JP2010006748A - Dipeptidyl peptidase iv inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a composition containing a high safety dipeptydyl peptidase IV inhibitor obtained from a material derived from a natural substance and its manufacturing method. <P>SOLUTION: The composition contains a substance having dipeptidyl peptidase IV inhibition activity derived from one or more plants belonging to a genus selected from the group consisting of Rosa, Rosaceae; Prunus, Rosaceae; Alchemilla, Rosaceae; Carthamus, Compositae; Chrysanthemum, Compositae; Origanum, Lamiaceae; Apocynum, Apocynaceae; Trifolium, Leguminosae; Aspalathus, Leguminosae; Cuminum, Apiaceae; Equisetum, Equisetaceae; Tremella, Tremellaceae; Syzygium, Myrtaceae; Phyllanthus, Euphorbiaceae; and Gentiana, Gentianaceae and its manufacturing method is described. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a composition containing a plant-derived peptidyl peptidase IV inhibitory activity and a method for producing the same.

  Dipeptidyl peptidase IV (EC 3.4.14.5; hereinafter referred to as “DPP-IV”) is derived from the amino terminus (N-terminus) of polypeptides and proteins from the X-Pro dipeptide (X is any amino acid). It is a non-classical serine aminodipeptidase that removes residues).

  DPP-IV is essentially expressed on epithelial and endothelial cells of a very wide range of animal tissues (eg intestine, liver, lung, kidney and placenta) and has also been found in body fluids. DPP-IV is also expressed on circulating T lymphocyte cells and has been shown to be synonymous with the cell surface antigen CD-26. It has been reported that DPP-IV is related to many disease states as described later (see Non-Patent Document 1).

  In recent years, it has been clarified that DPP-IV works on the metabolism of glucagon-like peptide (hereinafter referred to as “GLP-1”). GLP-1 is a peptide with 29 amino acids that is induced by post-translational processing of proglucagon in the small intestine. GLP-1 has many actions in vivo, such as promotion of insulin secretion, inhibition of glucagon secretion, feeding suppression action, enhancement of satiety, and reduction of gastric excretion rate (see Non-Patent Document 1).

  DPP-IV is inactivated by hydrolyzing the amino terminal dipeptide of GLP-1. Therefore, it has been clarified that the half-life of GLP-1 in vivo is about 1.5 minutes. In addition, it is known that the inactivated form acts as an antagonist of the GLP-1 receptor.

  Therefore, inhibiting DPP-IV enhances the action of GLP-1, promotes insulin secretion, improves glucose metabolism, and is useful for the treatment and prevention of type 2 diabetes, obesity, and insulin resistance. It is expected. In fact, in DPP-IV-deficient rats, a significant increase in blood GLP-1 and good glucose tolerance were observed compared to control animals, and obesity and inhibition of the development of insulin resistance were observed due to the addition of a high fat diet. (See Non-Patent Documents 2 and 3).

  In addition, administration of a substance having DPP-IV inhibitory activity to a spontaneously diabetic model rat has been observed to improve insulin sensitivity and lower blood glucose levels (see Non-Patent Document 4).

  DPP-IV is involved in the metabolism of neuropeptide Y, which is a neuropeptide, activation of T cells, which are immunocompetent cells, adhesion of cancer cells to the endothelium, and entry of HIV virus into lymphocytes. It is known (see Non-Patent Document 5). Therefore, inhibiting DPP-IV is considered useful for the treatment and prevention of immune diseases and the like related thereto.

  In addition, high expression of DPP-IV has been found in fibroblasts in the skin of patients with benign prostatic hypertrophy, psoriasis, rheumatoid arthritis, and lichen planus, and inhibition of DPP-IV is benign prostatic hypertrophy It is also considered useful for the treatment and prevention of these diseases including

  Until now, DPP-IV inhibitory compounds include fluoropyrrolidine, proline derivatives, pyrazines, etc., and some have already been put into practical use (see Patent Documents 1, 2, and 3). However, these known compounds are chemically synthesized products and are not sufficient in terms of safety.

  In addition, from natural products, it has been reported that peptides contained in cheese, paprika, rose red petal, and cats claw extract have DPP-IV inhibitory activity (see Patent Documents 4 and 5).

JP 2007-063286 A JP 2007-537231 A JP-T-2004-536115 JP 2007-039424 A JP 2007-277163 A Japanese pharmacology journal 2005 125 379-84 Biochem Biophys Res Commun. 2001 Jun 8; 284 (2): 501-6 Life Sci. 2002 May 31; 71 (2): 227-38. Diabetes. 2002 Apr; 51 943-50 Crit Rev Clin Lab Sci. 2003 Jun; 40 (3): 209-94

  An object of the present invention is to provide a composition containing a DPP-IV inhibitor obtained from a natural product-derived material and expected to have high safety, and a method for producing the same.

  As a result of intensive studies to solve the above problems, the present inventors have found that a specific plant, which is a material derived from a natural product that can be easily obtained or consumed daily, contains a substance having DPP-IV inhibitory activity. As a result, the present invention has been completed.

That is, the present invention includes the following features.
(1) Rosaceae, Genus Rosaceae, Rosaceae, Alchemilla, Asteraceae, Asteraceae, Chrysanthemum, Pteridocyticus, Pteridocyticum, Biculusmon, Leguminosae, Aspertus, Seri Peptidyl peptidase IV inhibitory activity derived from one or more plants belonging to the genus selected from the group consisting of genus Cumin, Toxaceae, Toxa, White jellyfish, White peach genus, Euphorbiaceae, Filanthus genus, Gentian genus Genus A composition comprising a substance having

  (2) The plants are Rosa multiflora, Rosa gallica, Carthamus tinctorius, Prunus domestica, Prunus armeniaca, Alchemia・ Alchemilla mollis, Origanum majorana, Apocynum venetum, Trifolium pratense, Chrysanthemum morifolium, Chrysanthemum numfo, umi cyme Equisetum arvense, Aspalathus linearis, Tremella fuciformis, Syzygium aramaticum, Phyllanthus niruri or Gentianella alboro A is (Gentianella alborosea) 1 or more, the (1) composition.

  (3) The composition according to (2) above, comprising a substance having peptidyl peptidase IV inhibitory activity derived from a bud of Rosa multiflora, Rosa gallica or Syzygium aramaticum object.

  (4) The composition according to any one of (1) to (3), wherein the substance having peptidyl peptidase IV inhibitory activity is an extract from the plant or a part thereof.

(5) The composition according to (4), wherein the extract is an organic solvent extract.
(6) The composition according to any one of (1) to (5) above, which is a food or drink.
(7) The composition according to any one of (1) to (5) above, which is a pharmaceutical product.

  (8) Rosaceae, Genus Rosaceae, Rosaceae Alchemilla, Chrysanthemum safflower, Asteraceae Chrysanthemum, Lamiaceae Hanahakka, Oleander Basiculum, Leguminous rhododendron, Legume Aspartus, Seri Solvent extraction of one or more plants belonging to the genus selected from the group consisting of the genus Cumin, Toxaceae, Toxa, White jellyfish, White jellyfish, Myrtaceae, Myrtaceae, Philanthus and Gentian And a method for producing a composition containing a dipeptidyl peptidase IV inhibitor, comprising collecting a substance having dipeptidyl peptidase IV inhibitory activity from the extracted fraction.

  (9) The plants are Rosa multiflora, Rosa gallica, Carthamus tinctorius, Prunus domestica, Prunus armeniaca, Alchemia・ Alchemilla mollis, Origanum majorana, Apocynum venetum, Trifolium pratense, Chrysanthemum morifolium, Chrysanthemum numfo, umi cyme Equisetum arvense, Aspalathus linearis, Tremella fuciformis, Syzygium aramaticum, Phyllanthus niruri or Gentianella alboro A is (Gentianella alborosea) 1 or more, (8) The method according.

  (10) The method according to (8) or (9) above, wherein the solvent is at least one selected from the group consisting of ethanol, methanol, ethyl acetate and acetone.

The composition of the present invention can significantly inhibit the activity of DPP-IV.
Moreover, since the composition of the present invention is a composition derived from a natural product based on a long-time eating experience, there is little concern about side effects and the like, and it can be taken continuously for a long time at a low cost.

  Hereinafter, the DPP-IV inhibitor-containing composition according to the present invention (hereinafter also referred to as “the composition of the present invention”) and the production method thereof will be described.

  The composition of the present invention comprises roses, roses, cherry blossoms, roses, alchemillas, chrysanthemum safflowers, chrysanthemum chrysanthemum, rhododendrons genus moth, oleanders basicurmon, leguminous rhododendrons, legumes asparagus. DPP derived from one or a plurality of species belonging to the genus selected from the group consisting of Tusus, Apiaceae Cumin, Toxaceae Toxa, White-clamaceae White-billed genus, Myrtaceae Futomomo, Euphorbiaceae Filansus and Gentian It contains a substance having -IV inhibitory activity.

  Examples of the plant belonging to the genus Rosaceae that can be used in the present invention include, but are not limited to, Rosa multiflora, Rosa gallica, Rosa wichuraiana. ), Rosa chinensis, Rosa damasccna and the like. In particular, it is preferable to use Rosa Multiflora or Rosa Galica.

  Examples of the plant belonging to the genus Rosaceae that can be used in the present invention include, but are not limited to, Prunus domestica, Prunus armeniaca, Prunus persica (Prunus). persica), Prunus dulcis, Prunus mume, Prunus cerasus and the like. In particular, it is preferable to use Prunus domestica or Prunus armenia.

  Plants belonging to the genus Alchemilla that can be used in the present invention include, but are not limited to, Alchemilla mollis, Alchemilla xanthoclora, Alchemilla vulgaris (Alchemilla vulgaris). ) And the like. In particular, it is preferable to use Alchemia Morris.

  Plants belonging to the genus Asteraceae that can be used in the present invention include, but are not limited to, Carthamus tinctorius, Carthamus lanatus, Carthamus arborescens. ) And the like. In particular, it is preferable to use Calsamus chinktorius.

  Plants belonging to the genus Asteraceae that can be used in the present invention include, but are not limited to, Chrysanthemum morifolium, Chrysanthemum coronarium, Chrysanthemum pacificum. ) And the like. It is particularly preferable to use chrysantemum molyfolium.

  Plants belonging to the genus Lamiaceae that can be used in the present invention include, but are not limited to, Origanum majorana, Origanum dictamnus, Origanum vulgare (Origanum vulgare). ) And the like. In particular, it is preferable to use Origanum marjorana.

  Examples of the plant belonging to the genus Basicummonaceae that can be used in the present invention include, but are not limited to, Apocynum venetum, Apocynum androsaemifolium, Apocynum canapinum (Apocynum cannabinum). It is particularly preferable to use apocynum venetum.

  Plants belonging to the genus Leguminosae that can be used in the present invention are not limited to these, but include trifolium pratense, trifolium repens, and trifolium arvense. (Trifolium arvense). In particular, it is preferable to use Trifolium pratense.

  Examples of the plant belonging to the genus Aspartus that can be used in the present invention include, but are not limited to, Aspalathus linearis, Aspalathus cedarbergensis, and the like. Can be mentioned. In particular, it is preferable to use Asparagus lineias.

  Examples of the plant belonging to the genus Cuminium that can be used in the present invention include, but are not limited to, Cuminum cyminum.

  Examples of the plant belonging to the genus Toxa family that can be used in the present invention include, but are not limited to, Equisetum arvense, Equisetum bogotense, Equisetum giganteum (Equisetum giganteum). ) And the like. In particular, it is preferable to use Equisetum albense.

  Examples of the plant belonging to the genus White jellyfish that can be used in the present invention include, but are not limited to, Tremella fuciformis, Tremella mesenterica and the like. It is particularly preferable to use Tremera fushiformis.

  Plants belonging to the genus Myrtaceae that can be used in the present invention include, but are not limited to, Syzygium aramaticum, Syzygium oleosum, Syzygium aqueum (Syzygium aqueum). ) And Syzygium forte. It is particularly preferred to use Siggium aramaticum (also called clove).

  Plants belonging to the genus Philanthus, which can be used in the present invention, include, but are not limited to, phyllanthus niruri, phyllanthus emblica, phyllanthus acidus (Phyllanthus acidus). ) And the like. It is particularly preferable to use Filansas nirrii.

  Plants belonging to the genus Gentianaceae that can be used in the present invention include, but are not limited to, Gentianella alborosea, Gentianella anglica, Gentianella germanica (Gentianella germanica). ) And the like. In particular, it is preferable to use gentianella alborosea.

  The plant as a material of the composition of the present invention may use the whole plant body or a part of the plant body. Examples of the plant body include, but are not limited to, buds, leaves, roots, stems, flowers, fruits, seeds, etc., and one or more of them can be used in the present invention. . In the present invention, it may be preferable to use a bud as a part of the plant body, and examples of such a bud include Rosa multiflora, Rosa galica, sigium aramaticum bud and the like. Moreover, the preservation | save state and form of the said plant or its part are not restrict | limited in particular, For example, you may use what dried the whole plant body or its part. Examples of such can include rooibos tea, which is a dried product of Asparagus linearis, clove, which is a dried product of buds of Sigigi Aramachicum, and cumin, which is a dried product of seeds of cuminum kiminum. .

  In the present invention, the varieties and production areas of the above plants are not particularly limited, and may be wild species or cultivars or horticultural varieties obtained by crossing.

  The “substance having DPP-IV inhibitory activity” (hereinafter, also referred to as “DPP-IV inhibitor”) refers to any substance having DPP-IV inhibitory activity derived from the plant of the plant or a part thereof. Point to. The DPP-IV inhibitor may be either a purified product or a crude product. The crude product includes, but is not limited to, for example, the plant itself or a dried product thereof or a crushed product thereof, a suspension or suspension of the crushed product, or a plant or a part thereof, or Examples thereof include extracts from those dried products or dried crushed materials.

  In the present invention, the DPP-IV inhibitor is preferably an extract derived from the plant or a part thereof. Such an extract can be obtained by an extraction method known to those skilled in the art. Examples of the extraction method include a solvent extraction method, a supercritical fluid extraction method, and a heated pressure extraction method. Each of the extraction methods may be used alone, or a plurality of extraction methods may be used in combination.

  In the present invention, the preferred extraction method is solvent extraction. Solvent extraction may be performed using a general method known to those skilled in the art, and it is not necessary to use a special method.

  For example, when hot water extraction is used as solvent extraction, the above-mentioned plant or a part of the crushed material or a dried crushed material is dispersed in a suitable water (for example, 1: 1 to 1: 100 weight ratio), and the homogenizer, stirrer is dispersed. Extraction can be carried out by heating after homogenization using a shaker or the like, adjusting the pH at around neutral. The temperature at the time of hot water extraction is usually 70 ° C to 100 ° C, preferably 80 ° C to 100 ° C, for example 90 ° C. The extraction time is 5 minutes to 24 hours, preferably 30 minutes to 3 hours, for example about 1 hour.

  Particularly preferred solvent extraction in the present invention is organic solvent extraction. In this case, water and an organic solvent are added to the pulverized product or dried crushed product of each plant material, and extraction is performed at room temperature or with heating (for example, 60 to 90 ° C.). There is no particular limitation on the ratio of water and organic solvent, and those skilled in the art can appropriately adopt an appropriate ratio, but it should be noted that a higher organic solvent concentration is preferable in terms of extraction efficiency. Examples of the organic solvent include, but are not limited to, ethanol, methanol, ethyl acetate, acetone, and the like. These organic solvents may be used as a mixture of two or more. The amount of the solvent to be added can be 1 to 100 times, preferably 5 to 50 times, for example, 10 times the weight of the plant material. The extraction time is the same as described above for hot water extraction. The DPP-IV inhibitor of the present invention is particularly preferably an organic solvent extract. The organic solvent extraction may be used in combination with the hot water extraction.

  After performing the solvent extraction as described above, the insoluble fraction containing the material residue is removed by filtration or centrifugation, and the soluble fraction or the organic solvent fraction (ie, the extracted fraction) is concentrated by, for example, evaporation under reduced pressure. Extracts containing DPP-IV inhibitors can be obtained in fluid or dry form.

  In some cases, the DPP-IV inhibitor of the present invention can be further obtained from the precipitate after solvent extraction by repeating hot water extraction or extraction with an organic solvent for the insoluble fraction after solvent extraction. The DPP-IV inhibitor of the present invention present in the extracted fraction may be further purified, and in this case, known separation and purification methods can be combined appropriately. For example, liquid-liquid partition, organic solvent precipitation, various column chromatography (for example, HPLC, silica gel chromatography, molecular sieve chromatography, ion exchange chromatography, reverse phase chromatography, etc.), crystallization and the like can be used.

  Although the DPP-IV inhibitory substance of the present invention can be obtained from each plant material as described above, the composition of the present invention is an extract preparation of each plant material that has already been commercialized. It may be included as an inhibitor. Examples of such extract preparations include rose oil, rose extract, safflower extract, and clove extract that are commercially available from various manufacturers.

  The composition of the present invention may be obtained by granulating, encapsulating, tableting or pasting the DPP-IV inhibitor of the present invention obtained as described above by a conventional method.

  Eur J Pharmacol. 2005 Jul l25; 518 (1): 63-70 and J Med Chem. 2005 Jan 13; 48 (1): 141-51 are in vitro evaluations for DPP-IV inhibitory activity. Reported to be correlated. Specifically, in the above document, DPP-IV inhibitory activity was confirmed by conducting an enzyme reaction test using a synthetic substrate as an in vitro evaluation, and orally administered to a mouse as an in vivo evaluation, and the blood DPP-IV activity decreased. The improvement effect with respect to an active GLP-1 density | concentration rise, an insulin concentration rise, and a glucose tolerance test is confirmed. The mechanism of action by which inhibition of DPP-IV in vivo is useful for the treatment and prevention of type 2 diabetes, obesity, insulin resistance, etc. is as described above. It focuses on the DPP-IV inhibition portion of the mechanism of action and reflects this. Thus, a component having DPP-IV inhibitory activity in vitro inhibits DPP-IV even in vivo, and as a result, is useful for the treatment and prevention of type 2 diabetes, obesity, insulin resistance, etc. It is known.

  The DPP-IV inhibitory substance of the present invention obtained as described above showed in vitro DPP-IV inhibitory activity as shown in the following examples as a result of in vitro evaluation in the same manner as in the above document. It is a thing. Therefore, the composition of the present invention can be provided as a food and drink and a medicine for treating and preventing diseases involving DPP-IV such as type 2 diabetes, obesity, and insulin resistance.

  When the composition of the present invention is provided as a food or drink, it can be a health food, a functional food, a food for specified health use, a dietary supplement, or a food with a disease risk reduction label.

  The form of the food or drink of the present invention is not particularly limited, and can be processed into various beverages and foods. Examples of such beverages and foods include, but are not limited to, beverages such as tea and liquor, dairy products such as dried fruits and yogurt, baked confectionery such as cookies, processed foods such as jams, seasonings, It can be processed, added or blended into various foods such as spices, or can be eaten as it is as a salad.

  Moreover, unless the DPP-IV inhibitory effect is suppressed, the food-drinks of this invention can contain other components, such as a nutritional supplement component, in addition to the DPP-IV inhibitor of this invention. Such ingredients include vitamins (eg vitamin A, vitamin B group, vitamin E, vitamin C, vitamin D, vitamin K, niacin, pantothenic acid, folic acid, etc.), carotenoids (eg β-carotene, lycopene, fucoxanthin, etc.), Minerals (eg seaweed components, CCM, heme iron, iron salt system, whey calcium, fermented calcium lactate, beef bone calcium, salmon calcium, eggshell calcium, etc.), various plants and their extracts, purified products and fractions (E.g. psyllium, chlorella, spirulina, garlic, ginkgo biloba, gymnema, tochu leaf, shiso leaves, pearl barley, soy globulin, rutin, green tea extract, theanine, polyphenols, licorice, yucca, soy saponin, caffeine, huahua Torberry extract, Champignon extract, Garcinia cambodia Etc.), microorganisms and their growth factors and microbial products (for example, lactic acid bacteria, yeast, lactic acid bacteria growth factors, etc.), dietary fiber and its enzymatic degradation products (for example, apple fiber, corn fiber, starch-derived dietary fiber, indigestible dextrin, Guar gum enzyme degradation product, sweet potato fiber, soybean fiber, seaweed fiber, mushroom fiber, tea fiber, acidic polysaccharide, plant mucilage, wheat bran, etc.), animal body and its extract, purified product, degraded product and product (for example, Royal jelly, propolis, oyster extract, chitin, chitosan, taurine, collagen, gelatin, etc.), various oligosaccharides (eg, galactooligosaccharide, xylooligosaccharide, soybean oligosaccharide, fructooligosaccharide, isomaltoligosaccharide, whey oligosaccharide, etc.), lipid ( For example, unsaturated fatty acids (DHA, EPA, etc.), phospholipids, salatrim, etc.) Seed proteins and proteolysates (eg corn protein, soy protein, TMP (total milk protein), lactalbumin, casein, whey, glutathione, soy peptide, egg white peptide, glutamine peptide, etc.), wheat germ such as defatted germ It is done.

  When the composition of the present invention is ingested as a food or drink, the content of the DPP-IV inhibitor of the present invention as an active ingredient in the food or drink is usually 0.01 to 50% by weight, preferably 1 to 30% by weight. It may be ingested so that the daily intake is 1 to 500 mg, preferably 1 to 200 mg per adult.

  When the composition of the present invention is provided as a pharmaceutical, the DPP-IV inhibitor of the present invention is orally or parenterally (for example, intravenous injection, intramuscular injection, subcutaneous administration, intraperitoneal administration, rectal administration, transdermal administration, etc. ) For any route of administration. Moreover, it can be set as a suitable dosage form according to an administration route. Specifically, injections such as intravenous injection and intramuscular injection, capsules, tablets, granules, powders, pills, fine granules, lozenges and other oral preparations, rectal administration agents, oily suppositories, aqueous suppositories It can be prepared in various preparation forms. The pharmaceutical product of the present invention is particularly preferably provided as an oral preparation.

  These various preparations are commonly used excipients, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, solubilizers, preservatives. , Coloring agents, flavoring agents, stabilizers, and the like, can be produced by known methods known to those skilled in the art.

  Examples of excipients include lactose, fructose, glucose, corn starch, sorbit, and crystalline cellulose. Examples of disintegrants include starch, sodium alginate, gelatin, calcium carbonate, calcium citrate, dextrin, magnesium carbonate, and synthetic silica. Magnesium acid and the like are binders such as methyl cellulose or a salt thereof, ethyl cellulose, gum arabic, gelatin, hydroxypropyl cellulose and polyvinyl pyrrolidone, etc. Lubricants are talc, magnesium stearate, polyethylene glycol, hydrogenated vegetable oil and the like However, other additives include syrup, petrolatum, glycerin, ethanol, propylene glycol, citric acid, sodium chloride, sodium nitrite and sodium phosphate, respectively. It is below.

  The dose or intake of the composition of the present invention as an active ingredient in the pharmaceutical of the present invention is an amount that can give a therapeutic effect for a given symptom or usage, and the amount is a test using an animal or a clinical test. However, the age, sex, body weight, applicable disease and symptoms thereof, dosage form, administration method and the like of the subject should be considered. When the pharmaceutical of the present invention is an oral preparation, the above dose or intake can be 0.01 to 20 mg / Kg / day, preferably 0.01 to 8 mg / Kg / day.

  As described above, the foods and beverages and pharmaceuticals of the present invention contain ingredients derived from natural products based on long-term dietary experience as active ingredients, so there are few concerns about side effects, etc., and inexpensively and continuously over a long period of time. Can be ingested.

  Hereinafter, the present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

[Example 1]
Ten times the amount of 80% acetone was added to 300 mg of dried rose (Rosa gallica) powder and stirred at room temperature for 1 hour. After centrifugation, the supernatant was collected and dried under reduced pressure to obtain 110 mg. This was dissolved in 80% methanol and partitioned three times with hexane. The obtained 80% methanol layer was dried under reduced pressure, dissolved in water, and partitioned three times with ethyl acetate. The ethyl acetate layer was dried under reduced pressure to obtain 29 mg of extract. This was dissolved in ethanol and DPP-IV inhibitory activity was evaluated.

  Evaluation of DPP-IV inhibitory activity was performed as follows. 50 μl of 40 mM Tris-HCl buffer (pH 7.4), 50 μl of DPP-IV crude enzyme solution, and 5 μl of ethanol or rosebud extract were mixed. The enzyme reaction was started by adding 25 μl of Glycyl-L-proline 4-methylcoumeric-7-amide (Gly-Pro-MCA) dissolved in 10% DMSO. After reaction at room temperature for 10 minutes, 130 μl of acetonitrile was added to stop the reaction.

  Here, the crude DPP-IV enzyme solution was prepared by adding 10 mM Tris-HCl buffer (0.15 M NaCl, 0.5% Nonidet-P40, pH 8.0) to the human colon cancer-derived cell line Caco-2 cells and centrifuging the supernatant. And the protein concentration adjusted to 0.4 mg / ml was used. In addition, the activity of DPP-IV in the reaction solution was evaluated by measuring the amount of 7-Amino-4-methylcoumarin (AMC) produced by cleavage of the substrate.

  The amount of AMC produced in the reaction solution was analyzed using high performance liquid chromatography under the following conditions.

  Column: Discovery C18 (manufactured by Spelco), flow rate: 1.25 ml / min, oven temperature: 40 degrees, injection volume: 10 μl, detection: fluorescence detector (excitation wavelength 380 nm, emission wavelength 460 nm), solvent: A) 0.1% Formic acid-water B) 0.1% formic acid-acetonitrile, elution conditions: B) 22-35% (0-7 minutes).

  The amount of AMC produced when ethanol was added was defined as 100, and the value obtained by subtracting the amount of AMC produced during addition of rose buds from this was expressed as a relative inhibition rate (%). As a result of setting and measuring several levels of the addition concentration of the rosebud extract, an increase in the inhibition rate depending on the addition concentration was observed (see FIG. 1).

[Example 2]
Extraction was performed in the same manner as in Example 1 from 300 mg of dried safflower (Carthamus tinctorius) powder to obtain 7.5 mg of extract. As a result of measuring the inhibition rate in the same manner as in Example 1, an inhibition rate dependent on the addition concentration was observed (see FIG. 1).

[Example 3]
Extraction was performed in the same manner as in Example 1 from 300 mg of dried prune (Prunus domestic) powder to obtain 5.4 mg of extract. As a result of measuring the inhibition rate in the same manner as in Example 1, an inhibition rate dependent on the addition concentration was observed (see FIG. 1).

[Example 4]
Extraction was performed in the same manner as in Example 1 from 300 mg of dried apricot (Prunus armeniaca) powder to obtain 13.9 mg of extract. As a result of measuring the inhibition rate in the same manner as in Example 1, an inhibition rate dependent on the addition concentration was observed (see FIG. 2).

[Example 5]
Extraction was performed in the same manner as in Example 1 from 300 mg of ladies mantle (Alchemilla mollis) dry powder to obtain 9.3 mg of extract. As a result of measuring the inhibition rate in the same manner as in Example 1, an inhibition rate dependent on the addition concentration was observed (see FIG. 2).

[Example 6]
Extraction was performed in the same manner as in Example 1 from 300 mg of dried marjoram (Origanum majorana) powder to obtain 14.5 mg of extract. As a result of measuring the inhibition rate in the same manner as in Example 1, an inhibition rate dependent on the addition concentration was observed (see FIG. 2).

[Example 7]
Extraction was performed in the same manner as in Example 1 from 300 mg of dry powder of Apocynum venetum to obtain 11.1 mg of extract. As a result of measuring the inhibition rate in the same manner as in Example 1, an inhibition rate dependent on the addition concentration was observed (see FIG. 3).

[Example 8]
Extraction was performed in the same manner as in Example 1 from 300 mg of dried red clover (Trifolium pratense) powder to obtain 9.0 mg of extract. As a result of measuring the inhibition rate in the same manner as in Example 1, an inhibition rate dependent on the addition concentration was observed (see FIG. 3).

[Example 9]
Extraction was performed in the same manner as in Example 1 from 300 mg of dried edible chrysanthemum (Chrysanthemum morifolium) powder to obtain 15.0 mg of extract. As a result of measuring the inhibition rate in the same manner as in Example 1, an inhibition rate dependent on the addition concentration was observed (see FIG. 3).

[Example 10]
Extraction was performed in the same manner as in Example 1 from 300 mg of dried cuminum cyminum powder to obtain 7.3 mg of extract. As a result of measuring the inhibition rate in the same manner as in Example 1, an inhibition rate dependent on the addition concentration was observed (see FIG. 4).

[Example 11]
Extraction was performed in the same manner as in Example 1 from 300 mg of dry powder of Equisetum arvense to obtain 8.9 mg of extract. As a result of measuring the inhibition rate in the same manner as in Example 1, an inhibition rate dependent on the addition concentration was observed (see FIG. 4).

[Example 12]
Extraction was performed in the same manner as in Example 1 from 300 mg of dry rooibos tea (Aspalathus linearis) powder to obtain 9.0 mg of extract. As a result of measuring the inhibition rate in the same manner as in Example 1, an inhibition rate dependent on the addition concentration was observed (see FIG. 4).

[Example 13]
Extraction was performed in the same manner as in Example 1 from 1 g of dried white jellyfish (Tremella fuciformis) powder to obtain 0.6 mg of extract. As a result of measuring the inhibition rate in the same manner as in Example 1, an inhibition rate dependent on the addition concentration was observed (see FIG. 5).

[Example 14]
Ten times the amount of water was added to 5 g of dry powder of Syzygium aramaticum, and extraction was performed by heating for 10 minutes while stirring. The centrifugal supernatant was collected and dried under reduced pressure to obtain 632 mg. This was dissolved in 10% methanol, and the inhibition rate was measured in the same manner as in Example 1. As a result, an inhibition rate dependent on the addition concentration was observed (see FIG. 5).

[Example 15]
Extraction was performed in the same manner as in Example 14 from 5 g of dried dry powder of Phyllanthus niruri to obtain 990 mg. This was dissolved in 10% methanol, and the inhibition rate was measured in the same manner as in Example 1. As a result, an inhibition rate dependent on the addition concentration was observed (see FIG. 5).

[Example 16]
Ten times the amount of 70% acetone was added to 5 g of dried dry powder of Gentianella alborosea and stirred at room temperature for 1 hour. The centrifugal supernatant was collected and dried under reduced pressure to obtain 1272 mg. This was dissolved again in 70% acetone, and the inhibition rate was measured in the same manner as in Example 1. As a result, an inhibition rate dependent on the addition concentration was observed (see FIG. 6).

FIG. 1 shows the results of measuring the inhibition rate in Examples 1, 2, and 3 by changing the concentration of the extract added. The abscissa represents the concentration of the extract in the reaction solution, and the ordinate represents the relative amount of AMC produced by the addition of the extract as an inhibition rate (%). FIG. 2 shows the results of measuring the inhibition rate in Examples 4, 5, and 6 by changing the concentration of the extract added. The abscissa represents the concentration of the extract in the reaction solution, and the ordinate represents the relative amount of AMC produced by the addition of the extract as an inhibition rate (%). FIG. 3 shows the results of measuring the inhibition rate in Examples 7, 8, and 9 by changing the concentration of the extract added. The abscissa represents the concentration of the extract in the reaction solution, and the ordinate represents the relative amount of AMC produced by the addition of the extract as an inhibition rate (%). FIG. 4 shows the results of measuring the inhibition rate in Examples 10, 11, and 12 by changing the concentration of the extract added. The abscissa represents the concentration of the extract in the reaction solution, and the ordinate represents the relative amount of AMC produced by the addition of the extract as an inhibition rate (%). FIG. 5 shows the results of measuring the inhibition rate in Examples 13, 14, and 15 by changing the concentration of the extract added. The abscissa represents the concentration of the extract in the reaction solution, and the ordinate represents the relative amount of AMC produced by the addition of the extract as an inhibition rate (%). FIG. 6 shows the results of measuring the inhibition rate in Example 16 by changing the concentration of the extract added. The abscissa represents the concentration of the extract in the reaction solution, and the ordinate represents the relative amount of AMC produced by the addition of the extract as an inhibition rate (%).

Claims (10)

  Rosaceae, Rosaceae, Sakura, Rosaceae, Alchemilla, Asteraceae, Asteraceae, Pteridocyta genus, Nymphalidae, Vesculumum, Leguminosae, Genus Asparatus, Sericidae, Cumin A substance having a peptidyl peptidase IV inhibitory activity derived from one or more plants belonging to the genus selected from the group consisting of genus Toxaaceae, Pteridomyceae, Pleurotus genus, Pleurosumidae, Pleurotus phyllanthus, and Gentianaceae A composition comprising   The plants include Rosa multiflora, Rosa gallica, Carthamus tinctorius, Prunus domestica, Prunus armeniaca, Alchemia Morris ( Alchemilla mollis), Origanum majorana, Apocynum venetum, Trifolium pratense, Chrysanthemum morifolium, Crynum umumumumum arvense), Aspalathus linearis, Tremella fuciformis, Syzygium aramaticum, Phyllanthus niruri, or Gentianella alborosea The composition of claim 1, wherein the composition is one or more of (Gentianella alborosea).   The composition of Claim 2 containing the substance which has peptidyl peptidase IV inhibitory activity derived from the bud of Rosa multiflora (Rosa multiflora), Rosa gallica (Rosa gallica), or Syzygium aramaticum (Syzygium aramaticum).   The composition according to any one of claims 1 to 3, wherein the substance having peptidyl peptidase IV inhibitory activity is an extract from the plant or a part thereof.   The composition according to claim 4, wherein the extract is an organic solvent extract.   It is food-drinks, The composition of any one of Claims 1-5.   It is a pharmaceutical, The composition of any one of Claims 1-5.   Rosaceae, Rosaceae, Sakura, Rosaceae, Alchemilla, Asteraceae, Asteraceae, Pteridocyta genus, Nymphalidae, Vesculumum, Leguminosae, Genus Asparatus, Sericidae, Cumin , One or more plants belonging to the genus selected from the group consisting of the genus Toxaaceae genus, white jellyfish genus white jellyfish, genus Futomida, genus Philanthus genus Philanthus and gentian genus, or a part thereof, is subjected to solvent extraction, A method for producing a dipeptidyl peptidase IV inhibitor-containing composition, comprising recovering a substance having dipeptidyl peptidase IV inhibitory activity from an extract fraction.   The plants are Rosa multiflora, Rosa gallica, Carthamus tinctorius, Prunus domestica, Prunus armeniaca, Alchemia Morris ( Alchemilla mollis), Origanum majorana, Apocynum venetum, Trifolium pratense, Chrysanthemum morifolium, Crynum umumumumum arvense), Aspalathus linearis, Tremella fuciformis, Syzygium aramaticum, Phyllanthus niruri, or Gentianella alborosea 9. The method of claim 8, wherein the method is one or more of (Gentianella alborosea).   The method according to claim 8 or 9, wherein the solvent is at least one selected from the group consisting of ethanol, methanol, ethyl acetate and acetone.

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