JP2012184208A - Sucrase inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new blood glucose level increase suppressor having sucrase inhibitory action.SOLUTION: There are provided new sucrase inhibitors (except fermented tea extracts) containing, as an active ingredient, epitheaflavic acid-3-O-gallate contained in fermented tea, and having wide versatility widely applicable to foods and drinks, medicines, quasi drugs, health foods, dietary supplements and the like. Further, based on suppression of blood glucose level increase after meals, it is expectable to involve diabetes prevention and/or improvement.

Description

The present invention includes a schuclase inhibitor containing epitheaflavic acid-3-O-gallate contained in fermented tea leaves as an active ingredient, and the prevention or prevention of diabetes. 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). Patent Document 5 discloses that a benzotropolone ring-containing compound has an α-glycosidase inhibitory activity.

JP-A-3-133828 JP-A-5-17364 JP 2009-268420 A JP 2010-222277 A WO / 2010/134595

However, there is no report that epitheaflavic acid-3-O-gallate, which is known as a component of oolong tea or black tea, which is a fermented tea, has an inhibitory effect on sucrose.

The inventors of the present invention have succeeded in synthesizing epiteaflavinic acid-3-O-gallate contained in fermented tea, and have examined the physiological activity of these components, and found that they exhibit an inhibitory effect on schuclase. It was.

That is, the invention described in claim 1 of the present application is a schuclase inhibitor characterized by containing epitheaflavic acid-3-O-gallate represented by the following formula (I) as an active ingredient (however, fermentation Excluding tea extract).
Formula (I)
The invention according to claim 2 of the present application is an object to be applied to the oral cavity containing the schulase inhibitor according to claim 1.
The present invention according to claim 3 is an object to be applied to the oral cavity according to claim 2 which is a food or drink.
The present invention according to claim 4 is the oral application object according to claim 2, which is a pharmaceutical product or a quasi-drug.

The present invention is a schuclase inhibitor containing epitheaflavic acid-3-O-gallate as an active ingredient, and is a versatile one that can be widely applied to food and drink, pharmaceuticals, quasi drugs, etc. By suppressing the increase, it can be expected to lead to prevention and / or improvement of diabetes.

Epitheaflavic acid-3-O-gallate according to the present invention can be synthesized easily by allowing tyrosinase to act on a mixed solution of catechins and gallic acid based on a known theaflavin enzymatic synthesis method (Patent Document 6). It can also be synthesized by allowing peroxidase to act on a mixed solution of epicatechin gallate and gallic acid. 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

In addition, theacinensins that can be expected to have a sucrose inhibitory effect can be obtained by a known organic chemical synthesis method (Patent Document 7). Moreover, it is also possible to manufacture using the enzyme in a pear fruit homogenate (nonpatent literature 1). Theacinensins include theacinensin A, theacinensin B, and theacinensin C represented by the following formula (II).

Formula (II)
JP 2010-138103 A Tetrahedron, 59, 7939-7947 (2003)

Epitheaflavic acid-3-O-gallate obtained by the above organic chemical synthesis method or enzyme synthesis method is not limited as long as it conforms to the standards acceptable on pharmaceuticals or foods and exhibits the effects of the present invention. It may be a purified product, and the resulting synthesized product or extract may be combined with known separation and purification methods as appropriate to increase the purity. For example, polar solvents such as water, hot water, alcohol, etc., or nonpolar solvents And a solvent extraction method performed using the above, and purification means such as centrifugation, high performance liquid chromatography, column chromatography and the like.

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

The schuclase inhibitor of the present invention further includes other schulase inhibitors, for example, catechins, theaflavins, guava polyphenols, flavonol aglycones, flavonol glycosides, sputum polyphenols, 1-deoxynojirimycin, polyphenols derived from mulberry, etc. It can also be used in combination.

In addition, when the schuclase inhibitor of the present invention is used as a medicine, it is not particularly limited as long as it can be contained in the mouth with a medicine contained in the Japanese Pharmacopoeia, and a pharmaceutically acceptable carrier for the above 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 the schuclase inhibitor of the present invention as an essential component may be in any form, for example, a liquid form such as an aqueous solution, a turbid substance, an emulsion, or a semi-solid gel or paste. Even if it is a shape form, solid form, such as supplements, such as powder, a granule, a capsule, and a tablet, may be sufficient.

Foods and drinks containing the schulase inhibitor of the present invention as an essential component include, for example, instant foods (instant noodles, cup noodles, retort / cooked foods, canned foods, microwave foods, instant miso soup / soup, canned soups, freeze-dried foods, etc. Food, etc.), carbonated beverages, citrus fruits (grapefruits, oranges, lemons, etc.), juices, fruit juices, soft drinks with fruit juices, citrus fruit beverages, fruit drinks with fruits, tomatoes, peppers, celery, cucumbers, carrots, potatoes, Vegetable drinks including vegetables such as asparagus, soy milk and soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks, sports drinks, nutrition drinks, liquor drinks and luxury goods such as alcoholic drinks and tobacco, macaroni spaghetti, Flour products such as noodles, cake mix, deep-fried flour, bread crumbs, gyoza skin, 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, mirin, Basic seasonings such as 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, cooked Frozen foods such as frozen foods, canned fish, canned fruits and pastes, fish ham and sausages, fish paste products, Seafood delicacies, marine products such as dried fish, boiled fish, canned livestock cans / pastes, canned meat, canned fruits, jams / marmalades, pickles / boiled beans, dried agricultural products, cereals (grain processed products) Agricultural processed products, baby food, and commercial foods such as sprinkles and tea pickles.

Although the compounding quantity of the schuclase inhibitor of this invention with respect to food-drinks is not restrict | limited in particular, A compounding quantity is suitably set with the food-drinks used as object. 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, and 0.1 to 1%. Most preferred is wt%.

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 epitheaflavinic 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 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 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. 1172 mg of the 40% methanol fraction concentrated and dried was dissolved in methanol (5 mL), and 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.

Test Example: Measurement of Sucralase Inhibitory Activity Preparation of Sucralase: Rat intestinal acetone powder (manufactured by SIGMA) was dissolved in 0.1 mM maleate buffer solution (pH 6.0) and centrifuged twice (4 ° C., 3000 rpm, 10 minutes) ) Was used as the sucrose enzyme solution (0.012 units / mL).
Preparation of substrate solution: Prepared by dissolving sucrose (manufactured by Wako Pure Chemical Industries, Ltd.) in water to a concentration of 100 mM.
For the sucrose enzyme reaction, 50 μL of a sample solution and 100 μL of a substrate solution (substrate: 100 mM sucrose solution) are added to 50 μL of an enzyme solution (0.012 units / mL), and reacted at 37 ° C. for 20 minutes, and then 250 μL of acetonitrile is added. The reaction was stopped. Further, 300 μL of water and 250 μL of acetonitrile were added for dilution, and the amount of glucose in the centrifugal supernatant was measured by HPLC-ELSD. HPLC analysis conditions were as follows: UK-Amino column (2.0 ID × 250 mm, manufactured by Intact Co.) was used to elute with 83% acetonitrile and ELSD (Waters Evaporative Light Scattering Detector ELSD2420) was used. Detected.
The schuclase inhibitory activity was determined from the following formula. The sample solution was prepared with a maleate buffer solution containing 5% ethanol, and a maleate buffer solution containing 5% ethanol was used as a control. Moreover, distilled water was used instead of the substrate solution as each blank.
Inhibition rate (%) = [(A−B) − (C−D)] / (A−B) × 100
However, A: Area value of the control solution (schuclase enzyme solution + maleic acid buffer solution containing 5% ethanol + substrate solution) B: Area value of the control solution blank (distilled water is used instead of the A sucrose enzyme solution) C: Sample Area value D of solution (schuclase enzyme solution + inventive inhibitor (maleic acid buffer solution containing 5% ethanol) + substrate) D: Area value of sample solution blank (use distilled water instead of C sucrose enzyme solution) From the results of the sucrose inhibitory activity, the 50% inhibitory concentration (IC ₅₀ value) for sucrose in each sample was determined.

The 50% inhibitory concentration (IC ₅₀ value) of epiteaflavic acid-3-O-gallate against sucrose was 668 μM, confirming the effect of inhibiting sucrose. It also examined similarly sucrase inhibitory effect with regard Teashinenshin B, Epi theaflavin acid -3-O-similar sucrase inhibitory effect as gallate was confirmed _{(IC 50} value: 826μM).

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 the epi obtained in Production Example 1 Theaflavic acid-3-O-gallate: 0.10% by weight was prepared by a conventional method using a candy formulation to obtain a candy containing epiteaflavic acid-3-O-gallate.

Production Example: Epiteaflavic acid-3-O-gallate obtained in Production Example 1 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, 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 Ion exchange water was added to the component by weight to make the total amount 200 parts by weight, and a sports beverage containing epiteaflavic acid-3-O-gallate was prepared.

Production example: 0.6 parts by weight of epiteaflavic acid-3-O-gallate obtained in Chewable tablet production example 1, 33.7 parts by weight of xylitol, 30.6 parts by weight of mannitol, 6.1 parts by weight of microcrystalline cellulose , 14.1 parts by weight of flavoring agent, 4.3 parts by weight of stearic acid, 0.6 parts by weight of talc, and 9.8 parts by weight of sorbitol were compressed by a tablet press, and epitheaflavic acid-3-O- Tablets containing gallate were obtained.

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 fragrance) 0.1 parts by weight and 0.1 part by weight of epiteaflavic acid-3-O-gallate obtained in Production Example 1 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: 87 parts by weight of tablet confectionery powder sugar, 2 parts by weight of lactose, 3 parts by weight of vitamin C, 1.5 parts by weight of citric acid powder, 4 parts by weight of gelatin 1% aqueous solution, epitheaflavic acid-3 obtained in Production Example 1 -1 part by weight of O-gallate, 1 part by weight of sucrose fatty acid ester, 0.5 part by weight of powdered lemon flavor were mixed uniformly, and 2 tablets having a diameter of 15 mmφ were tableted by a conventional method to produce epitheaflavic acid-3- Tablet confectionery containing O-gallate was obtained.

The schuclase inhibitor of the present invention can be advantageously used in industries such as health foods, nutritional supplements, foods for specified health use, foods for preparing ingredients, as health-oriented foods as well as pharmaceuticals.

Claims (4)

A schulase inhibitor (excluding a fermented tea extract) characterized by containing epitheaflavic acid-3-O-gallate represented by the formula (I) as an active ingredient.
Formula (I)
An oral application object containing the schuclase inhibitor according to claim 1. The object to be applied to the oral cavity according to claim 2, which is a food or drink. The oral application object according to claim 2, which is a pharmaceutical product or a quasi-drug.