WO2018179998A1 - Novel procyanidin and method for producing same - Google Patents
Novel procyanidin and method for producing same Download PDFInfo
- Publication number
- WO2018179998A1 WO2018179998A1 PCT/JP2018/005863 JP2018005863W WO2018179998A1 WO 2018179998 A1 WO2018179998 A1 WO 2018179998A1 JP 2018005863 W JP2018005863 W JP 2018005863W WO 2018179998 A1 WO2018179998 A1 WO 2018179998A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- procyanidins
- procyanidin
- novel
- water
- red pepper
- Prior art date
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
- C07D311/60—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2
- C07D311/62—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2 with oxygen atoms directly attached in position 3, e.g. anthocyanidins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
Definitions
- the present invention relates to a novel procyanidin and a method for producing the same.
- Controlling the transient hyperglycemia that occurs after meals is an important challenge in the prevention and treatment of diabetes.
- Carbohydrates ingested by meals are first decomposed into disaccharides by ⁇ -amylase contained in saliva and pancreatic juice. Subsequently, it is decomposed into monosaccharides by ⁇ -glucosidase localized on the membrane of small intestinal mucosal epithelial cells, taken into the cell via a transporter localized on the membrane, and then transported into the blood Is done.
- the intake of polysaccharides can be substantially suppressed by inhibiting the activity of ⁇ -amylase or ⁇ -glucosidase.
- Non-patent Document 1 Non-patent Document 1
- Plant-derived polyphenols include monomeric polyphenols and polymer polyphenols (generally called tannins), and polymer polyphenols are further divided into condensed and hydrolyzed types.
- Condensed tannin has a basic skeleton in which a flavonoid, which is a kind of monomeric polyphenol, is polymerized via a carbon-carbon bond.
- flavonoid which is a kind of monomeric polyphenol
- procyanidins those having flavan-3-ol as a structural unit of the basic skeleton are called procyanidins.
- Procyanidins are known to have an antioxidant action, an anti-inflammatory action, an anti-fat accumulation action, a vasorelaxant action, and the like, and their use as therapeutic agents for hypertension, heart disease and the like has been proposed (Patent Document 1). . Furthermore, in recent years, it has been reported to have an action of inhibiting polysaccharide digestion, and has attracted attention as a means for controlling postprandial hyperglycemia.
- Non-Patent Document 2 procyanidins extracted from grapes
- Non-Patent Document 3 tsurudokudami
- grapes Non-Patent Document 4
- cinnamon Non-Patent Document 5
- activities of ⁇ -amylase and ⁇ -glucosidase It has been reported to have an effect of inhibiting the above.
- the blood glucose level falls in the diabetes model rat which ingested grape origin procyanidin (nonpatent literature 6).
- procyanidins have a structure in which gallic acid, saccharides and the like are secondary ester-linked to a basic skeleton obtained by polymerizing two or three flavan-3-ols selected from catechin, epicatechin, and epigallocatechin.
- strength of the said polysaccharide digestion inhibitory effect originates in the difference in the structure.
- the present invention has been made in view of the above technical background, and an object thereof is to provide a novel procyanidin having an excellent polysaccharide digestion inhibitory action and a method for producing the same.
- procyanidins extracted from red pepper have been reported as novel procyanidins having a basic skeleton in which four types of flavan-3-ols have not been reported so far. It has been found that it has a very high ⁇ -amylase inhibitory activity, and the present invention has been completed.
- the present invention includes the following.
- the method includes the steps of adsorbing an extract of red pepper with water and / or a water-containing organic solvent with a synthetic adsorbent and eluting the synthetic adsorbent with a water-containing organic solvent.
- [1]-[ 4] The method for producing procyanidins according to any one of the above.
- a novel procyanidin excellent in polysaccharide digestion inhibiting action and a method for producing the same are provided.
- % means “% by volume” unless otherwise specified.
- the novel procyanidins according to the present invention are condensed tannins having a basic skeleton obtained by polymerizing four types of flavan-3-ols, epicatechin, catechin, epigallocatechin, and gallocatechin.
- procyanidins have been identified from various plants, many of which have a basic skeleton obtained by polymerizing 1-3 types of flavan-3-ols. No unit has been reported yet.
- Procyanidins are mainly of type A (having at least one bond of 4 ⁇ ⁇ 8 and 2 ⁇ ⁇ O ⁇ 7 or 4 ⁇ ⁇ 6 and 2 ⁇ ⁇ O ⁇ 7 between units) due to the difference in the bonding mode of the structural units of the basic skeleton.
- B carbon-bonded bonds between units are 4 ⁇ ⁇ 8 or 4 ⁇ ⁇ 6 only.
- the novel procyanidins according to the present invention are preferably B-type procyanidins.
- the novel procyanidins according to the present invention may have a molecular weight of 1,000-114,000, preferably 3,000-114,000, more preferably 8,000-15,000.
- the degree of polymerization of the structural unit may be 3 to 400, preferably 10 to 400, more preferably 25 to 50.
- one or more OH groups in one or more flavan-3-ols constituting the basic skeleton may be derivatized, for example, esterified.
- the flavan-3-ol may contain one or more ester groups, preferably a gallic acid ester group, at one or more of the 3-position, 5-position, 7-position, 3′-position and 4′-position of the ring. .
- one flavan-3-ol may be bound to 1, 2, 3, 4, or 5 gallate esters.
- the novel procyanidins according to the present invention may have an average of one galloyl group bonded per flavan-3-ol.
- the gallic acid ester include catechin gallate, epicatechin gallate, gallocatechin gallate, epigallocatechin gallate and the like.
- novel procyanidins according to the present invention can be extracted from red pepper.
- Akashoma (scientific name: Astilbe thunbergii var. Thunbergii) is a plant belonging to the genus Pleurotusaceae and is a perennial that grows naturally in mountainous areas in Honshu, Shikoku and Kyushu.
- the root of red ginger has long been used as a substitute for the rhizome of ginger (Ranunculaceae), which is used for antipyretic, detoxifying and anti-inflammatory effects.
- the rhizome of shouma is a pharmaceutical, but the root of red shouma is equivalent to a non-medicine (see ttps: //hfnet.nih.go.jp/usr/annzenn/image/iyakuhin2).
- the red pepper (Astilbe thunbergii var. Thunbergii) used in the present invention may be either native or cultivated red pepper, and the preferred site is the root and / or rhizome. Either raw or dried can be used, and from the viewpoint of improving extraction efficiency, it is preferably shredded or powdered.
- the extract of red pepper with water and / or water-containing organic solvent is adsorbed with a specific synthetic adsorbent, and the adsorbent is eluted with a specific extraction solvent to obtain a novel procyanidin at a high concentration. It is possible to obtain a fraction containing
- red pepper extract As the solvent for extracting red pepper, water, a water-containing organic solvent, or a combination thereof (specifically, extraction with a water-containing organic solvent and then extraction with water) is preferable.
- the water content in the water-containing organic solvent is 10-90%, preferably 20-80%, more preferably 30-70%, most preferably 40-60%.
- the organic solvent is not particularly limited, but examples thereof include lower alcohols such as methanol, ethanol, propanol, and butanol; esters such as ethyl acetate; glycols such as ethylene glycol, butylene glycol, propylene glycol, 1,3-butylene alcohol, and glycerin. Ethers such as diethyl ether and petroleum ether; polar solvents such as acetone and acetic acid; hydrocarbons such as benzene, hexane and xylene. Of these, lower alcohols and esters are preferable, and lower alcohols are more preferable. In addition, you may use these organic solvents individually or in combination of 2 or more types.
- lower alcohols such as methanol, ethanol, propanol, and butanol
- esters such as ethyl acetate
- glycols such as ethylene glycol, butylene glycol, propylene glycol, 1,3-butylene alcohol, and glycer
- the extraction temperature is a temperature within the range from room temperature to the boiling point of the solvent, and can be appropriately adjusted according to the type of solvent. You may carry out under pressurization, a normal pressure, and pressure reduction.
- the extraction time may also be adjusted as appropriate according to the type of solvent. For example, when water is used as the extraction solvent, it is 20 to 140 ° C., preferably 60 to 130 ° C., more preferably 80 to 125 ° C., within 1 minute to 1 hour, preferably 10 to 30 minutes. You may go. Further, when using water-containing ethanol as the extraction solvent, it may be carried out within a range of 20-100 ° C., preferably 40-80 ° C., 1 minute-1 hour, preferably 10-30 minutes. .
- the solvent is stirred or refluxed during the heat retention period.
- the amount of the solvent used for the extraction is not particularly limited. For example, it is 2 to 50 times, preferably 5 to 50 times, more preferably 10 to 30 times the dry matter of red pepper and / or rhizomes ( (Weight ratio) solvent may be used.
- the red pepper extract After extraction, the red pepper extract can be obtained by removing solids by filtration, centrifugation, or the like. Moreover, a commercial item can also be used as a red pepper extract. For example, “Akashoma extract powder (manufactured by BN Co., Ltd.), which is an aqueous ethanol extract of dry roots, may be used.
- the adsorbent for adsorbing the red pepper extract is preferably an aromatic synthetic adsorbent, more preferably a styrene-divinylbenzene synthetic adsorbent.
- Diaion HP20 manufactured by Mitsubishi Chemical Corporation
- Amberlite XAD-2, 4 manufactured by Dow Chemical Company
- the synthetic adsorbent obtained by adsorbing the red pepper extract can be washed with water and then eluted with a water-containing organic solvent.
- a water-containing methanol solution, a water-containing ethanol solution, or the like can be used as the organic solvent. Of these, a 50% aqueous methanol solution is particularly preferred.
- the obtained eluate may be further purified using a conventional purification method. Next, a method for purification using size exclusion chromatography is exemplified.
- a hydrophilic vinyl polymer system for example, Toyopearl HW-40F, manufactured by Tosoh Biosciences
- a dextran system for example Sepadex LH-20, manufactured by GE Healthcare Co., Ltd.
- a tip elution fraction more specifically, 0.2 to 0.4 column volume. It can be recovered (as an elution fraction with solvent).
- a dextran carrier When a dextran carrier is used, it may be washed with a 50% methanol aqueous solution and a 100% methanol sequentially and then eluted with a 70% acetone aqueous solution.
- the enriched fraction of novel procyanidins according to the present invention can be selected, for example, by conducting an ⁇ -amylase activity inhibition test (see Examples).
- the concentrated fraction of the novel procyanidin may be further subjected to reverse phase HPLC for purification.
- the procyanidins according to the present invention are excellent in the polysaccharide digestion inhibitory action, and more specifically, in the action of inhibiting ⁇ -amylase activity.
- the ⁇ -amylase inhibitory activity has an IC 50 value of about 1.7 to 2.6 ⁇ g / mL, and when converted to a weight concentration ratio, Acarbose, a typical postprandial hyperglycemia improving agent prescribed for diabetics Glucobay Co., Ltd., CAS Registry Number: 56180-94-0, IC 50 value is 10.2 ⁇ 83.33 ⁇ g / mL, Reference: Sudha P., et al, BMC Complement Altern Med.
- the procyanidins according to the present invention can be used as a polysaccharide digestion inhibitor for the purpose of improving postprandial hyperglycemia.
- polysaccharide refers to “two or more monosaccharides polymerized by glycosidic bonds (preferably ⁇ -1,4-glycosidic bonds)”.
- ⁇ -amylase inhibitory activity in the present invention means an activity that inhibits the ⁇ -1,4-glucoside bond cleavage activity of ⁇ -amylase (EC 3.2.1.1), and ⁇ -glucosidase inhibitory activity.
- ⁇ -glucosidase (EC 3.2.1.20) means the activity of inhibiting the ⁇ -1,4-glucoside bond cleavage activity of ⁇ -glucosidase (EC 3.2.1.20).
- procyanidins according to the present invention When procyanidins according to the present invention are used as a polysaccharide digesting agent, it is preferably taken orally during the meal from before taking the polysaccharide (before the meal), but if the time is shortly after the meal, the oral intake is sufficient. It is thought that there can be an effect. Therefore, 30 minutes before meal to 30 minutes after meal, preferably 15 minutes before meal to 15 minutes after meal, more preferably 10 minutes before meal to 10 minutes after meal, most preferably 5 minutes before meal.
- the amount of intake can be adjusted according to the content of the meal (the amount of polysaccharides).
- the content of the meal the amount of polysaccharides.
- the polysaccharide digestion inhibitor may be taken alone or in the form of an oral composition mixed with a pharmaceutically acceptable carrier, excipient, plasticizer, colorant, preservative, etc. May be.
- a pharmaceutically acceptable carrier examples include sugar alcohol (eg, mannitol), inorganic substance (eg, calcium carbonate), microcrystalline cellulose, cellulose (eg, carboxymethylcellulose), gelatin, sodium alginate, Examples include polyvinyl pyrrolidone, agar, magnesium stearate, talc and the like.
- the form of the oral composition is not particularly limited, and may be in the form of a tablet, pill, capsule, granule, powder, powder, troche, solution (beverage) or the like.
- the polysaccharide digestion inhibitor can be suitably ingested in a state where it is blended with general foods, health foods, insurance functional foods (food for specified health use, functional indication foods, etc.).
- the food include milk beverages, lactic acid bacteria beverages, soft drinks, carbonated beverages, fruit juice beverages, vegetable beverages, alcoholic beverages, powdered beverages, coffee beverages, tea beverages, green tea beverages and barley tea beverages; pudding, jelly , Bavaria, yogurt, ice cream, gum, chocolate, candy, caramel, biscuits, cookies, rice crackers, rice crackers and other confectionery; consommé soup, potage soup, etc .; miso, soy sauce, dressing, ketchup, sauce, sauce, Various seasonings such as sprinkles; jams such as strawberry jam, blueberry jam, marmalade, apple jam; fruit wine such as red wine; fruit for processing such as syrup pickled cherry, apricot, apple, strawberry, peach; udon, cold wheat, Somen, buckwheat, Chinese noodles, spaghetti, macaroni, bi
- Example 1 Method 1 for purifying novel procyanidins
- a red pepper extract powder (manufactured by BN Co., Ltd.) was suspended in water, and the filtrate obtained by suction filtration was adsorbed onto a styrene-divinylbenzene synthetic adsorbent (Diaion HP20, manufactured by Mitsubishi Chemical Corporation).
- the synthetic adsorbent was washed with water and eluted with 50% aqueous methanol.
- the obtained eluate was concentrated, suspended in water, centrifuged, and the supernatant was collected and fractionated by size exclusion chromatography (Toyopearl HW-40F, manufactured by Tosoh Bioscience) (elution) Use water as liquid).
- the fraction eluted at the leading edge (0.2-0.3 column volume) was collected as a procyanidin-enriched fraction.
- the result of analyzing the procyanidin-enriched fraction by 1 H-NMR is shown in FIG.
- the result of having used for reverse phase HPLC on the following conditions is shown in FIG.
- procyanidins according to the present invention are eluted with a retention time of 18.85 minutes as a peak.
- the procyanidin was measured for ⁇ -amylase inhibitory activity according to the following method. The results are shown in Table 1 together with the results of Example 2.
- ⁇ Amylase inhibitory activity test > ⁇ Reaction solution (500 ⁇ l) (1) Test substance (dried) in water, 100 ⁇ l (2) Starch azure 1.4mg in buffer, 350 ⁇ l (3) 0.5 U / mL porcine pancreatic ⁇ -amylase in buffer, 50 ⁇ l * Buffer: 0.1M Tris-HCl buffer solution (pH 6.9) containing 0.01M CaCl 2 ⁇ Method After placing (2) in a 2.0 ml microtube and incubating at 37 ° C for 5 minutes, add (1) and (3) in this order, and incubate at 37 ° C for an additional 15 minutes with shaking. .
- OD sample is a well to which a test substance is added
- OD sample blank is a well to which a buffer is added instead of an enzyme in the presence of the test substance
- OD control is a negative control well
- OD control blank is In the negative control, the absorbance of each well to which a buffer was added instead of the enzyme is shown.
- Example 2 Method 2 for purifying novel procyanidins
- a red pepper extract powder (manufactured by BN Co., Ltd.) was suspended in water, and the filtrate obtained by suction filtration was adsorbed onto a styrene-divinylbenzene synthetic adsorbent (Diaion HP20, manufactured by Mitsubishi Chemical Corporation).
- the synthetic adsorbent was washed with water and eluted with 50% aqueous methanol.
- the obtained eluate was directly adsorbed on Sephadex LH-20 (manufactured by GE Healthcare), washed sequentially with 50% methanol aqueous solution and 100% methanol, and eluted with 70% acetone aqueous solution to concentrate procyanidin-enriched fraction.
- Sephadex LH-20 manufactured by GE Healthcare
- Table 1 shows the yield of procyanidins obtained by the extraction methods of Examples 1 and 2 and the ⁇ -amylase inhibitory activity.
- Example 3 Analysis of constituent units of novel procyanidins
- thiol decomposition was performed and the decomposition products were analyzed using NMR.
- the procyanidin (concentrated fraction) 2.5 mg obtained in Example 2 was dissolved in 200 ⁇ L of 0.1 M hydrochloric acid / methanol solution, and 5 ⁇ L of benzyl mercaptan was added and mixed.
- the mixed solution was reacted by heating at 40 ° C. for 90 minutes.
- the reaction solution was concentrated to dryness, redissolved in methanol, and then subjected to reverse phase HPLC analysis under the following conditions.
- gallocatechin and epigallocatechin have a low absorbance at 280 nm
- detection was performed at 270 nm.
- calculating the molar ratio from the absorbance was calculated by multiplying the measured value (peak area) of gallocatechin and epigallocatechin by 1.57. The results are shown in FIG.
- procyanidins purified from red pepper by the above method are novel procyanidins having four flavan-3-ols of epicatechin, catechin, epigallocatechin, and gallocatechin as basic structural units. It was. Moreover, it became clear that the ratio (molar ratio) of each structural unit was 1.0: 0.56: 0: 76: 0.13 for epicatechin: catechin: epigallocatechin: gallocatechin. Furthermore, it has also been found that the terminal has mainly catechin, and the ratio of terminal catechin to the ratio of catechin (0.56) is 0.17 (the ratio of catechin in the extended portion is 0.39). As will be described later, since the procyanidin is a mixture of various degrees of polymerization, it is considered that there is a range in the ratio of each structural unit.
- Example 4 Analysis of molecular weight of novel procyanidins
- the procyanidins obtained in Examples 1 and 2 were subjected to gel filtration HPLC under the following conditions, and molecular weight distribution was analyzed using polystyrene as a standard sample.
- a calibration curve using polystyrene is shown in FIG. 4, and HPLC charts of procyanidins obtained in Examples 1 and 2 are shown in FIGS. 5 and 6, respectively.
- Table 3 shows the molecular weight distribution of each procyanidin calculated from the calibration curve.
- the molecular weight of procyanidin purified from red pepper by the method of Example 1 has a median of about 15,000, a maximum of about 114,000, and a minimum of about 2,000.
- the molecular weight of procyanidin purified by the method of Example 2 It can be seen that the median is about 8,300, the maximum is about 41,300, and the minimum is about 1,000.
- Example 1 Average degree of polymerization 50, maximum 378, minimum 6
- Example 2 Average degree of polymerization 27, maximum 138, minimum 3 It becomes.
- procyanidins contained in red pepper are a group of hetero molecules having a basic skeleton in which about 3 to 400 flavan-3-ols are polymerized and having a molecular weight in the range of about 1,000 to 114,000. became.
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The purpose of the present invention is to provide: a novel procyanidin having an excellent polysaccharide digestion inhibition effect; and a method for producing the same. Provided is a procyanidin characterized by having a basic skeleton in which epicatechin, catechin, epigallocatechin, and gallocatechin are polymerized.
Description
本出願は、２０１７年０３月２８日付け出願の日本国特許出願２０１７－０６３１６６号の優先権を主張しており、ここに折り込まれるものである。
This application claims the priority of Japanese Patent Application No. 2017-063166 filed on Mar. 28, 2017, and is incorporated herein.
本発明は、新規プロシアニジンとその製造方法に関する。
The present invention relates to a novel procyanidin and a method for producing the same.
食後に生じる一過的な高血糖状態（すなわち、食後過血糖）をコントロールすることは、糖尿病の予防及び治療における重要な課題である。
食事により摂取された炭水化物は、まず、唾液及び膵液に含まれるα-アミラーゼによって二糖類にまで分解される。その後、小腸粘膜上皮細胞の膜上に局在するα-グルコシダーゼによって単糖へと分解され、該膜上に局在するトランスポーターを介して該細胞内に取り込まれた後、血中へと輸送される。単糖にまで分解されないと小腸粘膜上皮細胞には取り込まれないため、α-アミラーゼ又はα-グルコシダーゼの活性を阻害することにより、多糖類の摂取を実質的に抑制することが可能となる。 Controlling the transient hyperglycemia that occurs after meals (ie, postprandial hyperglycemia) is an important challenge in the prevention and treatment of diabetes.
Carbohydrates ingested by meals are first decomposed into disaccharides by α-amylase contained in saliva and pancreatic juice. Subsequently, it is decomposed into monosaccharides by α-glucosidase localized on the membrane of small intestinal mucosal epithelial cells, taken into the cell via a transporter localized on the membrane, and then transported into the blood Is done. Since it is not taken up by small intestinal mucosal epithelial cells unless it is decomposed to monosaccharides, the intake of polysaccharides can be substantially suppressed by inhibiting the activity of α-amylase or α-glucosidase.
食事により摂取された炭水化物は、まず、唾液及び膵液に含まれるα-アミラーゼによって二糖類にまで分解される。その後、小腸粘膜上皮細胞の膜上に局在するα-グルコシダーゼによって単糖へと分解され、該膜上に局在するトランスポーターを介して該細胞内に取り込まれた後、血中へと輸送される。単糖にまで分解されないと小腸粘膜上皮細胞には取り込まれないため、α-アミラーゼ又はα-グルコシダーゼの活性を阻害することにより、多糖類の摂取を実質的に抑制することが可能となる。 Controlling the transient hyperglycemia that occurs after meals (ie, postprandial hyperglycemia) is an important challenge in the prevention and treatment of diabetes.
Carbohydrates ingested by meals are first decomposed into disaccharides by α-amylase contained in saliva and pancreatic juice. Subsequently, it is decomposed into monosaccharides by α-glucosidase localized on the membrane of small intestinal mucosal epithelial cells, taken into the cell via a transporter localized on the membrane, and then transported into the blood Is done. Since it is not taken up by small intestinal mucosal epithelial cells unless it is decomposed to monosaccharides, the intake of polysaccharides can be substantially suppressed by inhibiting the activity of α-amylase or α-glucosidase.
これまで、α-アミラーゼ及び／又はα-グルコシダーゼの活性阻害を作用機序とする薬剤が複数開発され、その一部は既に食後過血糖改善剤として糖尿病患者に処方されている。しかしながら、それらの薬剤には種々の副作用が知られるため、より副作用が少ないことを期待して、植物由来ポリフェノールへの期待が高まっている（非特許文献１）。
So far, several drugs have been developed that have an action mechanism that inhibits the activity of α-amylase and / or α-glucosidase, and some of them are already prescribed to diabetic patients as postprandial hyperglycemia improving agents. However, since various side effects are known for these drugs, anticipation for plant-derived polyphenols is increasing in anticipation of fewer side effects (Non-patent Document 1).
植物由来ポリフェノールには単量体ポリフェノールと重合体ポリフェノール（一般に、タンニンと呼ばれる）があり、重合体ポリフェノールはさらに縮合型と加水分解型に分けられる。縮合型タンニンは、単量体ポリフェノールの一種であるフラボノイドが炭素－炭素結合を介して重合した基本骨格を有するものである。そして、縮合型タンニンのうち、フラバン－３－オールを基本骨格の構成単位とするものは、プロシアニジンと呼ばれている。
Plant-derived polyphenols include monomeric polyphenols and polymer polyphenols (generally called tannins), and polymer polyphenols are further divided into condensed and hydrolyzed types. Condensed tannin has a basic skeleton in which a flavonoid, which is a kind of monomeric polyphenol, is polymerized via a carbon-carbon bond. Among condensed tannins, those having flavan-3-ol as a structural unit of the basic skeleton are called procyanidins.
プロシアニジンは、抗酸化作用、抗炎症作用、抗脂肪蓄積作用、血管弛緩作用等を有することが知られており、高血圧や心疾患等の治療薬としての使用が提案されている（特許文献１）。さらに、近年、多糖消化阻害作用を有することが報告され、食後過血糖をコントロールし得る手段として注目されている。
例えば、ブドウ（非特許文献２）、ツルドクダミ（非特許文献３）、柿（非特許文献４）、シナモン（非特許文献５）から抽出されたプロシアニジンには、α-アミラーゼ及びα-グルコシダーゼの活性を阻害する作用があることが報告されている。また、ブドウ由来プロシアニジンを摂取した糖尿病モデルラットでは、血糖値が低下することが報告されている（非特許文献６）。 Procyanidins are known to have an antioxidant action, an anti-inflammatory action, an anti-fat accumulation action, a vasorelaxant action, and the like, and their use as therapeutic agents for hypertension, heart disease and the like has been proposed (Patent Document 1). . Furthermore, in recent years, it has been reported to have an action of inhibiting polysaccharide digestion, and has attracted attention as a means for controlling postprandial hyperglycemia.
For example, procyanidins extracted from grapes (Non-Patent Document 2), tsurudokudami (Non-Patent Document 3), grapes (Non-Patent Document 4), and cinnamon (Non-Patent Document 5) have activities of α-amylase and α-glucosidase. It has been reported to have an effect of inhibiting the above. Moreover, it is reported that the blood glucose level falls in the diabetes model rat which ingested grape origin procyanidin (nonpatent literature 6).
例えば、ブドウ（非特許文献２）、ツルドクダミ（非特許文献３）、柿（非特許文献４）、シナモン（非特許文献５）から抽出されたプロシアニジンには、α-アミラーゼ及びα-グルコシダーゼの活性を阻害する作用があることが報告されている。また、ブドウ由来プロシアニジンを摂取した糖尿病モデルラットでは、血糖値が低下することが報告されている（非特許文献６）。 Procyanidins are known to have an antioxidant action, an anti-inflammatory action, an anti-fat accumulation action, a vasorelaxant action, and the like, and their use as therapeutic agents for hypertension, heart disease and the like has been proposed (Patent Document 1). . Furthermore, in recent years, it has been reported to have an action of inhibiting polysaccharide digestion, and has attracted attention as a means for controlling postprandial hyperglycemia.
For example, procyanidins extracted from grapes (Non-Patent Document 2), tsurudokudami (Non-Patent Document 3), grapes (Non-Patent Document 4), and cinnamon (Non-Patent Document 5) have activities of α-amylase and α-glucosidase. It has been reported to have an effect of inhibiting the above. Moreover, it is reported that the blood glucose level falls in the diabetes model rat which ingested grape origin procyanidin (nonpatent literature 6).
これらのプロシアニジンは、カテキン、エピカテキン、エピガロカテキンから選ばれる２種又は３種のフラバン－３－オールが重合した基本骨格に、没食子酸や糖類等が副次的にエステル結合した構造を有するものである（ブドウ：非特許文献７、ツルドクダミ：非特許文献３、柿：非特許文献８、シナモン：非特許文献５）。そして、当該多糖消化阻害作用の強弱は、その構造の違いに起因すると考えられている。
These procyanidins have a structure in which gallic acid, saccharides and the like are secondary ester-linked to a basic skeleton obtained by polymerizing two or three flavan-3-ols selected from catechin, epicatechin, and epigallocatechin. (Grape: Non-patent document 7, Tsurudukudami: Non-patent document 3, grape: Non-patent document 8, Cinnamon: Non-patent document 5). And it is thought that the intensity | strength of the said polysaccharide digestion inhibitory effect originates in the difference in the structure.
本発明は上記技術的背景を鑑みてなされたものであり、優れた多糖消化阻害作用を有する新規プロシアニジンと、その製造方法の提供を目的とする。
The present invention has been made in view of the above technical background, and an object thereof is to provide a novel procyanidin having an excellent polysaccharide digestion inhibitory action and a method for producing the same.
前記目的を達成するために本発明者が鋭意検討を行った結果、アカショウマから抽出されたプロシアニジンが、これまで報告されていない４種類のフラバン－３－オールが重合した基本骨格を有する新規プロシアニジンであり、且つ、非常に高いα-アミラーゼ阻害活性を有することを見出し、本発明を完成させるに至った。
As a result of intensive studies by the inventor in order to achieve the above object, procyanidins extracted from red pepper have been reported as novel procyanidins having a basic skeleton in which four types of flavan-3-ols have not been reported so far. It has been found that it has a very high α-amylase inhibitory activity, and the present invention has been completed.
すなわち、本発明は以下を包含する。
［１］ エピカテキン、カテキン、エピガロカテキン、及びガロカテキンが重合した基本骨格を有することを特徴とするプロシアニジン。
［２］ 前記構成単位間の結合様式がＢ型であることを特徴とする、［１］に記載のプロシアニジン。
［３］ 前記基本骨格に含まれる各構成単位のモル比が、エピカテキン：カテキン：エピガロカテキン：ガロカテキン＝0.50～2.00:0.28～1.12:0.38～1.52:0.07～0.26であることを特徴とする、［１］又は［２］に記載のプロシアニジン。
［４］ 分子量が1,000－114,000であることを特徴とする、［１］－［３］のいずれかに記載のプロシアニジン。
［５］ アカショウマの水及び／又は含水有機溶媒による抽出物を合成吸着剤で吸着処理し、該合成吸着剤を含水有機溶媒で溶出処理する工程を含むことを特徴とする、［１］－［４］のいずれかに記載のプロシアニジンの製造方法。 That is, the present invention includes the following.
[1] A procyanidin having a basic skeleton in which epicatechin, catechin, epigallocatechin, and gallocatechin are polymerized.
[2] The procyanidin according to [1], wherein the bonding mode between the structural units is B-type.
[3] The molar ratio of each structural unit contained in the basic skeleton is epicatechin: catechin: epigallocatechin: gallocatechin = 0.50 to 2.00: 0.28 to 1.12: 0.38 to 1.52: 0.07 to 0.26 Or procyanidins according to [1] or [2].
[4] The procyanidin according to any one of [1] to [3], which has a molecular weight of 1,000 to 114,000.
[5] The method includes the steps of adsorbing an extract of red pepper with water and / or a water-containing organic solvent with a synthetic adsorbent and eluting the synthetic adsorbent with a water-containing organic solvent. [1]-[ 4] The method for producing procyanidins according to any one of the above.
［１］ エピカテキン、カテキン、エピガロカテキン、及びガロカテキンが重合した基本骨格を有することを特徴とするプロシアニジン。
［２］ 前記構成単位間の結合様式がＢ型であることを特徴とする、［１］に記載のプロシアニジン。
［３］ 前記基本骨格に含まれる各構成単位のモル比が、エピカテキン：カテキン：エピガロカテキン：ガロカテキン＝0.50～2.00:0.28～1.12:0.38～1.52:0.07～0.26であることを特徴とする、［１］又は［２］に記載のプロシアニジン。
［４］ 分子量が1,000－114,000であることを特徴とする、［１］－［３］のいずれかに記載のプロシアニジン。
［５］ アカショウマの水及び／又は含水有機溶媒による抽出物を合成吸着剤で吸着処理し、該合成吸着剤を含水有機溶媒で溶出処理する工程を含むことを特徴とする、［１］－［４］のいずれかに記載のプロシアニジンの製造方法。 That is, the present invention includes the following.
[1] A procyanidin having a basic skeleton in which epicatechin, catechin, epigallocatechin, and gallocatechin are polymerized.
[2] The procyanidin according to [1], wherein the bonding mode between the structural units is B-type.
[3] The molar ratio of each structural unit contained in the basic skeleton is epicatechin: catechin: epigallocatechin: gallocatechin = 0.50 to 2.00: 0.28 to 1.12: 0.38 to 1.52: 0.07 to 0.26 Or procyanidins according to [1] or [2].
[4] The procyanidin according to any one of [1] to [3], which has a molecular weight of 1,000 to 114,000.
[5] The method includes the steps of adsorbing an extract of red pepper with water and / or a water-containing organic solvent with a synthetic adsorbent and eluting the synthetic adsorbent with a water-containing organic solvent. [1]-[ 4] The method for producing procyanidins according to any one of the above.
本発明により、多糖消化阻害作用に優れる新規プロシアニジンとその製造方法が提供される。
According to the present invention, a novel procyanidin excellent in polysaccharide digestion inhibiting action and a method for producing the same are provided.
以下、本発明の好適な実施形態について説明する。
本書では、別途記載がない限り、％は容量％を表す。 Hereinafter, preferred embodiments of the present invention will be described.
In this document, “%” means “% by volume” unless otherwise specified.
本書では、別途記載がない限り、％は容量％を表す。 Hereinafter, preferred embodiments of the present invention will be described.
In this document, “%” means “% by volume” unless otherwise specified.
［新規プロシアニジン］
本発明に係る新規プロシアニジンは、エピカテキン、カテキン、エピガロカテキン、ガロカテキンの４種類のフラバン－３－オールが重合した基本骨格を有する縮合型タンニンである。これまで、さまざまな植物からプロシアニジンが同定されているが、その多くは１－３種類のフラバン－３－オールが重合した基本骨格を有するものであり、４種類のフラバン－３－オールを基本骨格の構成単位とするものはまだ報告されていない。 [New procyanidins]
The novel procyanidins according to the present invention are condensed tannins having a basic skeleton obtained by polymerizing four types of flavan-3-ols, epicatechin, catechin, epigallocatechin, and gallocatechin. Up to now, procyanidins have been identified from various plants, many of which have a basic skeleton obtained by polymerizing 1-3 types of flavan-3-ols. No unit has been reported yet.
本発明に係る新規プロシアニジンは、エピカテキン、カテキン、エピガロカテキン、ガロカテキンの４種類のフラバン－３－オールが重合した基本骨格を有する縮合型タンニンである。これまで、さまざまな植物からプロシアニジンが同定されているが、その多くは１－３種類のフラバン－３－オールが重合した基本骨格を有するものであり、４種類のフラバン－３－オールを基本骨格の構成単位とするものはまだ報告されていない。 [New procyanidins]
The novel procyanidins according to the present invention are condensed tannins having a basic skeleton obtained by polymerizing four types of flavan-3-ols, epicatechin, catechin, epigallocatechin, and gallocatechin. Up to now, procyanidins have been identified from various plants, many of which have a basic skeleton obtained by polymerizing 1-3 types of flavan-3-ols. No unit has been reported yet.
本発明においては、前記基本骨格を構成する４種類のフラバン－３－オールの割合（モル比）が、エピカテキン：カテキン：エピガロカテキン：ガロカテキン＝0.50～2.00:0.28～1.12:0.38～1.52:0.07～0.26であることが好ましい。さらに好ましくは、エピカテキン：カテキン：エピガロカテキン：ガロカテキン＝0.75～1.50:0.42～0.84:0.57～1.14:0.10～0.20であり、最も好ましくは、0.9～1.2:0.50～0.67:0.68～0.91:0.12～0.16である。
In the present invention, the ratio (molar ratio) of the four types of flavan-3-ols constituting the basic skeleton is epicatechin: catechin: epigallocatechin: gallocatechin = 0.50 to 2.00: 0.28 to 1.12: 0.38 to 1.52: It is preferably 0.07 to 0.26. More preferably, epicatechin: catechin: epigallocatechin: gallocatechin = 0.75 to 1.50: 0.42 to 0.84: 0.57 to 1.14: 0.10 to 0.20, and most preferably 0.9 to 1.2: 0.50 to 0.67: 0.68 to 0.91: 0.12. ~ 0.16.
プロシアニジンは、基本骨格の構成単位の結合様式の違いから、主にＡ型（単位間に４β→８及び２β→Ｏ→７又は４β→６及び２β→Ｏ→７の結合を少なくとも１つ有する）、及びＢ型（単位間の炭素通しの結合が４β→８又は４β→６のみ）に分けられる。
本発明に係る新規プロシアニジンは、Ｂ型プロシアニジンであることが好ましい。 Procyanidins are mainly of type A (having at least one bond of 4β → 8 and 2β → O → 7 or 4β → 6 and 2β → O → 7 between units) due to the difference in the bonding mode of the structural units of the basic skeleton. , And B (carbon-bonded bonds between units are 4β → 8 or 4β → 6 only).
The novel procyanidins according to the present invention are preferably B-type procyanidins.
本発明に係る新規プロシアニジンは、Ｂ型プロシアニジンであることが好ましい。 Procyanidins are mainly of type A (having at least one bond of 4β → 8 and 2β → O → 7 or 4β → 6 and 2β → O → 7 between units) due to the difference in the bonding mode of the structural units of the basic skeleton. , And B (carbon-bonded bonds between units are 4β → 8 or 4β → 6 only).
The novel procyanidins according to the present invention are preferably B-type procyanidins.
本発明に係る新規プロシアニジンは、分子量が1,000－114,000のものであってよく、好ましくは3,000－114,000、さらに好ましくは8,000－15,000である。また、前記構成単位の重合度は、3－400、好ましくは10－400、さらに好ましくは25－50であってよい。また、末端に、主にカテキンを有していてもよい。
The novel procyanidins according to the present invention may have a molecular weight of 1,000-114,000, preferably 3,000-114,000, more preferably 8,000-15,000. The degree of polymerization of the structural unit may be 3 to 400, preferably 10 to 400, more preferably 25 to 50. Moreover, you may have catechin mainly at the terminal.
本発明に係る新規プロシアニジンは、前記基本骨格を構成する１以上のフラバン－３－オールにおける１以上のOH基が、誘導体化、例えば、エステル化されていてもよい。当該フラバン－３－オールは、環の３位、５位、７位、３’位及び４’位のうちの１以上で、１以上のエステル基、好ましくは、没食子酸エステル基を含んでもよい。また、１のフラバン－３－オールは、１、２、３、４、または５の没食子酸エステルと結合していてもよい。
本発明に係る新規プロシアニジンは、フラバン-3-オールあたり平均して一個のガロイル基が結合していてもよい。
前記没食子酸エステルの例としては、カテキンガレート、エピカテキンガレート、ガロカテキンガレート、エピガロカテキンガレート等が挙げられる。 In the novel procyanidins according to the present invention, one or more OH groups in one or more flavan-3-ols constituting the basic skeleton may be derivatized, for example, esterified. The flavan-3-ol may contain one or more ester groups, preferably a gallic acid ester group, at one or more of the 3-position, 5-position, 7-position, 3′-position and 4′-position of the ring. . Also, one flavan-3-ol may be bound to 1, 2, 3, 4, or 5 gallate esters.
The novel procyanidins according to the present invention may have an average of one galloyl group bonded per flavan-3-ol.
Examples of the gallic acid ester include catechin gallate, epicatechin gallate, gallocatechin gallate, epigallocatechin gallate and the like.
本発明に係る新規プロシアニジンは、フラバン-3-オールあたり平均して一個のガロイル基が結合していてもよい。
前記没食子酸エステルの例としては、カテキンガレート、エピカテキンガレート、ガロカテキンガレート、エピガロカテキンガレート等が挙げられる。 In the novel procyanidins according to the present invention, one or more OH groups in one or more flavan-3-ols constituting the basic skeleton may be derivatized, for example, esterified. The flavan-3-ol may contain one or more ester groups, preferably a gallic acid ester group, at one or more of the 3-position, 5-position, 7-position, 3′-position and 4′-position of the ring. . Also, one flavan-3-ol may be bound to 1, 2, 3, 4, or 5 gallate esters.
The novel procyanidins according to the present invention may have an average of one galloyl group bonded per flavan-3-ol.
Examples of the gallic acid ester include catechin gallate, epicatechin gallate, gallocatechin gallate, epigallocatechin gallate and the like.
［新規プロシアニジンの製造方法］
本発明に係る新規プロシアニジンは、アカショウマから抽出することができる。 [Method for producing new procyanidins]
The novel procyanidins according to the present invention can be extracted from red pepper.
本発明に係る新規プロシアニジンは、アカショウマから抽出することができる。 [Method for producing new procyanidins]
The novel procyanidins according to the present invention can be extracted from red pepper.
アカショウマ（学名：Astilbe thunbergii var. thunbergii）は、ユキノシタ科チダケサシ属の植物で、本州、四国、九州各地の山地に自生する多年草である。アカショウマの根は、解熱、解毒、消炎等の効用で用いられるショウマ（キンポウゲ科のサラシナショウマ）の根茎の代用品として古くから用いられてきた。なお、厚生労働省からの通達では、ショウマの根茎は医薬品だが、アカショウマの根は非医薬品に相当する（ttps://hfnet.nih.go.jp/usr/annzenn/image/iyakuhin2参照）。
Akashoma (scientific name: Astilbe thunbergii var. Thunbergii) is a plant belonging to the genus Pleurotusaceae and is a perennial that grows naturally in mountainous areas in Honshu, Shikoku and Kyushu. The root of red ginger has long been used as a substitute for the rhizome of ginger (Ranunculaceae), which is used for antipyretic, detoxifying and anti-inflammatory effects. According to a notification from the Ministry of Health, Labor and Welfare, the rhizome of shouma is a pharmaceutical, but the root of red shouma is equivalent to a non-medicine (see ttps: //hfnet.nih.go.jp/usr/annzenn/image/iyakuhin2).
本発明に用いるアカショウマ（Astilbe thunbergii var. thunbergii）は、自生又は栽培されたアカショウマのいずれであってもよく、好適な部位は根及び／又は根茎である。生及び乾燥させたもののいずれも用いることができ、抽出効率向上の観点からは、細断化又は粉末化されていることが好ましい。
The red pepper (Astilbe thunbergii var. Thunbergii) used in the present invention may be either native or cultivated red pepper, and the preferred site is the root and / or rhizome. Either raw or dried can be used, and from the viewpoint of improving extraction efficiency, it is preferably shredded or powdered.
本発明では、アカショウマの水及び／又は含水有機溶媒による抽出物を、特定の合成吸着剤で吸着処理し、該吸着剤を特定の抽出溶媒を用いて溶出処理することにより、新規プロシアニジンを高濃度で含む画分を得ることが可能である。
In the present invention, the extract of red pepper with water and / or water-containing organic solvent is adsorbed with a specific synthetic adsorbent, and the adsorbent is eluted with a specific extraction solvent to obtain a novel procyanidin at a high concentration. It is possible to obtain a fraction containing
・アカショウマ抽出物の調整
前記アカショウマを抽出する溶媒としては、水、含水有機溶媒、又はこれらの組み合わせ（具体的には、含水有機溶媒で抽出後に水で抽出）が好ましい。なお、含水有機溶媒における水分含量は、10-90％、好ましくは20-80％、さらに好ましくは30-70％、最も好ましくは40-60％である。 -Preparation of red pepper extract As the solvent for extracting red pepper, water, a water-containing organic solvent, or a combination thereof (specifically, extraction with a water-containing organic solvent and then extraction with water) is preferable. The water content in the water-containing organic solvent is 10-90%, preferably 20-80%, more preferably 30-70%, most preferably 40-60%.
前記アカショウマを抽出する溶媒としては、水、含水有機溶媒、又はこれらの組み合わせ（具体的には、含水有機溶媒で抽出後に水で抽出）が好ましい。なお、含水有機溶媒における水分含量は、10-90％、好ましくは20-80％、さらに好ましくは30-70％、最も好ましくは40-60％である。 -Preparation of red pepper extract As the solvent for extracting red pepper, water, a water-containing organic solvent, or a combination thereof (specifically, extraction with a water-containing organic solvent and then extraction with water) is preferable. The water content in the water-containing organic solvent is 10-90%, preferably 20-80%, more preferably 30-70%, most preferably 40-60%.
前記有機溶媒は特に制限されないが、例えば、メタノール、エタノール、プロパノール、ブタノール等の低級アルコール；酢酸エチル等のエステル；エチレングリコール、ブチレングリコール、プロピレングリコール、１，３-ブチレンアルコール、グリセリン等のグリコール類；ジエチルエーテル、石油エーテル等のエーテル；アセトン、酢酸等の極性溶媒；ベンゼン、ヘキサン、キシレン等の炭化水素等が挙げられる。このうち、低級アルコール、エステルが好ましく、さらに好ましくは低級アルコールである。なお、これらの有機溶媒は、単独又は二種以上を組み合わせて用いてもよい。
The organic solvent is not particularly limited, but examples thereof include lower alcohols such as methanol, ethanol, propanol, and butanol; esters such as ethyl acetate; glycols such as ethylene glycol, butylene glycol, propylene glycol, 1,3-butylene alcohol, and glycerin. Ethers such as diethyl ether and petroleum ether; polar solvents such as acetone and acetic acid; hydrocarbons such as benzene, hexane and xylene. Of these, lower alcohols and esters are preferable, and lower alcohols are more preferable. In addition, you may use these organic solvents individually or in combination of 2 or more types.
抽出温度は、常温から溶媒の沸点の範囲内の温度で、溶媒の種類に応じて適宜調整することができる。加圧、常圧、減圧下で行ってもよい。抽出時間も、溶媒の種類に応じて適宜調整してよい。例えば、抽出溶媒として水を用いる場合には、20-140℃、好ましくは60-130℃、さらに好ましくは80-125℃で、1分-1時間、好ましくは、10-30分間の範囲内で行ってもよい。また、抽出溶媒として含水エタノールを用いる場合には、20-100℃、好ましくは40-80℃の範囲内で、1分-1時間、好ましくは、10-30分間の範囲内で行ってもよい。さらに、前記保温期間中に、溶媒の攪拌又は還流を行うと一層好ましい。
抽出に用いる溶媒の量も特に制限されることはなく、例えば、アカショウマの根及び／又は根茎の乾燥物に対し、2-50倍、好ましくは5-50倍、さらに好ましくは10-30倍（重量比）の溶媒を用いてもよい。 The extraction temperature is a temperature within the range from room temperature to the boiling point of the solvent, and can be appropriately adjusted according to the type of solvent. You may carry out under pressurization, a normal pressure, and pressure reduction. The extraction time may also be adjusted as appropriate according to the type of solvent. For example, when water is used as the extraction solvent, it is 20 to 140 ° C., preferably 60 to 130 ° C., more preferably 80 to 125 ° C., within 1 minute to 1 hour, preferably 10 to 30 minutes. You may go. Further, when using water-containing ethanol as the extraction solvent, it may be carried out within a range of 20-100 ° C., preferably 40-80 ° C., 1 minute-1 hour, preferably 10-30 minutes. . Furthermore, it is more preferable that the solvent is stirred or refluxed during the heat retention period.
The amount of the solvent used for the extraction is not particularly limited. For example, it is 2 to 50 times, preferably 5 to 50 times, more preferably 10 to 30 times the dry matter of red pepper and / or rhizomes ( (Weight ratio) solvent may be used.
抽出に用いる溶媒の量も特に制限されることはなく、例えば、アカショウマの根及び／又は根茎の乾燥物に対し、2-50倍、好ましくは5-50倍、さらに好ましくは10-30倍（重量比）の溶媒を用いてもよい。 The extraction temperature is a temperature within the range from room temperature to the boiling point of the solvent, and can be appropriately adjusted according to the type of solvent. You may carry out under pressurization, a normal pressure, and pressure reduction. The extraction time may also be adjusted as appropriate according to the type of solvent. For example, when water is used as the extraction solvent, it is 20 to 140 ° C., preferably 60 to 130 ° C., more preferably 80 to 125 ° C., within 1 minute to 1 hour, preferably 10 to 30 minutes. You may go. Further, when using water-containing ethanol as the extraction solvent, it may be carried out within a range of 20-100 ° C., preferably 40-80 ° C., 1 minute-1 hour, preferably 10-30 minutes. . Furthermore, it is more preferable that the solvent is stirred or refluxed during the heat retention period.
The amount of the solvent used for the extraction is not particularly limited. For example, it is 2 to 50 times, preferably 5 to 50 times, more preferably 10 to 30 times the dry matter of red pepper and / or rhizomes ( (Weight ratio) solvent may be used.
抽出後に濾過、遠心分離等によって固形物を除去することで、アカショウマ抽出物を得ることができる。また、アカショウマ抽出物として、市販品を用いることもできる。例えば、乾燥根の含水エタノール抽出物である“アカショウマエキス末（ビーエイチエヌ株式会社製）を使用してもよい。
After extraction, the red pepper extract can be obtained by removing solids by filtration, centrifugation, or the like. Moreover, a commercial item can also be used as a red pepper extract. For example, “Akashoma extract powder (manufactured by BN Co., Ltd.), which is an aqueous ethanol extract of dry roots, may be used.
・アカショウマ抽出物の吸着処理
前記アカショウマ抽出物を吸着させる吸着剤は、芳香族系合成吸着剤が好ましく、さらに好ましくは、スチレン－ジビニルベンゼン系合成吸着剤である。例として、ダイヤイオンHP20（三菱化学株式会社製）、アンバーライトXAD-2、4（ダウ・ケミカル社製）等を好適に用いることができる。
前記アカショウマ抽出物を吸着処理した合成吸着剤は、水で洗浄した後、含水有機溶媒を用いて溶出処理することができる。当該有機溶媒としては、含水メタノール溶液、含水エタノール溶液等を用いることができる。このうち、特に好ましくは、50%メタノール水溶液である。
得られた溶出液は、さらに慣用の精製法を用いて精製してもよい。次に、サイズ排除クロマトグラフィーを用いて精製する方法を例示する。 -Adsorption treatment of red pepper extract The adsorbent for adsorbing the red pepper extract is preferably an aromatic synthetic adsorbent, more preferably a styrene-divinylbenzene synthetic adsorbent. As an example, Diaion HP20 (manufactured by Mitsubishi Chemical Corporation), Amberlite XAD-2, 4 (manufactured by Dow Chemical Company), or the like can be suitably used.
The synthetic adsorbent obtained by adsorbing the red pepper extract can be washed with water and then eluted with a water-containing organic solvent. A water-containing methanol solution, a water-containing ethanol solution, or the like can be used as the organic solvent. Of these, a 50% aqueous methanol solution is particularly preferred.
The obtained eluate may be further purified using a conventional purification method. Next, a method for purification using size exclusion chromatography is exemplified.
前記アカショウマ抽出物を吸着させる吸着剤は、芳香族系合成吸着剤が好ましく、さらに好ましくは、スチレン－ジビニルベンゼン系合成吸着剤である。例として、ダイヤイオンHP20（三菱化学株式会社製）、アンバーライトXAD-2、4（ダウ・ケミカル社製）等を好適に用いることができる。
前記アカショウマ抽出物を吸着処理した合成吸着剤は、水で洗浄した後、含水有機溶媒を用いて溶出処理することができる。当該有機溶媒としては、含水メタノール溶液、含水エタノール溶液等を用いることができる。このうち、特に好ましくは、50%メタノール水溶液である。
得られた溶出液は、さらに慣用の精製法を用いて精製してもよい。次に、サイズ排除クロマトグラフィーを用いて精製する方法を例示する。 -Adsorption treatment of red pepper extract The adsorbent for adsorbing the red pepper extract is preferably an aromatic synthetic adsorbent, more preferably a styrene-divinylbenzene synthetic adsorbent. As an example, Diaion HP20 (manufactured by Mitsubishi Chemical Corporation), Amberlite XAD-2, 4 (manufactured by Dow Chemical Company), or the like can be suitably used.
The synthetic adsorbent obtained by adsorbing the red pepper extract can be washed with water and then eluted with a water-containing organic solvent. A water-containing methanol solution, a water-containing ethanol solution, or the like can be used as the organic solvent. Of these, a 50% aqueous methanol solution is particularly preferred.
The obtained eluate may be further purified using a conventional purification method. Next, a method for purification using size exclusion chromatography is exemplified.
・プロシアニジンの精製
前記溶出液は、必要に応じて濃縮、溶媒置換等を行った後、親水性ビニルポリマー系（例として、Toyopearl HW-40F、東ソー バイオサイエンス社製）、又はデキストラン系（例として、Sephadex LH-20、GEヘルスケア株式会社製）の担体を用いて精製してもよい。例えば、親水性ビニルポリマー系担体を用いる場合には、水を溶出液とすることで、本発明に係る新規プロシアニジンを先端溶出画分として（さらに詳しくは、０．２～０．４カラムボリュームの溶媒による溶出画分として）回収することが可能である。また、デキストラン系担体を用いる場合には、50%メタノール水溶液、100%メタノールで順次洗浄後、70%アセトン水溶液で溶出させてもよい。本発明に係る新規プロシアニジンの濃縮画分は、例えば、α-アミラーゼ活性阻害試験（実施例参照）を行うことで選別可能である。
前記新規プロシアニジンの濃縮画分は、さらに逆相HPLCに供して精製してもよい。 -Purification of procyanidins The eluate is concentrated, solvent-substituted, etc., if necessary, and then a hydrophilic vinyl polymer system (for example, Toyopearl HW-40F, manufactured by Tosoh Biosciences) or a dextran system (for example Sepadex LH-20, manufactured by GE Healthcare Co., Ltd.) may be used for purification. For example, when using a hydrophilic vinyl polymer carrier, water is used as an eluent, and the novel procyanidin according to the present invention is used as a tip elution fraction (more specifically, 0.2 to 0.4 column volume). It can be recovered (as an elution fraction with solvent). When a dextran carrier is used, it may be washed with a 50% methanol aqueous solution and a 100% methanol sequentially and then eluted with a 70% acetone aqueous solution. The enriched fraction of novel procyanidins according to the present invention can be selected, for example, by conducting an α-amylase activity inhibition test (see Examples).
The concentrated fraction of the novel procyanidin may be further subjected to reverse phase HPLC for purification.
前記溶出液は、必要に応じて濃縮、溶媒置換等を行った後、親水性ビニルポリマー系（例として、Toyopearl HW-40F、東ソー バイオサイエンス社製）、又はデキストラン系（例として、Sephadex LH-20、GEヘルスケア株式会社製）の担体を用いて精製してもよい。例えば、親水性ビニルポリマー系担体を用いる場合には、水を溶出液とすることで、本発明に係る新規プロシアニジンを先端溶出画分として（さらに詳しくは、０．２～０．４カラムボリュームの溶媒による溶出画分として）回収することが可能である。また、デキストラン系担体を用いる場合には、50%メタノール水溶液、100%メタノールで順次洗浄後、70%アセトン水溶液で溶出させてもよい。本発明に係る新規プロシアニジンの濃縮画分は、例えば、α-アミラーゼ活性阻害試験（実施例参照）を行うことで選別可能である。
前記新規プロシアニジンの濃縮画分は、さらに逆相HPLCに供して精製してもよい。 -Purification of procyanidins The eluate is concentrated, solvent-substituted, etc., if necessary, and then a hydrophilic vinyl polymer system (for example, Toyopearl HW-40F, manufactured by Tosoh Biosciences) or a dextran system (for example Sepadex LH-20, manufactured by GE Healthcare Co., Ltd.) may be used for purification. For example, when using a hydrophilic vinyl polymer carrier, water is used as an eluent, and the novel procyanidin according to the present invention is used as a tip elution fraction (more specifically, 0.2 to 0.4 column volume). It can be recovered (as an elution fraction with solvent). When a dextran carrier is used, it may be washed with a 50% methanol aqueous solution and a 100% methanol sequentially and then eluted with a 70% acetone aqueous solution. The enriched fraction of novel procyanidins according to the present invention can be selected, for example, by conducting an α-amylase activity inhibition test (see Examples).
The concentrated fraction of the novel procyanidin may be further subjected to reverse phase HPLC for purification.
［新規プロシアニジンの用途］
本発明に係るプロシアニジンは、多糖消化抑制作用に優れ、さらに詳しくは、α-アミラーゼ活性を阻害する作用に優れるものである。当該α-アミラーゼ阻害活性は、IC50値が約1.7～2.6μg/mLであり、重量濃度比に換算すると、糖尿病患者に処方される食後過血糖改善剤の代表であるアカルボース（Acarbose；バイエル薬品株式会社のGlucobay、CAS登録番号：56180-94-0、IC50値は10.2～83.33μg/mL、参考文献：Sudha P., et al, BMC Complement Altern Med. 11:5, 2011、Tamil I. G., et al, Indian J. Pharmacol., 42(5):280-2, 2010）よりも高い。
よって、本発明に係るプロシアニジンは、食後過血糖の改善を目的とする多糖消化阻害剤として用いることが可能である。 [Use of new procyanidins]
The procyanidins according to the present invention are excellent in the polysaccharide digestion inhibitory action, and more specifically, in the action of inhibiting α-amylase activity. The α-amylase inhibitory activity has an IC 50 value of about 1.7 to 2.6 μg / mL, and when converted to a weight concentration ratio, Acarbose, a typical postprandial hyperglycemia improving agent prescribed for diabetics Glucobay Co., Ltd., CAS Registry Number: 56180-94-0, IC 50 value is 10.2 ~ 83.33μg / mL, Reference: Sudha P., et al, BMC Complement Altern Med. 11: 5, 2011, Tamil IG, et al, Indian J. Pharmacol., 42 (5): 280-2, 2010).
Therefore, the procyanidins according to the present invention can be used as a polysaccharide digestion inhibitor for the purpose of improving postprandial hyperglycemia.
本発明に係るプロシアニジンは、多糖消化抑制作用に優れ、さらに詳しくは、α-アミラーゼ活性を阻害する作用に優れるものである。当該α-アミラーゼ阻害活性は、IC50値が約1.7～2.6μg/mLであり、重量濃度比に換算すると、糖尿病患者に処方される食後過血糖改善剤の代表であるアカルボース（Acarbose；バイエル薬品株式会社のGlucobay、CAS登録番号：56180-94-0、IC50値は10.2～83.33μg/mL、参考文献：Sudha P., et al, BMC Complement Altern Med. 11:5, 2011、Tamil I. G., et al, Indian J. Pharmacol., 42(5):280-2, 2010）よりも高い。
よって、本発明に係るプロシアニジンは、食後過血糖の改善を目的とする多糖消化阻害剤として用いることが可能である。 [Use of new procyanidins]
The procyanidins according to the present invention are excellent in the polysaccharide digestion inhibitory action, and more specifically, in the action of inhibiting α-amylase activity. The α-amylase inhibitory activity has an IC 50 value of about 1.7 to 2.6 μg / mL, and when converted to a weight concentration ratio, Acarbose, a typical postprandial hyperglycemia improving agent prescribed for diabetics Glucobay Co., Ltd., CAS Registry Number: 56180-94-0, IC 50 value is 10.2 ~ 83.33μg / mL, Reference: Sudha P., et al, BMC Complement Altern Med. 11: 5, 2011, Tamil IG, et al, Indian J. Pharmacol., 42 (5): 280-2, 2010).
Therefore, the procyanidins according to the present invention can be used as a polysaccharide digestion inhibitor for the purpose of improving postprandial hyperglycemia.
なお、本発明における“多糖”とは、“二以上の単糖がグリコシド結合（好ましくは、α-１，４－グリコシド結合）によって重合したもの”を指す。また、本発明におけるα-アミラーゼ阻害活性とは、α-アミラーゼ（α-Amylase；EC 3.2.1.1）のα-1,4-グルコシド結合切断活性を阻害する活性を意味し、α-グルコシダーゼ阻害活性とは、α-グルコシダーゼ（α-glucosidase;EC 3.2.1.20）のα-1,4-グルコシド結合切断活性を阻害する活性を意味する。
In the present invention, “polysaccharide” refers to “two or more monosaccharides polymerized by glycosidic bonds (preferably α-1,4-glycosidic bonds)”. In addition, the α-amylase inhibitory activity in the present invention means an activity that inhibits the α-1,4-glucoside bond cleavage activity of α-amylase (EC 3.2.1.1), and α-glucosidase inhibitory activity. The term “α-glucosidase (EC 3.2.1.20)” means the activity of inhibiting the α-1,4-glucoside bond cleavage activity of α-glucosidase (EC 3.2.1.20).
本発明に係るプロシアニジンを多糖消化剤として用いる場合には、多糖を摂取する前（食事前）から食中に経口摂取されることが好ましいが、食後間もない時刻であれば経口摂取により十分に効果を奏し得ると考えられる。よって、食前30分～食後30分、好ましくは食前15分～食後15分、より好ましくは食前10分～食後10分、最も好ましくは食前5分～食中である。
When procyanidins according to the present invention are used as a polysaccharide digesting agent, it is preferably taken orally during the meal from before taking the polysaccharide (before the meal), but if the time is shortly after the meal, the oral intake is sufficient. It is thought that there can be an effect. Therefore, 30 minutes before meal to 30 minutes after meal, preferably 15 minutes before meal to 15 minutes after meal, more preferably 10 minutes before meal to 10 minutes after meal, most preferably 5 minutes before meal.
摂取量は食事の内容（多糖の量）に合わせて調節することができ、一般的な内容の食事の場合、ヒト（平均体重60kg）に対し、例えば、プロシアニジンとして11-33mg／回を目安とすることができる。
The amount of intake can be adjusted according to the content of the meal (the amount of polysaccharides). In the case of a general content meal, for example, for procyanidins 11-33mg / dose as a guide for humans (average body weight 60kg) can do.
前記多糖消化阻害剤は、単独で摂取してもよく、また、医薬的に許容される担体、賦形剤、可塑剤、着色剤、防腐剤等と混合して経口用組成物の形で摂取してもよい。当該経口用組成物に用いる担体としては、例えば、糖アルコール（例として、マンニトール）、無機物（例として、炭酸カルシウム）、微結晶性セルロース、セルロース（例として、カルボキシメチルセルロース）、ゼラチン、アルギン酸ナトリウム、ポリビニルピロリドン、寒天、ステアリン酸マグネシウム、タルク等が挙げられる。
前記経口用組成物の形態は特に限定されることはなく、錠剤、丸剤、カプセル剤、顆粒剤、散剤、粉末剤、トローチ剤、又は溶液（飲料）等の形態とすることができる。 The polysaccharide digestion inhibitor may be taken alone or in the form of an oral composition mixed with a pharmaceutically acceptable carrier, excipient, plasticizer, colorant, preservative, etc. May be. Examples of the carrier used for the oral composition include sugar alcohol (eg, mannitol), inorganic substance (eg, calcium carbonate), microcrystalline cellulose, cellulose (eg, carboxymethylcellulose), gelatin, sodium alginate, Examples include polyvinyl pyrrolidone, agar, magnesium stearate, talc and the like.
The form of the oral composition is not particularly limited, and may be in the form of a tablet, pill, capsule, granule, powder, powder, troche, solution (beverage) or the like.
前記経口用組成物の形態は特に限定されることはなく、錠剤、丸剤、カプセル剤、顆粒剤、散剤、粉末剤、トローチ剤、又は溶液（飲料）等の形態とすることができる。 The polysaccharide digestion inhibitor may be taken alone or in the form of an oral composition mixed with a pharmaceutically acceptable carrier, excipient, plasticizer, colorant, preservative, etc. May be. Examples of the carrier used for the oral composition include sugar alcohol (eg, mannitol), inorganic substance (eg, calcium carbonate), microcrystalline cellulose, cellulose (eg, carboxymethylcellulose), gelatin, sodium alginate, Examples include polyvinyl pyrrolidone, agar, magnesium stearate, talc and the like.
The form of the oral composition is not particularly limited, and may be in the form of a tablet, pill, capsule, granule, powder, powder, troche, solution (beverage) or the like.
前記多糖消化阻害剤は、一般食品、健康食品、保険機能食品（特定保健用食品、機能性表示食品等）に配合された状態で、好適に摂取することができる。
前記食品としては、例えば、乳飲料、乳酸菌飲料、清涼飲料、炭酸飲料、果汁飲料、野菜飲料、アルコール飲料、粉末飲料、コーヒー飲料、紅茶飲料、緑茶飲料、麦茶飲料等の飲料類；プリン、ゼリー、ババロア、ヨーグルト、アイスクリーム、ガム、チョコレート、キャンディー、キャラメル、ビスケット、クッキー、おかき、煎餅等の菓子類；コンソメスープ、ポタージュスープ等のスープ類；味噌、醤油、ドレッシング、ケチャップ、たれ、ソース、ふりかけなどの各種調味料；ストロベリージャム、ブルーベリージャム、マーマレード、リンゴジャム等のジャム類；赤ワイン等の果実酒；シロップ漬のチェリー、アンズ、リンゴ、イチゴ、桃等の加工用果実；うどん、冷麦、そうめん、ソバ、中華そば、スパゲッティ、マカロニ、ビーフン、はるさめ及びワンタン等の麺類；その他、各種加工食品等が挙げられる。 The polysaccharide digestion inhibitor can be suitably ingested in a state where it is blended with general foods, health foods, insurance functional foods (food for specified health use, functional indication foods, etc.).
Examples of the food include milk beverages, lactic acid bacteria beverages, soft drinks, carbonated beverages, fruit juice beverages, vegetable beverages, alcoholic beverages, powdered beverages, coffee beverages, tea beverages, green tea beverages and barley tea beverages; pudding, jelly , Bavaria, yogurt, ice cream, gum, chocolate, candy, caramel, biscuits, cookies, rice crackers, rice crackers and other confectionery; consommé soup, potage soup, etc .; miso, soy sauce, dressing, ketchup, sauce, sauce, Various seasonings such as sprinkles; jams such as strawberry jam, blueberry jam, marmalade, apple jam; fruit wine such as red wine; fruit for processing such as syrup pickled cherry, apricot, apple, strawberry, peach; udon, cold wheat, Somen, buckwheat, Chinese noodles, spaghetti, macaroni, bi Hung, noodles such as vermicelli and won ton; Etc processed foods and the like.
前記食品としては、例えば、乳飲料、乳酸菌飲料、清涼飲料、炭酸飲料、果汁飲料、野菜飲料、アルコール飲料、粉末飲料、コーヒー飲料、紅茶飲料、緑茶飲料、麦茶飲料等の飲料類；プリン、ゼリー、ババロア、ヨーグルト、アイスクリーム、ガム、チョコレート、キャンディー、キャラメル、ビスケット、クッキー、おかき、煎餅等の菓子類；コンソメスープ、ポタージュスープ等のスープ類；味噌、醤油、ドレッシング、ケチャップ、たれ、ソース、ふりかけなどの各種調味料；ストロベリージャム、ブルーベリージャム、マーマレード、リンゴジャム等のジャム類；赤ワイン等の果実酒；シロップ漬のチェリー、アンズ、リンゴ、イチゴ、桃等の加工用果実；うどん、冷麦、そうめん、ソバ、中華そば、スパゲッティ、マカロニ、ビーフン、はるさめ及びワンタン等の麺類；その他、各種加工食品等が挙げられる。 The polysaccharide digestion inhibitor can be suitably ingested in a state where it is blended with general foods, health foods, insurance functional foods (food for specified health use, functional indication foods, etc.).
Examples of the food include milk beverages, lactic acid bacteria beverages, soft drinks, carbonated beverages, fruit juice beverages, vegetable beverages, alcoholic beverages, powdered beverages, coffee beverages, tea beverages, green tea beverages and barley tea beverages; pudding, jelly , Bavaria, yogurt, ice cream, gum, chocolate, candy, caramel, biscuits, cookies, rice crackers, rice crackers and other confectionery; consommé soup, potage soup, etc .; miso, soy sauce, dressing, ketchup, sauce, sauce, Various seasonings such as sprinkles; jams such as strawberry jam, blueberry jam, marmalade, apple jam; fruit wine such as red wine; fruit for processing such as syrup pickled cherry, apricot, apple, strawberry, peach; udon, cold wheat, Somen, buckwheat, Chinese noodles, spaghetti, macaroni, bi Hung, noodles such as vermicelli and won ton; Etc processed foods and the like.
以下に、実施例を用いて本発明をさらに詳細に説明するが、これらの実施例により本発明の範囲が限定されるものではない。
Hereinafter, the present invention will be described in more detail with reference to examples, but the scope of the present invention is not limited by these examples.
実施例１：新規プロシアニジンの精製方法１
アカショウマエキス末（ビーエイチエヌ株式会社製）を水に懸濁し、吸引ろ過して得られたろ液を、スチレン－ジビニルベンゼン系合成吸着剤（ダイヤイオンHP20、三菱化学株式会社製）に吸着させた。当該合成吸着剤を水で洗浄し、50％メタノール水溶液を用いて溶出した。得られた溶出液を濃縮し、水に懸濁した後、遠心分離して上清を回収し、サイズ排除クロマトグラフィー（Toyopearl HW-40F、東ソー バイオサイエンス社製）に供して分画した（溶出液として水を使用）。最先端（0.2～0.3カラムボリューム）に溶出された画分を、プロシアニジン濃縮画分として回収した。
当該プロシアニジン濃縮画分に対し、1H-NMRにより解析した結果を図１に示す。また、下記条件下で逆相HPLCに供した結果を図２に示す。 Example 1:Method 1 for purifying novel procyanidins
A red pepper extract powder (manufactured by BN Co., Ltd.) was suspended in water, and the filtrate obtained by suction filtration was adsorbed onto a styrene-divinylbenzene synthetic adsorbent (Diaion HP20, manufactured by Mitsubishi Chemical Corporation). The synthetic adsorbent was washed with water and eluted with 50% aqueous methanol. The obtained eluate was concentrated, suspended in water, centrifuged, and the supernatant was collected and fractionated by size exclusion chromatography (Toyopearl HW-40F, manufactured by Tosoh Bioscience) (elution) Use water as liquid). The fraction eluted at the leading edge (0.2-0.3 column volume) was collected as a procyanidin-enriched fraction.
The result of analyzing the procyanidin-enriched fraction by 1 H-NMR is shown in FIG. Moreover, the result of having used for reverse phase HPLC on the following conditions is shown in FIG.
アカショウマエキス末（ビーエイチエヌ株式会社製）を水に懸濁し、吸引ろ過して得られたろ液を、スチレン－ジビニルベンゼン系合成吸着剤（ダイヤイオンHP20、三菱化学株式会社製）に吸着させた。当該合成吸着剤を水で洗浄し、50％メタノール水溶液を用いて溶出した。得られた溶出液を濃縮し、水に懸濁した後、遠心分離して上清を回収し、サイズ排除クロマトグラフィー（Toyopearl HW-40F、東ソー バイオサイエンス社製）に供して分画した（溶出液として水を使用）。最先端（0.2～0.3カラムボリューム）に溶出された画分を、プロシアニジン濃縮画分として回収した。
当該プロシアニジン濃縮画分に対し、1H-NMRにより解析した結果を図１に示す。また、下記条件下で逆相HPLCに供した結果を図２に示す。 Example 1:
A red pepper extract powder (manufactured by BN Co., Ltd.) was suspended in water, and the filtrate obtained by suction filtration was adsorbed onto a styrene-divinylbenzene synthetic adsorbent (Diaion HP20, manufactured by Mitsubishi Chemical Corporation). The synthetic adsorbent was washed with water and eluted with 50% aqueous methanol. The obtained eluate was concentrated, suspended in water, centrifuged, and the supernatant was collected and fractionated by size exclusion chromatography (Toyopearl HW-40F, manufactured by Tosoh Bioscience) (elution) Use water as liquid). The fraction eluted at the leading edge (0.2-0.3 column volume) was collected as a procyanidin-enriched fraction.
The result of analyzing the procyanidin-enriched fraction by 1 H-NMR is shown in FIG. Moreover, the result of having used for reverse phase HPLC on the following conditions is shown in FIG.
＜逆相HPLCの条件＞
カラム：InertSustain C18（直径4.6mm×長さ250mm、ジーエルサイエンス株式会社製）
移動相：
０～３０分間：0.1% トリフルオロ酢酸（TFA）、5% メタノール水溶液
－0.1% TFA、95% メタノール水溶液のグラジエント溶出
３０～４０分間：0.1% TFA、95% メタノール水溶液
流速：1.0mL/min
検出：UV254nm <Reverse phase HPLC conditions>
Column: InertSustain C18 (diameter 4.6mm x length 250mm, manufactured by GL Sciences Inc.)
Mobile phase:
0-30 minutes: 0.1% trifluoroacetic acid (TFA), 5% aqueous methanol solution -0.1% TFA, 95% aqueous methanol gradient elution 30-40 minutes: 0.1% TFA, 95% aqueous methanol flow rate: 1.0 mL / min
Detection: UV254nm
カラム：InertSustain C18（直径4.6mm×長さ250mm、ジーエルサイエンス株式会社製）
移動相：
０～３０分間：0.1% トリフルオロ酢酸（TFA）、5% メタノール水溶液
－0.1% TFA、95% メタノール水溶液のグラジエント溶出
３０～４０分間：0.1% TFA、95% メタノール水溶液
流速：1.0mL/min
検出：UV254nm <Reverse phase HPLC conditions>
Column: InertSustain C18 (diameter 4.6mm x length 250mm, manufactured by GL Sciences Inc.)
Mobile phase:
0-30 minutes: 0.1% trifluoroacetic acid (TFA), 5% aqueous methanol solution -0.1% TFA, 95% aqueous methanol gradient elution 30-40 minutes: 0.1% TFA, 95% aqueous methanol flow rate: 1.0 mL / min
Detection: UV254nm
図２において、保持時間18.85分をピークとして溶出されたものが本発明に係るプロシアニジンである。当該プロシアニジンについて、下記手法に従ってα-アミラーゼ阻害活性を測定した。結果は実施例２の結果と合わせて表１に示す。
In FIG. 2, procyanidins according to the present invention are eluted with a retention time of 18.85 minutes as a peak. The procyanidin was measured for α-amylase inhibitory activity according to the following method. The results are shown in Table 1 together with the results of Example 2.
＜アミラーゼ阻害活性試験＞
・反応溶液（500μl）
（１）被験物質（乾燥させたもの） in 水、100μl
（２）Starch azure 1.4mg in buffer、350μl
（３）0.5U/mL ブタ膵臓由来α-アミラーゼ in buffer、50μl
＊buffer：0.01M CaCl2含有0.1M トリス塩酸緩衝液（pH6.9）
・方法
2.0ml容マイクロチューブに（２）を入れて37℃で5分間インキューベーションした後、（１）、（３）の順に添加し、振盪しながら37℃でさらに15分間インキューベーションした。50%酢酸水溶液を加えて反応を停止させた後、遠心を行い（4℃、1500×g、5分間）、上清200μlを96ウェルマイクロプレートに移して595nmの吸光度を測定した。なお、blankには前記酵素液の代わりにバッファーを、negative controlには被験物質の代わりに水、positive controlには被験物質としてアカルボース（10μM）を用いた。
各被験物質について2回の反復測定を行い、平均値を算出し、下記式（１）に従ってα-アミラーゼ活性阻害率を算出した。なお、下記式（１）において、ODsampleは被験物質を添加したウェル、ODsample blankは被験物質の存在下で酵素の代わりにバッファーを添加したウェル、ODcontrolはnegative controlウェル、ODcontrol blankはnegative controlにおいてさらに酵素の代わりにバッファーを添加したウェルの吸光度をそれぞれ表す。 <Amylase inhibitory activity test>
・ Reaction solution (500μl)
(1) Test substance (dried) in water, 100 μl
(2) Starch azure 1.4mg in buffer, 350μl
(3) 0.5 U / mL porcine pancreatic α-amylase in buffer, 50 μl
* Buffer: 0.1M Tris-HCl buffer solution (pH 6.9) containing 0.01M CaCl 2
・ Method After placing (2) in a 2.0 ml microtube and incubating at 37 ° C for 5 minutes, add (1) and (3) in this order, and incubate at 37 ° C for an additional 15 minutes with shaking. . After stopping the reaction by adding a 50% aqueous acetic acid solution, centrifugation was performed (4 ° C., 1500 × g, 5 minutes), 200 μl of the supernatant was transferred to a 96-well microplate, and the absorbance at 595 nm was measured. Note that a buffer was used instead of the enzyme solution for blank, water was used for negative control, and acarbose (10 μM) was used as a test substance for positive control.
Each test substance was measured twice, the average value was calculated, and the α-amylase activity inhibition rate was calculated according to the following formula (1). In the following formula (1), OD sample is a well to which a test substance is added, OD sample blank is a well to which a buffer is added instead of an enzyme in the presence of the test substance, OD control is a negative control well, and OD control blank is In the negative control, the absorbance of each well to which a buffer was added instead of the enzyme is shown.
・反応溶液（500μl）
（１）被験物質（乾燥させたもの） in 水、100μl
（２）Starch azure 1.4mg in buffer、350μl
（３）0.5U/mL ブタ膵臓由来α-アミラーゼ in buffer、50μl
＊buffer：0.01M CaCl2含有0.1M トリス塩酸緩衝液（pH6.9）
・方法
2.0ml容マイクロチューブに（２）を入れて37℃で5分間インキューベーションした後、（１）、（３）の順に添加し、振盪しながら37℃でさらに15分間インキューベーションした。50%酢酸水溶液を加えて反応を停止させた後、遠心を行い（4℃、1500×g、5分間）、上清200μlを96ウェルマイクロプレートに移して595nmの吸光度を測定した。なお、blankには前記酵素液の代わりにバッファーを、negative controlには被験物質の代わりに水、positive controlには被験物質としてアカルボース（10μM）を用いた。
各被験物質について2回の反復測定を行い、平均値を算出し、下記式（１）に従ってα-アミラーゼ活性阻害率を算出した。なお、下記式（１）において、ODsampleは被験物質を添加したウェル、ODsample blankは被験物質の存在下で酵素の代わりにバッファーを添加したウェル、ODcontrolはnegative controlウェル、ODcontrol blankはnegative controlにおいてさらに酵素の代わりにバッファーを添加したウェルの吸光度をそれぞれ表す。 <Amylase inhibitory activity test>
・ Reaction solution (500μl)
(1) Test substance (dried) in water, 100 μl
(2) Starch azure 1.4mg in buffer, 350μl
(3) 0.5 U / mL porcine pancreatic α-amylase in buffer, 50 μl
* Buffer: 0.1M Tris-HCl buffer solution (pH 6.9) containing 0.01M CaCl 2
・ Method After placing (2) in a 2.0 ml microtube and incubating at 37 ° C for 5 minutes, add (1) and (3) in this order, and incubate at 37 ° C for an additional 15 minutes with shaking. . After stopping the reaction by adding a 50% aqueous acetic acid solution, centrifugation was performed (4 ° C., 1500 × g, 5 minutes), 200 μl of the supernatant was transferred to a 96-well microplate, and the absorbance at 595 nm was measured. Note that a buffer was used instead of the enzyme solution for blank, water was used for negative control, and acarbose (10 μM) was used as a test substance for positive control.
Each test substance was measured twice, the average value was calculated, and the α-amylase activity inhibition rate was calculated according to the following formula (1). In the following formula (1), OD sample is a well to which a test substance is added, OD sample blank is a well to which a buffer is added instead of an enzyme in the presence of the test substance, OD control is a negative control well, and OD control blank is In the negative control, the absorbance of each well to which a buffer was added instead of the enzyme is shown.
実施例２：新規プロシアニジンの精製方法２
アカショウマエキス末（ビーエイチエヌ株式会社製）を水に懸濁し、吸引ろ過して得られたろ液を、スチレン－ジビニルベンゼン系合成吸着剤（ダイヤイオンHP20、三菱化学株式会社製）に吸着させた。当該合成吸着剤を水で洗浄し、50％メタノール水溶液を用いて溶出した。得られた溶出液をSephadex LH-20（GEヘルスケア株式会社製）に直接吸着させ、50％メタノール水溶液、100%メタノールにより順次洗浄後、70％アセトン水溶液を用いて溶出してプロシアニジン濃縮画分を得た。 Example 2:Method 2 for purifying novel procyanidins
A red pepper extract powder (manufactured by BN Co., Ltd.) was suspended in water, and the filtrate obtained by suction filtration was adsorbed onto a styrene-divinylbenzene synthetic adsorbent (Diaion HP20, manufactured by Mitsubishi Chemical Corporation). The synthetic adsorbent was washed with water and eluted with 50% aqueous methanol. The obtained eluate was directly adsorbed on Sephadex LH-20 (manufactured by GE Healthcare), washed sequentially with 50% methanol aqueous solution and 100% methanol, and eluted with 70% acetone aqueous solution to concentrate procyanidin-enriched fraction. Got.
アカショウマエキス末（ビーエイチエヌ株式会社製）を水に懸濁し、吸引ろ過して得られたろ液を、スチレン－ジビニルベンゼン系合成吸着剤（ダイヤイオンHP20、三菱化学株式会社製）に吸着させた。当該合成吸着剤を水で洗浄し、50％メタノール水溶液を用いて溶出した。得られた溶出液をSephadex LH-20（GEヘルスケア株式会社製）に直接吸着させ、50％メタノール水溶液、100%メタノールにより順次洗浄後、70％アセトン水溶液を用いて溶出してプロシアニジン濃縮画分を得た。 Example 2:
A red pepper extract powder (manufactured by BN Co., Ltd.) was suspended in water, and the filtrate obtained by suction filtration was adsorbed onto a styrene-divinylbenzene synthetic adsorbent (Diaion HP20, manufactured by Mitsubishi Chemical Corporation). The synthetic adsorbent was washed with water and eluted with 50% aqueous methanol. The obtained eluate was directly adsorbed on Sephadex LH-20 (manufactured by GE Healthcare), washed sequentially with 50% methanol aqueous solution and 100% methanol, and eluted with 70% acetone aqueous solution to concentrate procyanidin-enriched fraction. Got.
実施例１及び２の抽出方法によって得られたプロシアニジンの収量とα-アミラーゼ阻害活性を表１に示す。
Table 1 shows the yield of procyanidins obtained by the extraction methods of Examples 1 and 2 and the α-amylase inhibitory activity.
表１より、本発明に係る方法でアカショウマから精製されたプロシアニジンのα-アミラーゼ阻害活性は非常に高く、食後過血糖改善剤として糖尿病患者に処方されるアカルボースの当該活性（IC50=10.2～83.33μg/mL）よりも重量濃度比において高いことが明らかとなった。
これまで、ブドウの皮（IC50=12.5～27.4μg/mL、非特許文献２）、ツルドクダミの塊茎（IC50=2.9±0.15μg/mL、非特許文献３）、柿の葉（IC50=約50μg/mL、非特許文献４）、シナモン（IC50=4.8～1,320μg/mL、非特許文献５）、アカシア樹皮（IC50=38μg/mL、Kusano R., et al, J. Nat. Prod., 74:119-128, 2011）、サポジラ（IC50=4.2μg/mL、Wang H., et al, J. Agric. Food Chem., 60:3098-3104, 2012）等、さまざまな植物から精製されたプロシアニジンについてα-アミラーゼ阻害活性が報告されているが、本発明で見出されたアカショウマ由来プロシアニジンの当該活性は、これらのものと比較しても傑出したものである。
よって、アカショウマ由来プロシアニジンは、既知の薬剤及び植物由来プロシアニジンと比べて、非常に高いα-アミラーゼ阻害活性を有することが示された。 From Table 1, procyanidin purified from red pepper by the method of the present invention has very high α-amylase inhibitory activity, and the activity of acarbose prescribed to diabetic patients as a postprandial hyperglycemia improving agent (IC 50 = 10.2 to 83.33). It was revealed that the weight concentration ratio was higher than that of μg / mL.
So far, grape skin (IC 50 = 12.5-27.4 μg / mL, Non-Patent Document 2), tuber of tubers (IC 50 = 2.9 ± 0.15 μg / mL, Non-Patent Document 3), bamboo leaves (IC 50 = About 50 μg / mL, Non-Patent Document 4), Cinnamon (IC 50 = 4.8 to 1,320 μg / mL, Non-Patent Document 5), Acacia bark (IC 50 = 38 μg / mL, Kusano R., et al, J. Nat. Prod., 74: 119-128, 2011), sapodilla (IC 50 = 4.2 μg / mL, Wang H., et al, J. Agric. Food Chem., 60: 3098-3104, 2012) Α-Amylase inhibitory activity has been reported for procyanidins purified from the above, but the activity of procyanidins derived from red pepper found in the present invention is outstanding even in comparison with these.
Therefore, it was shown that red pepper-derived procyanidins have very high α-amylase inhibitory activity as compared with known drugs and plant-derived procyanidins.
これまで、ブドウの皮（IC50=12.5～27.4μg/mL、非特許文献２）、ツルドクダミの塊茎（IC50=2.9±0.15μg/mL、非特許文献３）、柿の葉（IC50=約50μg/mL、非特許文献４）、シナモン（IC50=4.8～1,320μg/mL、非特許文献５）、アカシア樹皮（IC50=38μg/mL、Kusano R., et al, J. Nat. Prod., 74:119-128, 2011）、サポジラ（IC50=4.2μg/mL、Wang H., et al, J. Agric. Food Chem., 60:3098-3104, 2012）等、さまざまな植物から精製されたプロシアニジンについてα-アミラーゼ阻害活性が報告されているが、本発明で見出されたアカショウマ由来プロシアニジンの当該活性は、これらのものと比較しても傑出したものである。
よって、アカショウマ由来プロシアニジンは、既知の薬剤及び植物由来プロシアニジンと比べて、非常に高いα-アミラーゼ阻害活性を有することが示された。 From Table 1, procyanidin purified from red pepper by the method of the present invention has very high α-amylase inhibitory activity, and the activity of acarbose prescribed to diabetic patients as a postprandial hyperglycemia improving agent (IC 50 = 10.2 to 83.33). It was revealed that the weight concentration ratio was higher than that of μg / mL.
So far, grape skin (IC 50 = 12.5-27.4 μg / mL, Non-Patent Document 2), tuber of tubers (IC 50 = 2.9 ± 0.15 μg / mL, Non-Patent Document 3), bamboo leaves (IC 50 = About 50 μg / mL, Non-Patent Document 4), Cinnamon (IC 50 = 4.8 to 1,320 μg / mL, Non-Patent Document 5), Acacia bark (IC 50 = 38 μg / mL, Kusano R., et al, J. Nat. Prod., 74: 119-128, 2011), sapodilla (IC 50 = 4.2 μg / mL, Wang H., et al, J. Agric. Food Chem., 60: 3098-3104, 2012) Α-Amylase inhibitory activity has been reported for procyanidins purified from the above, but the activity of procyanidins derived from red pepper found in the present invention is outstanding even in comparison with these.
Therefore, it was shown that red pepper-derived procyanidins have very high α-amylase inhibitory activity as compared with known drugs and plant-derived procyanidins.
実施例３：新規プロシアニジンの構成単位の解析
前記アカショウマ由来プロシアニジンの基本骨格を構成する単位の種類とその比率を明らかにするために、チオール分解を行い、分解産物をNMRを用いて解析した。
実施例２で得られたプロシアニジン（濃縮画分）2.5mgを0.1M塩酸／メタノール溶液200μLに溶解し、ベンジルメルカプタン5μLを加えて混合した。当該混合溶液を、40℃で90分間加熱して反応させた。当該反応液を濃縮乾固し、メタノールに再溶解させた後、下記条件下での逆相HPLCに供して分析した。
なお、ガロカテキン、エピガロカテキンは280nmの吸光度が小さいので、検出は270nmで行った。等モル濃度のエピカテキン及びエピガロカテキン標品を解析したところ、270nm（検出）のピーク面積比がエピカテキン：エピガロカテキン＝1.57:1.00であったので、吸光度からモル比を計算する際には、ガロカテキン及びエピガロカテキンの実測値（ピーク面積）に1.57を乗じて補正した。結果を図３に示す。 Example 3: Analysis of constituent units of novel procyanidins In order to clarify the types and ratios of units constituting the basic skeleton of the aforementioned procyanidins derived from red pepper, thiol decomposition was performed and the decomposition products were analyzed using NMR.
The procyanidin (concentrated fraction) 2.5 mg obtained in Example 2 was dissolved in 200 μL of 0.1 M hydrochloric acid / methanol solution, and 5 μL of benzyl mercaptan was added and mixed. The mixed solution was reacted by heating at 40 ° C. for 90 minutes. The reaction solution was concentrated to dryness, redissolved in methanol, and then subjected to reverse phase HPLC analysis under the following conditions.
Since gallocatechin and epigallocatechin have a low absorbance at 280 nm, detection was performed at 270 nm. When analyzing equimolar concentrations of epicatechin and epigallocatechin samples, the peak area ratio at 270 nm (detection) was epicatechin: epigallocatechin = 1.57: 1.00. When calculating the molar ratio from the absorbance, Was corrected by multiplying the measured value (peak area) of gallocatechin and epigallocatechin by 1.57. The results are shown in FIG.
前記アカショウマ由来プロシアニジンの基本骨格を構成する単位の種類とその比率を明らかにするために、チオール分解を行い、分解産物をNMRを用いて解析した。
実施例２で得られたプロシアニジン（濃縮画分）2.5mgを0.1M塩酸／メタノール溶液200μLに溶解し、ベンジルメルカプタン5μLを加えて混合した。当該混合溶液を、40℃で90分間加熱して反応させた。当該反応液を濃縮乾固し、メタノールに再溶解させた後、下記条件下での逆相HPLCに供して分析した。
なお、ガロカテキン、エピガロカテキンは280nmの吸光度が小さいので、検出は270nmで行った。等モル濃度のエピカテキン及びエピガロカテキン標品を解析したところ、270nm（検出）のピーク面積比がエピカテキン：エピガロカテキン＝1.57:1.00であったので、吸光度からモル比を計算する際には、ガロカテキン及びエピガロカテキンの実測値（ピーク面積）に1.57を乗じて補正した。結果を図３に示す。 Example 3: Analysis of constituent units of novel procyanidins In order to clarify the types and ratios of units constituting the basic skeleton of the aforementioned procyanidins derived from red pepper, thiol decomposition was performed and the decomposition products were analyzed using NMR.
The procyanidin (concentrated fraction) 2.5 mg obtained in Example 2 was dissolved in 200 μL of 0.1 M hydrochloric acid / methanol solution, and 5 μL of benzyl mercaptan was added and mixed. The mixed solution was reacted by heating at 40 ° C. for 90 minutes. The reaction solution was concentrated to dryness, redissolved in methanol, and then subjected to reverse phase HPLC analysis under the following conditions.
Since gallocatechin and epigallocatechin have a low absorbance at 280 nm, detection was performed at 270 nm. When analyzing equimolar concentrations of epicatechin and epigallocatechin samples, the peak area ratio at 270 nm (detection) was epicatechin: epigallocatechin = 1.57: 1.00. When calculating the molar ratio from the absorbance, Was corrected by multiplying the measured value (peak area) of gallocatechin and epigallocatechin by 1.57. The results are shown in FIG.
＜逆相HPLCの条件＞
カラム：InertSustain C18 HP 3μm
（直径4.6mm×長さ150mm、ジーエルサイエンス株式会社製）
移動相：
０～６０分間：0.1% トリフルオロ酢酸（TFA）、20% メタノール水溶液
－0.1% TFA、95% メタノール水溶液のグラジエント溶出
６０分後～：0.1% TFA、95% メタノール水溶液
流速：1.0mL/min
検出：UV270nm <Reverse phase HPLC conditions>
Column: InertSustain C18 HP 3μm
(Diameter 4.6mm x Length 150mm, manufactured by GL Sciences Inc.)
Mobile phase:
0-60 minutes: 0.1% trifluoroacetic acid (TFA), 20% aqueous methanol solution -0.1% TFA, 95% aqueousmethanol gradient elution 60 minutes later: 0.1% TFA, 95% aqueous methanol flow rate: 1.0 mL / min
Detection: UV270nm
カラム：InertSustain C18 HP 3μm
（直径4.6mm×長さ150mm、ジーエルサイエンス株式会社製）
移動相：
０～６０分間：0.1% トリフルオロ酢酸（TFA）、20% メタノール水溶液
－0.1% TFA、95% メタノール水溶液のグラジエント溶出
６０分後～：0.1% TFA、95% メタノール水溶液
流速：1.0mL/min
検出：UV270nm <Reverse phase HPLC conditions>
Column: InertSustain C18 HP 3μm
(Diameter 4.6mm x Length 150mm, manufactured by GL Sciences Inc.)
Mobile phase:
0-60 minutes: 0.1% trifluoroacetic acid (TFA), 20% aqueous methanol solution -0.1% TFA, 95% aqueous
Detection: UV270nm
図３において複数の溶出ピークが認められ、複数の分解産物が生じたことがわかる。これらの分解産物に対し、NMRを行って構造を同定した。結果を表２に示す。
In FIG. 3, a plurality of elution peaks are recognized, and it can be seen that a plurality of degradation products are generated. These decomposition products were subjected to NMR to identify the structure. The results are shown in Table 2.
表２に示されるように、アカショウマから上記方法によって精製されたプロシアニジンは、エピカテキン、カテキン、エピガロカテキン、ガロカテキンの4種類のフラバン－３－オールを基本骨格の構成単位とする新規プロシアニジンであった。また、各構成単位の比率（モル比）は、エピカテキン：カテキン：エピガロカテキン：ガロカテキンが1.0:0.56:0:76:0.13であることも明らかとなった。さらに、末端に主にカテキンを有しており、前記カテキンの比率（0.56）に占める末端カテキンの割合は0.17（伸長部分のカテキンの割合は0.39）であることも明らかとなった。なお、後述するように、当該プロシアニジンは種々の重合度のものの混合物であるため、各構成単位の比率には幅があると考えられる。
As shown in Table 2, procyanidins purified from red pepper by the above method are novel procyanidins having four flavan-3-ols of epicatechin, catechin, epigallocatechin, and gallocatechin as basic structural units. It was. Moreover, it became clear that the ratio (molar ratio) of each structural unit was 1.0: 0.56: 0: 76: 0.13 for epicatechin: catechin: epigallocatechin: gallocatechin. Furthermore, it has also been found that the terminal has mainly catechin, and the ratio of terminal catechin to the ratio of catechin (0.56) is 0.17 (the ratio of catechin in the extended portion is 0.39). As will be described later, since the procyanidin is a mixture of various degrees of polymerization, it is considered that there is a range in the ratio of each structural unit.
結果は割愛するが、本解析においてA型の結合を有したプロシアニジン分解産物は検出されず、LC-MSを行っても該当する質量イオンは見つからなかった。このことから、本発明に係る新規プロシアニジンは、B型の結合のみ有するB型プロシアニジンであることが明らかとなった。
Although the results are omitted, no procyanidin degradation product having an A-type bond was detected in this analysis, and no corresponding mass ion was found by LC-MS. From this, it became clear that the novel procyanidins according to the present invention are B-type procyanidins having only B-type bonds.
実施例４：新規プロシアニジンの分子量の解析
実施例１及び２で得られたプロシアニジンに対し、下記条件によるゲル濾過HPLCを行い、ポリスチレンを標準試料として分子量分布を解析した。ポリスチレンによる検量線を図４に、実施例１及び２で得られたプロシアニジンのHPLCチャートを図５及び図６にそれぞれ示す。また、検量線から計算された各プロシアニジンの分子量分布を表３に表す。 Example 4: Analysis of molecular weight of novel procyanidins The procyanidins obtained in Examples 1 and 2 were subjected to gel filtration HPLC under the following conditions, and molecular weight distribution was analyzed using polystyrene as a standard sample. A calibration curve using polystyrene is shown in FIG. 4, and HPLC charts of procyanidins obtained in Examples 1 and 2 are shown in FIGS. 5 and 6, respectively. Table 3 shows the molecular weight distribution of each procyanidin calculated from the calibration curve.
実施例１及び２で得られたプロシアニジンに対し、下記条件によるゲル濾過HPLCを行い、ポリスチレンを標準試料として分子量分布を解析した。ポリスチレンによる検量線を図４に、実施例１及び２で得られたプロシアニジンのHPLCチャートを図５及び図６にそれぞれ示す。また、検量線から計算された各プロシアニジンの分子量分布を表３に表す。 Example 4: Analysis of molecular weight of novel procyanidins The procyanidins obtained in Examples 1 and 2 were subjected to gel filtration HPLC under the following conditions, and molecular weight distribution was analyzed using polystyrene as a standard sample. A calibration curve using polystyrene is shown in FIG. 4, and HPLC charts of procyanidins obtained in Examples 1 and 2 are shown in FIGS. 5 and 6, respectively. Table 3 shows the molecular weight distribution of each procyanidin calculated from the calibration curve.
＜ゲル濾過HPLCの条件＞
カラム：Shodex GF-510 HQ（直径7.5mm×長さ300mm、昭和電工株式会社製）
移動相：10mM 臭化リチウム（LiBr） in N,N-ジメチルホルムアミド（DMF）
流速：0.4mL/min
測定温度：室温
試料濃度：4mg/mL
試料注入量：10μL <Gel filtration HPLC conditions>
Column: Shodex GF-510 HQ (diameter 7.5 mm x length 300 mm, Showa Denko KK)
Mobile phase: 10 mM lithium bromide (LiBr) in N, N-dimethylformamide (DMF)
Flow rate: 0.4mL / min
Measurement temperature: Room temperature Sample concentration: 4mg / mL
Sample injection volume: 10 μL
カラム：Shodex GF-510 HQ（直径7.5mm×長さ300mm、昭和電工株式会社製）
移動相：10mM 臭化リチウム（LiBr） in N,N-ジメチルホルムアミド（DMF）
流速：0.4mL/min
測定温度：室温
試料濃度：4mg/mL
試料注入量：10μL <Gel filtration HPLC conditions>
Column: Shodex GF-510 HQ (diameter 7.5 mm x length 300 mm, Showa Denko KK)
Mobile phase: 10 mM lithium bromide (LiBr) in N, N-dimethylformamide (DMF)
Flow rate: 0.4mL / min
Measurement temperature: Room temperature Sample concentration: 4mg / mL
Sample injection volume: 10 μL
表３より、アカショウマから実施例１の方法によって精製されたプロシアニジンの分子量は、中央値が約15,000、最大が約114,000、最少が約2,000であり、実施例２の方法によって精製されたプロシアニジンの分子量は、中央値が約8,300、最大が約41,300、最少が約1,000であることがわかる。当該値を重合度に換算（1ユニットを分子量300として計算）すると、概算値として、
実施例１：平均重合度が50、最大378、最少6
実施例２：平均重合度が27、最大138、最少3
となる。
よって、アカショウマに含まれるプロシアニジンは、約3～400個のフラバン－３－オールが重合した基本骨格を有し、分子量が約1,000～114,000の範囲にあるヘテロな分子の集団であることが明らかとなった。 From Table 3, the molecular weight of procyanidin purified from red pepper by the method of Example 1 has a median of about 15,000, a maximum of about 114,000, and a minimum of about 2,000. The molecular weight of procyanidin purified by the method of Example 2 It can be seen that the median is about 8,300, the maximum is about 41,300, and the minimum is about 1,000. When the value is converted into the degree of polymerization (calculated as 1 unit of molecular weight 300),
Example 1: Average degree ofpolymerization 50, maximum 378, minimum 6
Example 2: Average degree of polymerization 27, maximum 138,minimum 3
It becomes.
Therefore, it is clear that procyanidins contained in red pepper are a group of hetero molecules having a basic skeleton in which about 3 to 400 flavan-3-ols are polymerized and having a molecular weight in the range of about 1,000 to 114,000. became.
実施例１：平均重合度が50、最大378、最少6
実施例２：平均重合度が27、最大138、最少3
となる。
よって、アカショウマに含まれるプロシアニジンは、約3～400個のフラバン－３－オールが重合した基本骨格を有し、分子量が約1,000～114,000の範囲にあるヘテロな分子の集団であることが明らかとなった。 From Table 3, the molecular weight of procyanidin purified from red pepper by the method of Example 1 has a median of about 15,000, a maximum of about 114,000, and a minimum of about 2,000. The molecular weight of procyanidin purified by the method of Example 2 It can be seen that the median is about 8,300, the maximum is about 41,300, and the minimum is about 1,000. When the value is converted into the degree of polymerization (calculated as 1 unit of molecular weight 300),
Example 1: Average degree of
Example 2: Average degree of polymerization 27, maximum 138,
It becomes.
Therefore, it is clear that procyanidins contained in red pepper are a group of hetero molecules having a basic skeleton in which about 3 to 400 flavan-3-ols are polymerized and having a molecular weight in the range of about 1,000 to 114,000. became.
Claims (5)
- エピカテキン、カテキン、エピガロカテキン、及びガロカテキンが重合した基本骨格を有することを特徴とするプロシアニジン。 Procyanidins characterized by having a basic skeleton in which epicatechin, catechin, epigallocatechin, and gallocatechin are polymerized.
- 前記構成単位間の結合様式がＢ型であることを特徴とする、請求項１に記載のプロシアニジン。 The procyanidin according to claim 1, wherein the bonding mode between the structural units is B type.
- 前記基本骨格に含まれる各構成単位のモル比が、エピカテキン：カテキン：エピガロカテキン：ガロカテキン＝0.50～2.00:0.28～1.12:0.38～1.52:0.07～0.26であることを特徴とする、請求項１又は２に記載のプロシアニジン。 The molar ratio of each structural unit contained in the basic skeleton is epicatechin: catechin: epigallocatechin: gallocatechin = 0.50 to 2.00: 0.28 to 1.12: 0.38 to 1.52: 0.07 to 0.26, The procyanidin according to 1 or 2.
- 分子量が1,000－114,000であることを特徴とする、請求項１－３のいずれかに記載のプロシアニジン。 Procyanidins according to any of claims 1-3, characterized in that the molecular weight is 1,000-114,000.
- アカショウマの水及び／又は含水有機溶媒による抽出物を合成吸着剤で吸着処理し、該合成吸着剤を含水有機溶媒で溶出処理する工程を含むことを特徴とする、請求項１－４のいずれかに記載のプロシアニジンの製造方法。 The method according to any one of claims 1 to 4, further comprising a step of adsorbing an extract of water and / or water-containing organic solvent of red pepper with a synthetic adsorbent and eluting the synthetic adsorbent with a water-containing organic solvent. A method for producing procyanidins according to 1.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2017063166A JP6491685B2 (en) | 2017-03-28 | 2017-03-28 | Novel procyanidins and process for producing the same |
| JP2017-063166 | 2017-03-28 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2018179998A1 true WO2018179998A1 (en) | 2018-10-04 |
Family
ID=63675126
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/JP2018/005863 WO2018179998A1 (en) | 2017-03-28 | 2018-02-20 | Novel procyanidin and method for producing same |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JP6491685B2 (en) |
| WO (1) | WO2018179998A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114712386A (en) * | 2021-03-31 | 2022-07-08 | 贝尔克斯生技股份有限公司 | High polymeric proanthocyanidin composition and its application |
| JP7520395B2 (ja) | 2021-03-31 | 2024-07-23 | ベルクス・バイオ－ファーマシューティカル（タイワン）・コーポレーション | 高分子プロアトシアニジン組成物及びその応用 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007085868A (en) * | 2005-09-21 | 2007-04-05 | Asahi Breweries Ltd | Quantitative analysis method of polyphenol in food and drink, quantitative analyzer and planning method of food and drink |
| JP2009156813A (en) * | 2007-12-27 | 2009-07-16 | Asahi Breweries Ltd | Quantitative determination method of procyanidins |
-
2017
- 2017-03-28 JP JP2017063166A patent/JP6491685B2/en active Active
-
2018
- 2018-02-20 WO PCT/JP2018/005863 patent/WO2018179998A1/en active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007085868A (en) * | 2005-09-21 | 2007-04-05 | Asahi Breweries Ltd | Quantitative analysis method of polyphenol in food and drink, quantitative analyzer and planning method of food and drink |
| JP2009156813A (en) * | 2007-12-27 | 2009-07-16 | Asahi Breweries Ltd | Quantitative determination method of procyanidins |
Non-Patent Citations (2)
| Title |
|---|
| HAN, L. K. ET AL., NATURAL MEDICINES, vol. 60, no. 2, 2006, pages 68 - 72 * |
| KARONEN, M. ET AL., PHYTOCHEMICAL ANALYSIS, vol. 18, 2007, pages 378 - 386 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114712386A (en) * | 2021-03-31 | 2022-07-08 | 贝尔克斯生技股份有限公司 | High polymeric proanthocyanidin composition and its application |
| EP4066828A1 (en) * | 2021-03-31 | 2022-10-05 | Belx Bio-Pharmaceutical (Taiwan) Corporation | Polymeric proathocyanidin composition and application thereof |
| JP2022159123A (en) * | 2021-03-31 | 2022-10-17 | ベルクス・バイオ－ファーマシューティカル（タイワン）・コーポレーション | Polymeric proathocyanidin composition and application thereof |
| AU2022202161B2 (en) * | 2021-03-31 | 2023-07-20 | BELX Bio-Pharmaceutical (Taiwan) Corporation | Polymeric proathocyanidin composition and application thereof |
| JP7520395B2 (ja) | 2021-03-31 | 2024-07-23 | ベルクス・バイオ－ファーマシューティカル（タイワン）・コーポレーション | 高分子プロアトシアニジン組成物及びその応用 |
Also Published As
| Publication number | Publication date |
|---|---|
| JP6491685B2 (en) | 2019-03-27 |
| JP2018165252A (en) | 2018-10-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Hua et al. | Inhibition of α-glucosidase and α-amylase by flavonoid glycosides from Lu'an GuaPian tea: molecular docking and interaction mechanism | |
| Jiao et al. | Characterization of a new heteropolysaccharide from green guava and its application as an α-glucosidase inhibitor for the treatment of type II diabetes | |
| Asghari et al. | Flavonoids from Salvia chloroleuca with α-amylsae and α-glucosidase inhibitory effect | |
| Saltos et al. | Inhibitors of α-amylase and α-glucosidase from Andromachia igniaria Humb. & Bonpl. | |
| Wang et al. | Chemical constituents with antioxidant activities from litchi (Litchi chinensis Sonn.) seeds | |
| Higbee et al. | The emerging role of dark berry polyphenols in human health and nutrition | |
| de Araújo et al. | Enzymatic de-glycosylation of rutin improves its antioxidant and antiproliferative activities | |
| Li et al. | Antioxidant activity of proanthocyanidins-rich fractions from Choerospondias axillaris peels using a combination of chemical-based methods and cellular-based assay | |
| Wold et al. | Bioactive triterpenoids and water-soluble melanin from Inonotus obliquus (Chaga) with immunomodulatory activity | |
| Lakshmanasenthil et al. | Fucoidan—a novel α-amylase inhibitor from Turbinaria ornata with relevance to NIDDM therapy | |
| Bule et al. | Tannins (hydrolysable tannins, condensed tannins, phlorotannins, flavono-ellagitannins) | |
| Spínola et al. | Polyphenols of Myrica faya inhibit key enzymes linked to type II diabetes and obesity and formation of advanced glycation end-products (in vitro): Potential role in the prevention of diabetic complications | |
| Xu et al. | Isolation, purification, characterization and bioactivities of a glucan from the root of Pueraria lobata | |
| Orqueda et al. | Chemical and functional characterization of skin, pulp and seed powder from the Argentine native fruit mistol (Ziziphus mistol). Effects of phenolic fractions on key enzymes involved in metabolic syndrome and oxidative stress | |
| Fischer et al. | Determination of lignans in edible and nonedible parts of pomegranate (Punica granatum L.) and products derived therefrom, particularly focusing on the quantitation of isolariciresinol using HPLC-DAD-ESI/MS n | |
| Dang et al. | α-Glucosidase inhibitors from the leaves of Embelia ribes | |
| Xiong et al. | Phenolic content, anti-inflammatory properties, and dermal wound repair properties of industrially processed and non-processed acai from the Brazilian Amazon | |
| Zhang et al. | Flavonoids and its derivatives from Callistephus chinensis flowers and their inhibitory activities against alpha-glucosidase | |
| Jain et al. | Isolation and standardization of various phytochemical constituents from methanolic extracts of fruit rinds of Punica granatum | |
| Ighodaro et al. | FT-IR analysis of Sapium ellipticum (Hochst) pax ethanol leaf extract and its inhibitory effects on pancreatic α-amylase and intestinal α-glucosidase activities in vitro | |
| WO2017159679A1 (en) | Polysaccharide digestion inhibitor | |
| JP6491685B2 (en) | Novel procyanidins and process for producing the same | |
| Vu et al. | Flavonoids from the peels of Citrus unshiu Markov. and their inhibitory effects on RANKL-induced osteoclastogenesis through the downregulation of c-Fos signaling in vitro | |
| JP2010202558A (en) | Antiobestic agent and diabetes-ameliorating agent | |
| Meyre-Silva et al. | Phytochemical and pharmacological analysis of Bauhinia microstachya (Raddi) Macbr.(Leguminosae) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 18774801 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 18774801 Country of ref document: EP Kind code of ref document: A1 |