JP5744507B2 - α-Amylase inhibitor - Google Patents

Description

The present invention comprises an amylase inhibitor containing at least one of theasinensins and epitheaflavic acids contained in fermented tea leaves as an active ingredient, as well as diabetes prevention or The present invention relates to an oral application target used for improving blood glucose level.

Excessive calorie intake due to changes in dietary life has long been said to increase the risk of lifestyle-related diseases such as obesity, arteriosclerosis, and diabetes. Suppressing the digestion of carbohydrates such as starch and sucrose is thought to reduce the risk of obesity, including diabetes, and inhibits the action of α-amylase, a starch digestive enzyme, and sucrose, a degrading enzyme of sucrose. Research has been made to find substances to be found in food materials.
In particular, regarding tea components, epicatechin gallate, which is a green tea catechin, epigallocatechin gallate, and free theaflavin, theaflavin monogallate, and theaflavin digallate, which are polyphenol components of black tea (Patent Document 1), It is disclosed that there is a schuclase inhibitory action (Patent Document 2). In addition, it is disclosed that a composition containing theaflavin, theaflavin-3-gallate, theaflavin-3′-gallate, theaflavin-3,3′-digallate at a specific ratio has an inhibitory effect on α-amylase and α-glucosidase. (Patent Document 3). Furthermore, it has also been reported that a polyphenol composition containing a flavonol aglycone derived from Camellia sinensis and a flavonol glycoside exhibits an α-amylase inhibitory activity (Patent Document 4).

JP-A-3-133828 JP-A-5-17364 JP 2009-268420 A JP 2010-222277 A

However, there is no report that theacinensins and epiteaflavic acids known as components of oolong tea and black tea, which are fermented teas, have an α-amylase inhibitory action.

The present inventors have succeeded in synthesizing theacinensins and epitheaflavic acids contained in fermented tea, and have examined the physiological activity of these components, and found that they have an inhibitory effect on α-amylase. .

That is, the invention described in claim 1 of the present application contains at least one or more of theacinensins represented by the following formula (I) and epitheaflavic acids represented by the following formula (II) as active ingredients. wherein, Ru amylase inhibitor der except that the fermented tea extract.
Formula (I)
(R ₁ and R _{2 in} formula (I) each independently represent hydrogen (H) or a galloyl group.)

Formula (II)
(R _{1 in} formula (II) represents hydrogen (H) or a galloyl group.)
The invention according to claim 2 of the present application is the amylase inhibitor according to claim 1, wherein the theacinensins represented by the formula (I) are theasinensin A, theasinensin B and theacinensin C.
The invention according to claim 3 of the present application is the amylase inhibitor according to claim 1, wherein the epiteaflavic acids represented by the formula (II) are epiteaflavic acid and epiteaflavic acid-3-O-gallate.
The invention according to claim 4 of the present application is the amylase inhibitor according to claim 1, wherein the theasinens are theasinensin A, theasinensin B, and theasinensin C, and the epiflavic acids are epitheaflavic acid and epitheaflavic acid-3-O-gallate. is there.
Furthermore, the invention according to claim 5 of the present application is an oral application object containing the amylase inhibitor according to claims 1 to 4.
The present invention according to claim 6 is an object to be applied to the oral cavity according to claim 5 which is a food or drink.
The present invention according to claim 7 is the object to be applied to the oral cavity according to claim 5, which is a pharmaceutical product or a quasi-drug.

The present invention is an α-amylase inhibitor containing theacinensins and epitheaflavic acids as active ingredients, and is highly versatile that can be widely applied to foods and drinks, pharmaceuticals, quasi drugs, etc. It can be expected to prevent and / or improve diabetes by suppressing.

Theasinensins in the present invention can be obtained by a known organic chemical synthesis method (Patent Document 5). Moreover, it is also possible to manufacture using the enzyme in a pear fruit homogenate (nonpatent literature 1). Examples of theacinensins of the present invention include theacinensin A, theacinensin B, and theacinensin C represented by the following formula (III).
Formula (III)
JP 2010-138103 A Tetrahedron, 59, 7939-7947 (2003)

Epitheaflavic acids in the present invention can be easily synthesized by allowing tyrosinase to act on a mixed solution of catechins and gallic acid based on a known enzyme synthesis method of theaflavin (Patent Document 6), as well as epicatechin and gallic acid. It is also possible to synthesize it by allowing peroxidase to act on the mixed solution. The concentration and ratio of catechins and gallic acid, reaction temperature, reaction time, pH, enzyme amount and the like can be appropriately adjusted so that the yield is improved.
JP 2010-35548 A

Theasinensins and epitheaflavic acids obtained by the above organic chemical synthesis method and enzyme synthesis method are crudely purified products that conform to the pharmaceutically or food acceptable standards and exhibit the effects of the present invention. Further, the obtained synthetic product or extract may be appropriately combined with known separation and purification methods to increase the purity. For example, using a polar solvent such as water, hot water, alcohol, or a nonpolar solvent. Solvent extraction methods to be performed and purification means such as centrifugation, high performance liquid chromatograph, column chromatograph, etc.

The form of use of the amylase inhibitor of the present invention is not limited, but examples thereof include liquid, powder, granule, etc., and forms usually used as food or nutritional supplements, such as liquids, suspensions, powders, granules It can be used in preparations, fine granules, tablets, capsules, syrups, elixirs and spirits.

The amylase inhibitor of the present invention is still another α-amylase inhibitor, for example, catechins, theaflavins, guava polyphenols, flavonol aglycones, flavonol glycosides, sputum polyphenols, 1-deoxynojirimycin, polyphenols derived from mulberry It can also be used in combination with.

In addition, when the amylase inhibitor of the present invention is used as a pharmaceutical, it is not particularly limited as long as it can be contained in the mouth with a pharmaceutical stored in the Japanese Pharmacopoeia, and a pharmaceutically acceptable carrier for the active ingredient. Can be added to form an oral preparation. Examples of the dosage form include tablets, granules, fine granules, pills, powders, capsules, troches, chewables, liquids (drinks) and the like.

The quasi-drug is not particularly limited as long as it is a quasi-drug specified by the Minister of Health, Labor and Welfare and can be contained in the mouth. Examples thereof include oral liquids, health drinks, vitamin-containing health agents, and the like.

The food containing at least one of the amylase inhibitors of the present invention as an essential component may be in any form, for example, in a liquid form such as an aqueous solution, a turbid product, an emulsion, or a gel or paste Even a semi-solid form in the form of a solid, such as supplements such as powders, granules, capsules and tablets may be used.

Examples of foods and drinks containing at least one of the amylase inhibitors of the present invention as essential components include instant foods (immediate noodles, cup noodles, retort / cooked foods, cooking cans, microwave foods, instant miso soup / soup, soups) Canned foods, freeze-dried foods, etc.), carbonated beverages, citrus fruits (grapefruits, oranges, lemons, etc.) fruit juices, fruit juice beverages and fruit juice soft drinks, citrus fruit meat beverages and fruit juice beverages, tomatoes, peppers, celery, cucumbers, Vegetable beverages including vegetables such as carrots, potatoes, asparagus, soy milk and soy milk beverages, coffee beverages, tea beverages, powdered beverages, concentrated beverages, sports beverages, nutritional beverages, alcoholic beverages and tobacco, etc. Macaroni spaghetti, noodles, cake mix, fried flour, bread crumbs, gyoza skin Flour products, caramel candy, chewing gum, chocolate, cookies, biscuits, cake pie, snack crackers, Japanese confectionery, rice confectionery, bean confectionery, dessert confectionery, soy sauce, miso, sauces, tomato processed seasonings, Basic seasonings such as mirin, vinegars, sweeteners, flavor seasonings, cooking mixes, curry ingredients, sauces, dressings, noodle soups, spices and other complex seasonings and foods, butter, margarines , Mayonnaises, oils and fats such as vegetable oil, milk / processed milk, milk drinks, yogurt, lactic acid bacteria drinks, cheese, ice cream, prepared milk powder, milk and dairy products such as cream, frozen food, semi-cooked frozen food , Frozen foods such as cooked frozen foods, canned fish, canned fruits and pastes, fish ham and sausages, Freshly processed products, marine products such as marine delicacies, dried marine products, boiled fish, livestock canned and pasted products, canned meat, canned fruit, jams, marmalades, pickles and boiled beans, dried agricultural products, cereals ) And other processed agricultural products, baby foods, and commercial foods such as sprinkles and green tea paste.

Although the compounding quantity of the amylase inhibitor of this invention with respect to food / beverage products is not restrict | limited in particular, A compounding quantity is suitably set with the food / beverage products used as object. Generally, it is preferably 0.001 to 20% by weight in the final product, more preferably 0.005 to 10% by weight, still more preferably 0.01 to 5% by weight, Most preferably, it is 0.1 to 1% by weight.

Hereinafter, the present invention will be described in more detail based on production examples and test examples, but the present invention is not limited to these examples.

Production Example 1 Synthesis of Teasinensin A 1833 mg (4.00 mmol) of epigallocatechin gallate (EGCg; manufactured by SIGMA) was dissolved in 60 mL of water and 60 mL of McIlvine buffer (pH 5). To each of four 50 mL conical beakers, 30 mL of this solution and 170 mg of 10% palladium / carbon (about 50% water-wet product, manufactured by Aldrich) were added, and stirred at room temperature for 180 minutes in an air atmosphere. After the catalyst was filtered off, 463 mg (3.00 mmol) of dithiothreitol was added to the filtrate and stirred at room temperature for 45 minutes. The reaction solution was adjusted to pH 6 by adding 1M aqueous sodium hydroxide solution. The reaction solution was extracted 5 times with 50 mL of ethyl acetate, and the ethyl acetate layer was dehydrated with sodium sulfate. After sodium sulfate was filtered off, the filtrate was concentrated to dryness on a rotary evaporator. The ethyl acetate fraction 1552 mg was dissolved in methanol (5 mL), and then subjected to Toyopearl HW-40S column chromatography (manufactured by Tosoh Corporation; 40 mm ID × 450 mm, mobile phase; methanol, flow rate 15 mL / min) to obtain crude theasinensin 374 mg of A was obtained. The obtained crude theasinensin A was dissolved in 15 mL of 5% acetonitrile, and then MC eye gel CHP55Y column chromatography (Mitsubishi Chemical Corporation; 20 mm ID × 300 mm, mobile phase; water: acetonitrile: formic acid = 870: 130: 1 and a flow rate of 7.5 mL / min) to obtain 286 mg (312 μmol, yield 15.6%) of theacinensin A as an off-white powder. The yield of theacinensin A was calculated by the following formula: Yield (%) = {(number of moles of obtained theacinensin A) / (number of moles of raw material catechins × 0.5)} × 100.

Production Example 2 Synthesis of Teasinensin B Epigallocatechin (EGC; manufactured by SIGMA) 613 mg (2.00 mmol) and epigallocatechin gallate (EGCg; manufactured by SIGMA) 917 mg (2.00 mmol) in 60 mL of water and McIlvaine buffer ( pH 5) Dissolved in 60 mL. To each of four 50 mL conical beakers, 30 mL of this solution and 250 mg of 10% palladium / carbon (about 50% water wet product, manufactured by Aldrich) were added, and the mixture was stirred at room temperature for 210 minutes in an air atmosphere. After the catalyst was filtered off, 860 mg (3.00 mmol) of tris (2-carboxyethyl) phosphine hydrochloride was added to the filtrate, and the mixture was stirred at room temperature for 10 minutes. The reaction solution was directly subjected to Diaion HP-20 column chromatography (manufactured by Mitsubishi Chemical Corporation; 30 mm ID × 140 mm), washed with 350 mL of water, and eluted with 500 mL of 40% methanol. After dissolving 1172 mg of the 40% methanol fraction concentrated and dried with methanol (5 mL), Toyopearl HW-40S column chromatography (manufactured by Tosoh Corporation; 40 mm ID × 450 mm, mobile phase; methanol, flow rate 15 mL / min) 294 mg of crude theasinensin B was obtained. The obtained crude theasinensin B was dissolved in 7.5 mL of 5% acetonitrile, and then MC eye gel CHP55Y column chromatography (manufactured by Mitsubishi Chemical Corporation; 20 mm ID × 300 mm, mobile phase; water: acetonitrile: formic acid = 900: 100: 1, flow rate 7.5 mL / min) to obtain 188 mg (247 μmol, 12.3% yield) of theasinensin B as an off-white powder. The yield of theacinensin B was calculated by the following formula: Yield (%) = {(number of moles of obtained theacinensin B) / (number of moles of raw material catechins × 0.5)} × 100.

Production Example 3: Synthesis of theasinensin C 1225 mg (4.00 mmol) of epigallocatechin (EGC; manufactured by SIGMA) was dissolved in 60 mL of water and 60 mL of McIlvine buffer (pH 5). To each of four 50 mL conical beakers, 30 mL of this solution and 170 mg of 10% palladium / carbon (about 50% water wet product, manufactured by Aldrich) were added, and the mixture was stirred at room temperature for 240 minutes in an air atmosphere. After the catalyst was filtered off, 860 mg (3.00 mmol) of tris (2-carboxyethyl) phosphine hydrochloride was added to the filtrate, and the mixture was stirred at room temperature for 10 minutes. The reaction solution was directly subjected to MC eye gel CHP55Y column chromatography (manufactured by Mitsubishi Chemical Corporation; 20 mm ID × 300 mm, mobile phase water: methanol: formic acid = 950: 50: 1, flow rate 7.5 mL / min), 369 mg of theasinensin C (604 μmol, yield 30.2%) was obtained as an off-white powder. The yield of theacinensin C was calculated by the following formula: Yield (%) = {(number of moles of obtained theacinensin C) / (number of moles of raw material catechins × 0.5)} × 100.

Production Example 4: Synthesis of epitheaflavic acid-3-O-gallate Epicatechin gallate (ECg; manufactured by SIGMA) 451 mg (1.02 mmol) and gallic acid (manufactured by SIGMA) 1.42 g (8.37 mmol) in 500 mL Dissolved in MacIlvaine buffer (pH 5.0). 100 mL of this solution was dispensed into five 1 L Erlenmeyer flasks, and 100 mL of water was added to each. To this solution, 200,000 U of tyrosinase manufactured by SIGMA was added per Erlenmeyer flask and incubated at 40 ° C. for 10 minutes at 100 rpm. The enzyme reaction solution was extracted by adding an equal amount of ethyl acetate. The ethyl acetate extraction was repeated twice more. The collected organic layer was distilled off to obtain 1.64 g of a fraction containing 15.3% of the desired epitheaflavic acid-3-O-gallate with an area value purity of 280 nm. The obtained fraction was dissolved in 30 mL of 95% methanol, and then subjected to Toyopearl HW-40S column chromatography (manufactured by Tosoh Corporation; 40 mm ID × 450 mm, 565 mL, mobile phase: 95% methanol, flow rate 10 mL / min). The whole amount was applied and eluted with methanol. After further concentration to about 5 mL, the entire amount was dissolved in 120% acetonitrile. The total amount was applied to MC eye gel CHP55Y column chromatography (Mitsubishi Chemical Corporation; 20 mm ID × 290 mm, 91 mL, mobile phase; 12% → 22% acetonitrile gradient, flow rate 10 mL / min), and the area value at 280 nm. 52.5 mg (90.5 μmol) of epitheaflavic acid-3-O-gallate having a purity of 99% or more was obtained. The yield based on ECg used was 8.86%. In addition, the yield of epiteaflavic acid-3-O-gallate is the following formula, yield (%) = {(number of moles of obtained epiteaflavic acid-3-O-gallate) / (mol of ECg of raw material) Number)} × 100.

Production Example 5: Synthesis of Epiteaflavic Acid Two 100 mL Erlenmeyer flasks containing 290 mg (1.00 mmol) of epicatechin (EC; manufactured by SIGMA) and 180 mg (1.00 mmol) of gallic acid (manufactured by SIGMA) were prepared. After adding 5 mL of acetone to this, 0.1M
Sodium acetate buffer (pH 4.5) 45 mL was added and dissolved. To each of these solutions, 4500 U of Peroxidase Type IV-A manufactured by SIGMA was added and stirred at room temperature. Next, about 1.1 mL of 3% hydrogen peroxide solution was slowly dropped and reacted, and then 3 mL of 1M hydrochloric acid was added to stop the reaction. The reaction solution was all applied to a Diaion HP-20 column (Mitsubishi Chemical Corporation; 50 mm ID × 150 mm, 290 mL) equilibrated with water, washed with 30% aqueous methanol solution, and eluted with 100% methanol. The 100% methanol-eluted fraction was concentrated with an evaporator. When the liquid volume became 1/4 to 1/5, the concentration was stopped and placed in a refrigerator to stand overnight for crystallization. The recovered crystals were further dissolved in methanol and then subjected to Toyopearl HW-40S column chromatography (manufactured by Tosoh Corporation, 25 mm ID × 270 mm, 130 mL, mobile phase: methanol) to obtain the desired fraction. Water was added to the target fraction, and the mixture was concentrated with an evaporator. When the liquid volume became 1/4 to 1/5, the concentration was stopped, and the mixture was placed in a refrigerator and allowed to stand overnight for crystallization. 141 mg (329 μmol) of epitheaflavic acid having an area purity of 280 nm of 99% or more was obtained. The yield was 32.9%. The yield of epitheaflavic acid was calculated by the following formula, yield (%) = {(number of moles of obtained epiteaflavic acid) / (number of moles of starting EC)} × 100.

Test Example: α-Amylase Inhibitory Activity Measurement Porcine pancreatic α-amylase (Type VI-B, manufactured by SIGMA) was dissolved in a 0.01 mM calcium chloride (CaCl ₂ ) solution to obtain an α-amylase enzyme solution. The enzyme reaction was carried out using an amylase assay kit manufactured by Kanto Chemical Co., Inc .: Shikari Liquid-NAMY (substrate: 2-chloro-4-nitrophenyl-4-O-β-D-galactopyranosyl maltoside (Gal-G2- CNP)) and followed its manual. That is, 50 μL of an α-amylase enzyme solution (3 units / ml) and 50 μL of a sample solution (prepared with the α-amylase inhibitor of the present invention, 30% methanol) and R1 solution [50 mM MES (2-morpholinoethanesulfonic acid) pH 6.0, 37 ° C.), 140 mM KSCN, 5 mM CaCl ₂ , 300 mM NaCl] 750 μL, R2 solution [50 mM MES (pH 6.0, 37 ° C.), 140 mM KSCN, 5 mM CaCl ₂ , 300 mM NaCl, 10.6 mM Gal-G2 -CNP] 250 μL was added and allowed to react at 37 ° C. for 5 minutes, and the reaction was stopped by incubation in a boiling water bath for 5 minutes. The amount of 2-chloro-4-nitrophenol (CNP) produced in the reaction solution was measured with a high performance liquid chromatograph (HPLC) apparatus. The HPLC analysis conditions were a Mightysil RP-18 GP column (manufactured by Kanto Chemical Co., Inc .; 4.6 mm ID × 150 mm), eluted with 40% acetonitrile containing 0.05% phosphoric acid, and a wavelength of 317 nm. Detected with.
The α-amylase inhibitory activity was determined from the following formula. All sample solutions were prepared with 30% methanol, and 30% methanol was used as a control. As a comparative sample, EGCg (manufactured by Mitsui Norin Co., Ltd.) was used.
As each blank, a 0.01 M CaCl ₂ solution was used instead of the α-amylase enzyme solution.
Inhibition rate (%) = [(A−B) − (C−D)] / (A−B) × 100
However, A: Area value of control solution (α-amylase solution + 30% methanol solution + R1 + R2) B: Area value of control solution blank (using 0.01 M CaCl ₂ solution instead of α-amylase solution of A) C: Sample solution Area value D of (α-amylase solution + inventive inhibitor (30% methanol solution) + R1 + R2) D: area value of sample solution blank (using 0.01 M CaCl ₂ solution instead of α-amylase solution of C) From the results of the inhibitory activity test, the 50% inhibitory concentration (IC ₅₀ value) for α-amylase of each sample was determined.

From the results in Table 1, it was found that epitheaflavic acid has a very strong α-amylase inhibitory action. A strong inhibitory action was also confirmed on epiteaflavic acid-3-O-gallate. Α-Amylase inhibitory activity was also observed in theasinensin A, theasinensin B, and theasinensin C. Theasinensin B has an inhibitory activity equivalent to that of EGCg, and it was found that theasinensin A and theasinensin C inhibit α-amylase more strongly than those.

Production example: Candy sugar: 33% by weight, starch syrup: 66% by weight, citric acid: 0.67% by weight, fragrance: 0.16% by weight, colorant: 0.07% by weight and theasinensin obtained in Production Example 1 A: 0.10% by weight was prepared by a conventional method using a candy formulation, and a candy containing theacinensin A was obtained.

Production Example: Theasinensin B obtained in Production Example 2 for sports beverages: 0.2 parts by weight, vitamin B1 hydrochloride: 0.0002 parts by weight, vitamin B2: 0.0001 parts by weight, vitamin C: 0.005 parts by weight, Ion exchange into components of niacin: 0.0004 parts by weight, Ca pantothenate: 0.0001 parts by weight, ammonium iron citrate: 0.006 parts by weight, citric acid: 0.05 parts by weight and fructose: 1.25 parts by weight Water was added to make the total amount 200 parts by weight, and a sports drink containing theacinensin B was prepared.

Production Example: Chewable Tablets Teasinensin C 0.6 parts by weight, xylitol 33.7 parts by weight, mannitol 30.6 parts by weight, microcrystalline cellulose 6.1 parts by weight, flavoring 14.1 parts by weight, stearic acid 4.3 parts by weight Part, 0.6 parts by weight of talc, and 9.8 parts by weight of sorbitol were compressed by a tablet press to obtain tablets containing theacinensin C.

Production example: Soft drink water fructose glucose sugar 13 parts by weight, grapefruit straight fruit juice 2.0 parts by weight, citric acid 0.3 parts by weight, vitamin C 0.01 parts by weight, sodium citrate 0.02 parts by weight, flavor (grapefruit Like a fragrance) 0.1 parts by weight and 0.1 parts by weight of epiteaflavic acid-3-O-gallate obtained in Production Example 4 were dissolved by adding water to prepare 100 parts by weight of a beverage. This was dispensed into a glass bottle container and sterilized by a conventional method to obtain a soft drink containing epiteaflavic acid-3-O-gallate.

Production example: tablet confectionery sugar 87 parts by weight, lactose 2 parts by weight, vitamin C 3 parts by weight, citric acid powder 1.5 parts by weight, gelatin 1% aqueous solution 4 parts by weight, epitheaflavic acid obtained in Production Example 5 1 part by weight 1 part by weight, 1 part by weight of sucrose fatty acid ester and 0.5 part by weight of powdered lemon flavor were uniformly mixed, and 2 tablets having a diameter of 15 mmφ were tableted by a conventional method to obtain a tablet confection containing epitheaflavic acid.

The amylase inhibitor of the present invention can be advantageously used in industries such as health foods, dietary supplements, foods for specified health use, foods for ingredient preparation, as health-oriented foods, as well as pharmaceuticals.

Claims (7)

Inhibiting amylase other than fermented tea extract, comprising at least one type of theacinensins represented by formula (I) and epitheaflavic acids represented by formula (II) as active ingredients Agent.
Formula (I)
(R ₁ and R _{2 in} formula (I) each independently represent hydrogen (H) or a galloyl group.)

Formula (II)
(R _{1 in} formula (II) represents hydrogen (H) or a galloyl group.) 2. The amylase inhibitor according to claim 1, wherein the theasinensins represented by the formula (I) are theasinensin A, theasinensin B and theasinensin C. 2. The amylase inhibitor according to claim 1, wherein the epitheaflavic acids represented by the formula (II) are epiteaflavic acid and epiteaflavic acid-3-O-gallate. The amylase inhibitor according to claim 1, wherein the theasinens are theasinensin A, theasinensin B, and theasinensin C, and the epitheaflavic acids are epitheaflavic acid and epitheaflavinic acid-3-O-gallate. An oral application object containing the amylase inhibitor according to claim 1. The object to be applied to the oral cavity according to claim 5, which is a food or drink. The oral application object according to claim 5, which is a pharmaceutical product or a quasi-drug.