 Riichiro Abe et al., "Regulation of Human Melanoma Growth and Metastasis by AGE-AGE Receptor Interactions", Journal of Investigative Dermatology, (UK), Nature Publishing Group, February 2004, Vol. 122, No. 2, p. 461-467 Sean M. Culbertson et al., "Paradoxical Impact of Antioxidants on Post-Amadori Glycoxidation: COUNTERINTUITIVE INCREASE IN THE YIELDS OF PENTOSIDINE AND Nε-CARBOXYMETHYLLYSINE USING A NOVEL MULTIFUNCTIONAL PYRIDOXAMINE DERIVATIVE, U.S.A., Chemistry, U.S.A. American Society for Biochemistry and Molecular Biology, October 2003, Volume 273, p. 38384-38394

  However, regarding the Maillard reaction inhibitor described in Patent Document 1, the safety at the time of oral ingestion is unknown. The composition for inhibiting AGEs production described in Patent Document 2 is a synthetic product and contains aminoguanidine and the like, which are likely to cause side effects. Further, in the AGEs adsorption and removal agent described in Patent Document 3, there is a possibility that useful nutrient components may be simultaneously adsorbed and removed due to nonspecific adsorption.

  The present invention has been made in view of such circumstances, and an object of the present invention is to provide a terminal glycation end product production inhibitor that is safe and has high activity.

The present invention along the object, Rugoshin include D and one or both of Korunushiin G as an active ingredient, inhibition of production of advanced glycation end products from the protein and sugar, advanced glycation end products and its inhibitory Gainami beauty α- glucosidase The object is achieved by providing an advanced saccharified product formation inhibitor for the degradation of one or both of the precursors.

  In the advanced saccharified product production inhibitor according to the present invention, the active ingredient may be derived from a plant belonging to the family Rubiaceae.

  The compound which is an active ingredient of the advanced glycation end product production inhibitor according to the present invention is, for example, one contained in a plant belonging to the family of the arboreal family, which has been eaten, and whose safety has been confirmed. As has been found in the invention, it has a high activity of inhibiting advanced glycation end product formation. Thus, according to the present invention, a safe and highly active advanced glycation end product formation inhibitor is provided.

It is a figure which shows the procedure of isolation | separation of a compound from the peel of abalone. It is a figure which shows the procedure of isolation | separation of a compound from the peel of a brassica.

The advanced glycation end product production inhibitor according to one embodiment of the present invention (hereinafter, may be abbreviated as “end glycation end product generation inhibitor”) belongs to gallotannins or ellagitannins, and has a β-D-glucose skeleton. comprising as one or the active ingredient one or both of Rugoshin D and Korunushiin G of the plurality of compounds and one or more compounds selected from the group consisting of ellagic acid containing the product of advanced glycation end products from the protein and sugar It has an inhibitory activity, an α-glucosidase inhibitory activity, and an activity of degrading one or both of advanced glycation end products and their precursors .

  Gallotannins and ellagitannins are a kind of hydrolyzable tannins that are hydrolyzed to polyhydric phenolic acids and polyhydric alcohols with acids, alkalis and enzymes.The former is gallic acid as polyhydric phenolic acid, and the latter is gallic acid. It produces ellagic acid as a polyhydric phenolic acid. Compounds belonging to gallotannins and ellagitannins used as an active ingredient of the terminal saccharification product production inhibitor generate D-glucose as a polyhydric alcohol.

  As a compound belonging to gallotannins or ellagitannins and having a β-D-glucose skeleton, for example, among the gallotannins and ellagitannins represented by the following formulas (I) and (I ′), for example, the following formula ( Lugocin D represented by II), cornusin G represented by the following formula (III), and terimaglandin II represented by the following formula (IV).

  Ellagic acid is a compound represented by the following formula (V).

  In the formulas (I) and (I '), R, R' and R "each independently represent a hydrogen atom or a galloyl group represented by the following formula.

  Specific examples of compounds belonging to gallotannins include 2,6-di-O-galloyl-β-D-glucose, 1,2,3-tri-O-galloyl-β-D-glucose, 1,2,6 -Tri-O-galloyl-β-D-glucose, 2,3,6-tri-O-galloyl-β-D-glucose, 1,2,3,6-tetra-O-galloyl-β-D-glucose , 1,2,4,6-tetra-O-galloyl-β-D-glucose and 1,2,3,4,6-penta-O-galloyl-β-D-glucose. Is 1,2,3-tri-O-galloyl-β-D-glucose, 1,2,6-tri-O-galloyl-β-D-glucose, 2,3,6-tri-O-galloyl- β-D-glucose, 1,2,3,6-tetra-O-galloyl-β-D-glucose, 1,2,4,6-tetra-O-galloyl-β-D-glucose and 1,2,3,4,6-penta-O-galloyl-β-D-glucose. These compounds and Lugocin D, Cornusin G and Terimaglandin II may be used alone or in any combination of two or more.

  These compounds may be isolated from any plant or the like, or may be produced by chemical synthesis. Examples of plants containing these compounds include plants of the arboreal family. Although there are no particular restrictions on the arborea family, specific examples include burdock (wabishi) (Trapa japonica), eastern bark (Trapa natans L. ver. .

  These compounds can be extracted from each part of the Luminoceae plant, for example, flowers, spikes, pericarp, fruits, pulp, stems, leaves, roots, seeds, and the like. Can be The extraction can be performed by a commonly used method. Specifically, it can be carried out by cutting the pericarp of a plant as it is or by cutting it into an appropriate size, and extracting with a solvent or homogenizing in a solvent. As the extraction solvent, for example, water, various organic solvents, or a mixed solvent thereof can be used. Examples of the organic solvent for extraction include lower alcohols (for example, methanol and ethanol), chloroform, ethyl acetate, and n-hexane. Among the extraction solvents, water, methanol and ethanol are particularly preferred. These solvents can be used alone or in combination of two or more. The amount of the extraction solvent to be used varies depending on the site to be used, the extraction solvent, and the like, but the weight ratio is appropriately in the range of 1: 2 to 1:30 (plant material: extraction solvent), and 1: 3 to 1:20. Is preferable, and the range of 1: 5 to 1:10 is more preferable. The extraction time is suitably in the range of 1 hour to 15 days. The extraction temperature is suitably in the range of 5 to 100 ° C. The extraction method is not particularly limited, and any method such as batch extraction and continuous extraction using a column can be applied.

  Before the separation of one or more compounds from the solvent extract of pericarp of perennial plants, pretreatment by dialysis, ultrafiltration, filtration, column chromatography, etc. to remove high molecular weight components, insolubles, etc. May be performed.

  When removing insolubles or the like by filtration, an adsorbent such as activated carbon, bentonite, or celite, or a filter aid may be added as necessary to remove impurities. In particular, when used in the state of an extract, it is preferable to perform sterilization filtration using a membrane filter or the like.

  Separation of the extract that has been subjected to the pretreatment as described above, if necessary, can be performed using any known method such as column chromatography, reverse phase chromatography, or ion chromatography. It can be performed by fractionating fractions having high inhibitory activity.

  The terminal glycation product production inhibitory activity is determined, for example, by determining the ratio (inhibition rate) of the terminal glycation product production inhibitor in the absence of the terminal glycation product production inhibitor to that in the presence of the terminal glycation product production inhibitor. Can be evaluated. The measurement of the amount of the advanced glycation end product can be performed, for example, by measuring a physical quantity such as the fluorescence intensity unique to the advanced glycation end product.

  The terminal glycation product to be inhibited by the terminal glycation product production inhibitor is a terminal glycation product derived from any protein and any sugar, and specific examples include serum proteins such as albumin and globulin, collagen, elastin and the like. And extracellular matrix proteins, membrane proteins such as G proteins, and advanced glycation end products formed by saccharification of intracellular proteins. Typical examples of sugars include glucose and fructose, which are present in high concentrations in blood and body fluids.

  Reactions to be inhibited by the terminal glycation end product formation inhibitor include formation of Schiff bases by reaction of amino groups of proteins with reducing sugars, formation of 1,2-enaminol or 2,3-enediol by Amadori rearrangement, Amadori transfer It may be any one or more reactions, such as decomposition of the product and formation of a polymerization product of the degradation product with an amino acid, peptide or protein.

  The terminal glycation end product production inhibitor is mixed with a carrier or the like to form a pharmaceutical composition having one or both of a therapeutic effect and a preventive effect on diseases involving terminal glycation end products such as diabetes and diseases and symptoms related thereto. Can be used. The administration form of the pharmaceutical composition to humans or animals includes oral, rectal, parenteral (for example, intravenous administration, intramuscular administration, subcutaneous administration, etc.) and the like. It is difficult to unambiguously determine the dose by appropriately setting it according to the method of administration, the purpose of use and the age, weight, and symptoms of the subject to which it is applied, but in the case of humans, generally the active ingredient contained in the formulation And preferably 0.1 to 2000 mg / day for an adult. Of course, since the dosage varies depending on various conditions, a dosage smaller than the above-mentioned dosage may be sufficient, or may be required to exceed the above-mentioned range.

  When prepared as an oral administration preparation, it can be prepared in the form of tablets, granules, powders, capsules, coatings, solutions, suspensions, and the like, and when prepared into a parenteral administration preparation, injections, drops, It can be prepared in the form of a suppository or the like. Any known method can be used for formulation. For example, the above-mentioned desired dosage form can be prepared by blending an advanced glycation end product formation inhibitor with a pharmaceutically acceptable carrier or diluent, stabilizer, and other desired additives.

  The food containing the advanced saccharified product formation inhibitor can be in a form in which the advanced saccharified product formation inhibitor is blended with the food, or in any form usually used for foods or health foods such as capsules and tablets. There is no particular limitation on the type of food to be mixed, and for example, liquid (fluid) food such as coffee, fruit juice, soft drink, beer, milk, miso soup, soup, black tea, tea, nutritional supplement, syrup, margarine, jam, etc. , Rice, bread, potato products, rice cake, candy, chocolate, sprinkle, ham, sausage, candy, and other solid foods, and other staple foods, side dishes, confectioneries, and seasonings. Depending on the application, it may be formed into a powder, granule, tablet or the like. Further, if necessary, it may be appropriately mixed with excipients, extenders, binders, thickeners, emulsifiers, coloring agents, flavors, food additives, seasonings and the like.

  In addition to foods to be consumed by humans, the terminal glycation end product production inhibitor is mixed in the feed, and when administered to animals such as livestock and pets, it is mixed in advance with the raw material of the feed to function. It can be prepared as a feed with added properties. Further, an advanced glycation end product formation inhibitor can be added to the feed and administered. That is, foods containing an advanced glycation end product production inhibitor as an active ingredient can be safely added to livestock such as pigs, chickens, cows, horses, sheep, and feeds such as fish and pets (dogs, cats, birds). Thus, it can be used as a functional feed having one or both of a therapeutic effect and a preventive effect on diabetes and diseases and symptoms related thereto.

  The terminal glycation end product formation inhibitor may have an α-glucosidase inhibitory activity, an activity of inhibiting fructose absorption into the body, and the like. These activities do not directly inhibit the chemical reactions involved in the production of advanced glycation end products, but having these activities, by suppressing the absorption of glucose and fructose into the body, This is preferable in that generation can be suppressed indirectly. Evaluation of these activities can be performed using any known method. Further, the advanced glycation end product formation inhibitor may also have an activity of decomposing one or both of the advanced glycation end product and its precursor. In addition to the activity of inhibiting the chemical reaction involved in the production of advanced glycation end products, having the activity of degrading one or both of the produced advanced glycation end products and their precursors, such as diabetes and diseases and conditions related thereto, It is more preferable from the viewpoint of treatment and prevention for diseases associated with advanced glycation end products.

Next, an example performed to confirm the operation and effect of the present invention will be described.
Example 1 Separation and Identification of Compound from Waxy Peel The outline of the procedure for separating the compound is as shown in FIG. After the dried pericarp of the abalone (Trapa japonica) was homogenized in 70% acetone ("Homogenized in 70% aq. Acetone" in FIG. 1), the solution was filtered through a filter ("filtd." In FIG. 1) and centrifuged. Was. The obtained acetone-extracted fraction was subjected to diethyl ether (“Et ₂ O ext.” In FIG. 1), ethyl acetate (“AcOEt ext.” In FIG. 1), water-saturated butanol (“n-BuOH” in FIG. 1). ext. "). Next, the ethyl acetate-extracted fraction was applied to an HPLC column (Toyopearl HW-40 (inner diameter 2.2 cm × 50 cm)) to separate and purify the components. Elution is 30% methanol → 40% methanol → 50% methanol → 60% methanol → 70% methanol → 100% methanol → methanol: acetone: water (7: 1: 2) → methanol: acetone: water (7: 2: 1) Performed under the condition of 70% acetone. From a 30% methanol eluate, 1,2,3-tri-O-galloyl-β-D-glucose, 1,2,6-tri-O-galloyl-β-D-glucose, 1,2,3,6- Tetra-O-galloyl-β-D-glucose was obtained. Lugocin D was obtained from a methanol: acetone: water (7: 1: 2) eluate, and cornuciin G was obtained from a methanol: acetone: water (7: 2: 1) eluate. The isolated compound was identified by comparing physicochemical and spectroscopic data using MS, NMR and the like with a pure product or a known compound.

Example 2 Separation and Identification of Compound from Peel Peel The outline of the compound separation procedure is as shown in FIG. A hot water extract of the pericarp (Trapa bispinosa) peel was extracted using ether, ethyl acetate and butanol. Next, the obtained ethyl acetate fraction was applied to an HPLC column (Toyopearl HW-40C) to separate and purify the components. From the 40% methanol eluate, 2,6-di-O-galloyl-β-D-glucose, from the 50% methanol eluate, 1,2,3-tri-O-galloyl-β-D-glucose, 1, From 2,6-tri-O-galloyl-β-D-glucose, 2,3,6-tri-O-galloyl-β-D-glucose, 60% methanol eluate, 1,2,3,6-tetra From the eluate of -O-galloyl-β-D-glucose and 70% methanol, 1,2,4,6-tetra-O-galloyl-β-D-glucose and terimaglandin II were obtained. The presence of ellagic acid was confirmed by HPLC analysis of ethyl acetate and butanol extract, and comparative analysis with a standard product. The isolated compound was identified by comparing the physicochemical and spectroscopic data using MS, NMR and the like with those of a genuine product or a known compound.

Example 3 Measurement of α-Glucosidase Inhibitory Activity Lugocin D, Cornusinin G, 1,2,3,4,6-penta-O-galloyl-β-D-glucose and α-glucosidase inhibitory activity as a positive control The a-glucosidase inhibitory activity of acarbose, an oral hypoglycemic agent known to have been evaluated, was evaluated.

  After suspending rat intestinal acetone powder in 20 mL of 100 mM sodium phosphate buffer (pH 7.0), centrifugation (10,000 × g, 15 minutes) was performed, and the supernatant was mixed with α-glucosidase solution (enzyme solution). did. As a test method, first, 250 mM maltose, 100 mM sodium phosphate buffer (pH 7.0), an enzyme solution, and a measurement sample (Lugosine D, Cornusin G, 1,2,3,4,6-penta-O-galloyl-β- D-glucose and acarbose as a positive control) were mixed to prepare a reaction solution (200 μL in total). After incubation at 37 ° C. for 15 minutes, the enzyme reaction was stopped by heating at 100 ° C. for 5 minutes. The amount of glucose generated by the action of α-glucosidase was quantified by F-kit Glucose (JK International Inc.) to calculate the α-glucosidase inhibitory activity of each measurement sample.

Table 1 shows the measurement results of the α-glucosidase inhibitory activity. Rugoshin D in α- glucosidase inhibitory activity measurement, respectively the _{IC 50} values of Korunushiin G, 5.1MyuM, a 6.3MyuM, acarbose was used as a positive control _{(IC 50} value: 4.0 [mu] M) equivalent to a strong inhibitory activity Was found. In addition, 1,2,3,4,6-penta-O-galloyl-β-D-glucose was also found to exhibit α-glucosidase inhibitory activity measurement, although weaker than acarbose (IC ₅₀ value: 59 μM).

Example 4: Measurement of anti-glycation activity 2,6-di-O-galloyl-β-D-glucose, 1,2,3-tri-O-galloyl-β-D-glucose, 1,2,6-tri —O-galloyl-β-D-glucose, 2,3,6-tri-O-galloyl-β-D-glucose, 1,2,3,6-tetra-O-galloyl-β-D-glucose, , 2,4,6-tetra-O-galloyl-β-D-glucose, 1,2,3,4,6-penta-O-galloyl-β-D-glucose, rugosine D, cornusiin G, terrimaglandin II and ellagic acid and, as a positive control, aminoguanidine, which is known to inhibit the reaction of producing a terminal glycation product from 3-deoxyglucosone, which is an intermediate of the saccharification reaction, was used to prepare human serum albumin and glucose. Activity to suppress the production of advanced saccharification products Were evaluated (anti-glycation activity).

  Measurement sample (2,6-di-O-galloyl-β-D-glucose, 1,2,3-tri-O-galloyl-β-D-glucose, 1,2,6-tri-O-galloyl-β -D-glucose, 2,3,6-tri-O-galloyl-β-D-glucose, 1,2,3,6-tetra-O-galloyl-β-D-glucose, 1,2,4,6 -Tetra-O-galloyl-β-D-glucose, 1,2,3,4,6-penta-O-galloyl-β-D-glucose, rugosine D, cornucinin G, terimaglandin II and ellagic acid) , 0.3, 1, 3, 10, 30, 100 μg / mL, aminoguanidine as a positive control was added at 0.01, 0.03, 0.1, 0.3, 1, 3 mg / mL. It was dissolved in distilled water at a concentration to obtain a sample solution. The sample solution and the reagent were dispensed into the microtube in an amount shown in Table 2 below, mixed well, and incubated at 60 ° C. for 40 hours. Each reaction solution was diluted 8-fold with distilled water, 200 μL was transferred to a 96-well black plate, and the fluorescence intensity (Ex: 370 nm, Em derived from advanced glycation end products produced from human serum albumin and glucose) was measured using a plate reader. : 465 nm).

The reaction rate and the inhibition rate (reaction inhibition rate) were calculated according to the following equations.
Reaction rate (%) = ((AB) / (CD)) × 100
Inhibition rate (%) = 100-reaction rate A: Relative fluorescence intensity of sample B: Relative fluorescence intensity of sample blank C: Relative fluorescence intensity of control D: Relative fluorescence intensity of control blind

An approximate curve was created by plotting the sample concentration on the horizontal axis and the inhibition rate on the vertical axis. The concentration (IC ₅₀ ) showing the inhibition rate of 50% for each sample was calculated from the equation of the approximate curve. Table 3 shows the results.

As compared with the aminoguanidine used as a positive control, all the measured samples showed a very strong anti-glycation activity, especially 1,2,3-tri-O-galloyl-β-D-glucose, 1,2,6 -Tri-O-galloyl-β-D-glucose, 2,3,6-tri-O-galloyl-β-D-glucose, 1,2,3,6-tetra-O-galloyl-β-D-glucose , 1,2,4,6-tetra-O-galloyl-β-D-glucose, 1,2,3,4,6-penta-O-galloyl-β-D-glucose, rugosine D, cornusin G, terima Grangein II was found to have a significantly higher activity when the IC ₅₀ was less than 1 μM. For the advanced glycation end products produced from human serum albumin and fructose, the anti-glycation activity was evaluated using the same procedure. Table 4 shows the results. It can be seen that all the measured samples show an anti-glycation activity even stronger than that of glucose, as compared to aminoguanidine used as a positive control. Therefore, it was confirmed that all the measurement samples showed strong anti-glycation activity for both glucose and fructose.

Example 5: Measurement of Degradation Rate of Advanced Saccharified Product Precursor 1. 900 μL of a 13 mM PPD (1-phenyl-1,2-propanedione) solution and 100 μL of various sample solutions were added to a 1.5 mL tube and mixed. Thereafter, the reaction was carried out in a 37 ° C. water bath for 4 hours. After stopping the reaction by adding 200 μL of a 2N HCl solution, each solution was centrifuged and then filtered through a HPLC filter to obtain a sample for HPLC analysis. The peak area of benzoic acid generated in proportion to the decomposition of PPD was detected under the following conditions using HPLC.

-Column: InertStainC18 (inner diameter 4.6 mm x 150 mm)
Eluent A: 50 mM phosphate buffer solution (pH 2.2)
Eluent B: 100% acetonitrile Gradient (concentration of eluent B (%))
0 minutes 25%
20 minutes 25%
21 minutes 100%
25 minutes 100%
26 minutes 25%
36 minutes 25%
・ Flow rate: 1 mL / min ・ Temperature: 40 ° C
・ Injection volume: 10 μL

  The concentration of the generated benzoic acid was calculated using a standard curve prepared using a benzoic acid reagent. The AGEs precursor decomposition rate was calculated from the following equation.

  AGEs precursor decomposition rate (%) = benzoic acid formation concentration (mM) / PPD concentration (1 mM) × 100

  In addition, a system to which 100 μL of distilled water was added instead of the sample solution was set as a control group. Table 5 shows the results. All the measured samples showed a higher decomposition rate than PTB (N-phenacylthiazolium bromide) used as a positive control.

Claims (2)

Wherein one or both of Rugoshin D and Korunushiin G as an active ingredient, inhibition of production of advanced glycation end products from the protein and sugar, one or both of the advanced glycation end products and its precursors inhibitory Gainami beauty α- glucosidase decomposition advanced glycation endproducts formation inhibitor for.   The terminal glycation end product formation inhibitor according to claim 1, wherein the active ingredient is derived from a plant belonging to the family Rubiaceae.

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