HK1108889B - Novel compound having lipase inhibitory activity - Google Patents

Description

Novel compound having lipase inhibitory activity

Technical Field

The present invention provides a novel polyphenol derived from oolong tea having lipase inhibitory activity, a process for producing the same, and foods, drinks and pharmaceuticals containing the same.

Background

In recent years, the intake of high-fat diets has been increasing with the westernization of japanese lifestyle patterns. According to the 11 year-by-year national nutrition survey, it is reported that although the energy intake is reduced year by year, the percentage of people with higher lipid energy than the normal percentage of 25% and higher neutral fat value and cholesterol value is 5 to 6 among people over 60 years old (the 11 year-by-year thicknessing labor of Japan, summary clinical nutrition 2001, 98 (5): 577 588, which is the result of the 11 year-by-year national nutrition survey, is shown in (11 year-by-year , thicknessing , bed 2001, 98 (5): 577) -588).

Obesity is one of the most important diseases in modern society, and the main cause thereof is excessive intake of fat. It is also known that excessive intake of fat not only causes obesity, but also induces diabetes, hyperlipidemia, hypertension, arteriosclerosis, and the like, which are caused by obesity. As a drug for treating such obesity, the appetite suppressant, Mazindol (registered trademark), has been exclusively approved in Japan, but it has been reported that it has side effects such as thirst, constipation, stomach discomfort, nausea, vomiting, etc. (clinical evaluation 1985; 13 (2): 419-. Further, Xenical (registered trademark), which has been marketed in Japan as an obesity-ameliorating agent having an effect of inhibiting The absorption of fat from The intestinal tract by lipase inhibitory activity, is still reported to have side effects such as fatty stools, increased frequency of defecation, soft stools, diarrhea, abdominal pain, etc., and is hardly said to be safe (The Lancet 1998; 352: 67-172).

To prevent obesity, restricting diet to reduce intake of calories is an effective means, but must be subjected to strict nutritional guidelines and is difficult to implement in many cases in daily life. Therefore, it is considered to be a practical and useful measure for the purpose of safely and healthily inhibiting the absorption of postprandial fat in the body and treating obesity and diseases related thereto or improving health.

Under such a background, development of specific health foods which are safe and proved to be effective for human bodies has been receiving attention. As a food material for suppressing the increase of the serum neutral fat value after meal, hitherto, globin decomposition products which inhibit fat absorption by inhibiting pancreatic lipase (J.Nutr.1988; 128: 56-60, 1988, Japanese society for clinical food society 1999; 52 (2): 71-77, health and nutritional food research 2002; 5 (3): 131-.

On the other hand, recently, there have been paid attention to lipase inhibitory active substances derived from plants, and in particular, regarding polyphenols having lipase inhibitory activity, there have been reported tannin derived from plant bark (Japanese examined patent publication No. 60-11912), tannins, flavonoids and their glycosides contained in Japanese honeylocust fruit, a food product (Japanese examined patent publication No. 8-259557) containing epigallocatechin gallate and epicatechin gallate, which are main components of green tea, for inhibiting lipid absorption, a lipase inhibitor (Japanese examined patent publication No. 3-228664) containing water extracts of green pepper, Tricholoma caespitosum, pumpkin, Grifola frondosa, Hizikia fusiforme, green tea, oolong tea, etc. (Japanese examined patent publication No. 3-219872), flavones and flavonols (Japanese examined patent publication No. 7-61927), and hydroxyamphatic acids (gallic acid) (Japanese examined patent publication No. 1-102022), triterpenes and their derivatives (Japanese patent application laid-open No. Hei 9-40689), and antiobesity agent (Japanese patent application laid-open No. Hei 9-291039) containing tamarind Procyanidin (Procyanidin) as effective component. Further, lipase inhibitory action of grape seed extract (Nutrition vol.19, (10), 876-.

However, the above reported plant-derived lipase inhibitors have insufficient effects, and for example, even if an extract of a certain plant has an effect, it is difficult to maintain a stable lipase inhibitory activity because the amount of an active ingredient contained therein is not determined and the lipase inhibitory activity is derived from a natural product. In addition, if the inhibitor is derived from an undesirable plant, it has a problem of affecting the flavor when used as a food or drink. For example, as reports describing the lipid-improving effect of oolong tea, there are reports that 1330ml of commercially available oolong tea taken daily showed a significant decrease in the blood neutral fat value after 6 weeks of administration (Japanese society of Nutrition and diet 1991; 44 (4): 251-; as a result of orally ingesting 102 simple obese men and women for 6 consecutive weeks (2 g.times.4/day), it was found that 67% of subjects had a weight reduction of 1kg or more and that subjects having a high blood neutral fat value had a significant improvement effect after ingesting oolong tea (Japanese journal of clinical Nutrition society 1998; 20 (1): 83-90). Although such a large amount of drinking of the effective fruit of oolong tea proves to be difficult to continue in daily life, and even if a pure concentrated oolong tea is provided, it is not suitable as a practical countermeasure because of strong bitterness and astringency and an increase in the amount of caffeine.

[ patent document 1]

Japanese examined patent publication No. 60-11912

[ patent document 2]

Japanese Kokai Hei 8-259557

[ patent document 3]

Japanese patent application laid-open No. Hei 3-228664

[ patent document 4]

Japanese patent application laid-open No. Hei 3-219872

[ patent document 5]

Japanese unexamined patent publication Hei 7-61927

[ patent document 6]

Japanese patent application laid-open No. Hei 1-102022

[ patent document 7]

Japanese patent application laid-open No. 9-40689

[ patent document 8]

Japanese patent application laid-open No. Hei 9-291039

[ non-patent document 1]

Summary of national nutrition survey results of 11 years of Chinese average labor province in the great birth of China

[ non-patent document 2]

Clinical nutrition 2001; 98(5): 577-588

[ non-patent document 3]

Clinical evaluation 1985; 13(2): 419-459, clinical evaluation 1985; 13(2): 461-515

[ non-patent document 4]

The Lancet 1998；352：67-172

[ non-patent document 5]

J.Nutr.1988；128：56-60，1988

[ non-patent document 6]

Japan society of clinical food and diet 1999; 52(2): 71-77

[ non-patent document 7]

Health nutritional food research 2002; 5(3): 131-144

[ non-patent document 8]

J.Am.Coll.Nutr.2000；19(6)：789-796

[ non-patent document 9]

Clin.Chim.Acta.2001；11(2)：109-117

[ non-patent document 10]

Nutrition vol.19，(10)，876-879，2003

[ non-patent document 11]

J.Nutr.132，1819-1824，2002

[ non-patent document 12]

Int.J.Obes.23 98-105，1999

[ non-patent document 13]

Japan society of nutrition and food science 1991; 44(4): 251-259

[ non-patent document 14]

Journal of the japanese clinical nutrition society 1998; 20(1): 83-90

Disclosure of Invention

The present invention concerns the components contained in tea which is popular, and provides a novel polyphenol compound having lipase inhibitory activity derived from tea, and a process for producing the same.

The present invention also provides a food or drink which contains the novel polyphenol compound of the present invention having lipase inhibitory activity and which inhibits postprandial fat absorption and inhibits the increase in neutral fat in blood.

The present invention further provides a pharmaceutical composition which contains the novel polyphenol compound of the present invention having lipase inhibitory activity and which inhibits postprandial fat absorption and inhibits elevation of neutral fat in blood.

The present inventors have found that a novel compound, oolong theanine-3 '-O-gallate (oolongthanin-3' -O-gallate), which is a dimer represented by the following formula and has a strong function of inhibiting pancreatic lipase which is essential for fat absorption, is obtained by oxidative polymerization of epigallocatechin-3-O-gallate (epigallocatechin-3-O-gallate), which is a main catechin component of oolong tea, using a tea enzyme (polyphenol oxidase).

[ chemical formula 1]

Manufacturing method

The compound of the present invention can be obtained by oxidative polymerization of epigallocatechin-3-O-gallate (epigallocatechin-3-O-gallate) using polyphenol oxidase. epigallocatechin-3-O-gallate (epigallocatechin-3-O-gallate), which is a starting material, is known and commercially available, and can also be extracted from natural materials such as green tea, black tea, oolong tea, and the like. Polyphenol oxidase used for oxidative polymerization can be extracted from tea leaves as described in example 1, but is not limited to the enzyme derived from tea leaves, and can be an enzyme derived from horseradish, for example, as long as it can catalyze oxidative polymerization of epigallocatechin-3-O-gallate (epigallocatechin-3-O-gallate) to oolong theanine-3' -O-gallate (OTNG).

Making starting material (epigallocatechin-3-O-gallate: epigallocatechin-3-O-gallate), and oxidant (such as H)₂O₂Etc.) and polyphenol oxidase in an aqueous buffer solution having a pH of 4 to 7, preferably 5 to 6, and carrying out an oxidative polymerization reaction at 20 to 40 ℃, preferably 25 to 35 ℃ for 1 to 4 hours, preferably 3 hours. The amounts of the oxidizing agent and polyphenol oxidase used are, for example, 2mg of the oxidizing agent and 100g of the enzyme obtained from raw tea leaves, respectively, based on 100mg of the starting material.

The product obtained by the oxidative polymerization can be purified by a usual method such as chromatography. The purified oolong theanine-3 '-O-gallate (oolongthanin-3' -O-gallate) is "white powder, soluble in water, ethanol, DMSO, neutral", and is preferably used as a lipase inhibitory active ingredient for foods, beverages, pharmaceuticals, and the like, because it is highly safe and can inhibit postprandial fat absorption, inhibit the increase in blood neutral fat, or reduce the increased blood neutral fat.

Oolong theanine-3 '-O-gallate (oolongtheanin-3' -O-gallate) is a novel compound provided by the above oxidative polymerization reaction, but may be present in natural materials such as tea leaves, or may be obtained by extracting and purifying these natural materials.

Method for measuring lipase inhibitory activity

The compounds of the present invention have a strong inhibitory activity against lipases, particularly pancreatic lipase, and the inhibitory activity thereof can be measured according to the method specifically described in example 2.

Lipase inhibitors

The compound of the present invention can be used alone or together with a solvent or a solid carrier as a lipase inhibitor. The solvent or the carrier is preferably a substance that can be safely used for foods or medicines, considering that it is used as a food, drink and/or medicine described below. The lipase inhibitor of the present invention has various uses, and can be used, for example, as an active ingredient for foods and medicines for experimental research and for preventing accumulation of neutral fat. Food or drink containing oolong theanine-3 '-O-gallate (oolongtheanin-3' -O-gallate)

The compound of the present invention or a lipase inhibitor containing the compound can be added to foods and beverages as a lipase inhibitory active ingredient, and can prevent an undesirable increase in blood neutral fat and/or a decrease in blood neutral fat that has increased due to postprandial ingestion of a fat component. The preferred food and drink is a food and drink for daily intake, such as green tea, barley tea, oolong tea, black tea, coffee, sports drink, drinking water, seasoning, and dressing (seasoning). The food or drink may be a food or drink that is normally consumed, or a refreshing drink, a cocktail, beer, whisky, distilled liquor, wine, sake, seasoning, sauce (dressing), seasoned rice, processed food, snack food, retort pouch food, chocolate, whipped cream, snack food, dairy product, health food, nutritional supplement, or the like.

The amount of the compound of the present invention added to foods and beverages is 0.1mg to 1000mg per meal. However, since the compound of the present invention is derived from food, the safety is very high, and there is no practical upper limit of the amount of the compound added to food or drink.

A medicinal preparation containing oolong theanine-3 '-O-gallate (oolongtheanin-3' -O-gallate)

The compound or lipase inhibitor of the present invention can also be used as an active ingredient of a medicament for inhibiting postprandial fat absorption, preventing an increase in undesirable neutral fat in blood, and/or reducing an increase in undesirable neutral fat in blood. Preferably, the preparation is oral preparation, such as health beverage, tablet, capsule, granule, powder, dripping pill, etc. The dosage of the compound of the invention contained in the medicament is 0.1 mg-1000 mg per dose.

The pharmaceutical product of the present invention is safe for long-term administration because of the high safety of oolong theanine-3' -O-gallate (an active ingredient for inhibiting lipase), and therefore can be daily administered for preventing or eliminating obesity which is a lifestyle-related disease.

The present invention can provide a food or drink which is added with polyphenols derived from oolong tea, does not spoil the flavor, is popular, and has the purpose of reducing neutral fat and promoting health. In order to suppress the absorption of fat after meals, a beverage in which the effective ingredient obtained from tea is enriched by taking the beverage at the same time as eating is desired is significant.

The compound of the present invention is produced by a simple method using epigallocatechin-3-O-gallate (epigallocatechin-3-O-gallate) contained in oolong tea in a large amount as a starting material, and is easily purified.

Drawings

FIG. 1 shows the mass spectrum of oolong theanine-3' -O-gallate (OTNG).

FIG. 2 shows OTNG¹H NMR。

FIG. 3 shows OTNG¹³C NMR。

FIG. 4 shows the chemical structure of OTNG.

Examples

Example 1 enzymatic Synthesis of oolong theanine-3' -O-gallate (OTNG)

Preparation of enzymes

600g of Tea variety, Jing Ming No. 129 (supplied by Kyotopprefectural Tea Industry Institute) was pulverized in liquid nitrogen, and 1800ml of extraction buffer (0.01M KH with 0.01M) was added₂PO₄And 0.02M K₂HPO₄pH 7.0) and 300ml of polyamide, stirring and filtering with gauze. The filtrate was centrifuged at 8000rpm for 20 minutes, 1500ml of acetone previously cooled to-20 ℃ was added to 1500ml of the supernatant, and the mixture was stirred and allowed to stand at 4 ℃ for 1 hour. This solution was centrifuged at 8000rpm and 4 ℃ for 20 minutes to obtain a white precipitate. The precipitate was dissolved in 600ml reaction buffer (0.01M citric acid and 0.02M H₃PO₄Adjusting pH to 5.6) to prepare an enzyme solution.

Enzyme reaction

600mg of epigallocatechin-3-O-gallate (Epigallocatechin-3-O-gate) (Wako pure chemical industries, Ltd.) and 8.8mM of H were added to 600ml of the enzyme solution₂O₂After stirring, the reaction was carried out at 32 ℃. After 3 hours the reaction was stopped by adding 600ml of 90% acetonitrile containing 1% trifluoroacetic acid (TFA). The solution was diluted 5 times with water and supported on an adsorption resin HP-20(1000ml, Mitsubishi chemical corporation), and after washing with water, the reaction product was eluted with 2000ml of 90% acetonitrile containing 0.1% TFA, and then concentrated under reduced pressure and lyophilized. The reaction product was purified by preparative HPLC as described below.

Refining

Column: develosil ODS-UG-5(50 mm. phi. times.500 mm, Nomura chemical)

Mobile phase: a: 0.05% TFA/H₂O，B：90％CH₃CN、0.05％TFA

And (3) detection: a280nm

Flow rate: 32ml/min

Gradient: linear gradient elution from B20% to B50% for 100 min

By this chromatography, elution was carried out for 52 minutes to obtain oolong theanine gallate (oolongtheanin-gallate), and in order to further increase the purity, preparative HPLC separation was carried out again.

Column: develosil C30-UG-5(20 mm. phi. times.250 mm, Nomura chemical)

Mobile phase: a: 0.1% TFA/H₂O，B：90％CH₃CN、0.1％TFA

And (3) detection: a280nm

Flow rate: 6ml/min

Gradient: elution was linear gradient from B10% to B40% for 40 min. By this chromatography, the elution time was 34 minutes to obtain 25mg of oolong theanine-3 '-O-gallate (oolongtheanin-3' -O-gallate).

Oolong theanine (olonggtheanin) (no Gallate) was reported to be isolated from tea leaves in chem. pharm. Bull 36(5), 1676. sup. 1684, 1988, but the currently obtained oolong theanine-3 '-O-Gallate (Oolongtheanin-3' -O-Gallate) is a novel compound.

MS measurements were performed in Positive ion mode (Positive mode) using an ESI probe on a Q-TOF (Micromass, Manchester, UK). Confirmation of [ M + H ] at M/z 885]⁺Ion absorption peak, [ M + Na ] at M/z 907]⁺Ion absorption peak. Fig. 1 shows a spectrum.

¹H NMR、¹³C NMR、¹H[¹³C]-HSQC、¹H[¹³C]-HMBC, TOCSY and DQF-COSY are dissolved in CD₃OD was determined on DMX-750(BRUKER BIOSPIN). FIG. 2 shows¹H NMR, FIG. 3 shows¹³C NMR, FIG. 4 shows the structural formula.

EXAMPLE 2 measurement of Lipase inhibitory Activity

The lipase activity was measured by measuring the fluorescence of 4-methylumbelliferone produced by the reaction using a fluorescent 4-methylumbelliferone oleate (4-UMO) as a substrate.

For the assay, the buffer used contained 150mM NaCl, 1.36mM CaCl₂13mM Tris-HCl (pH8.0), substrate 4-UMO (manufactured by Sigma) was prepared by diluting the prepared 0.1M DMSO solution 1000 times with the above buffer solution, and lipase was prepared as a 400U/ml solution with the above buffer solution for enzyme assay.

The enzyme reaction was started by adding 50. mu.l of 4-UMO buffer solution and 25. mu.l of distilled water (or an aqueous sample solution) to a 96-well microplate and mixing the mixture at 25 ℃ and then adding 25. mu.l of lipase buffer solution, and after the reaction proceeded for 30 minutes, the reaction was terminated by adding 100. mu.l of 0.1M citric acid buffer solution (pH 4.2). The fluorescence (excitation wavelength: 355nm, fluorescence wavelength: 460nm) of 4-methylumbelliferone produced by the reaction was measured with a fluorescence analyzer (Fluoroskan asset CF, manufactured by Labsystems).

The inhibitory activity of the test sample was determined as a sample amount IC that inhibited the activity of the test sample by 50% relative to the activity of the control (distilled water)₅₀And (4) obtaining. Results of determination of Lipase inhibitory Activity of OTNG, IC₅₀0.06. mu.g/ml (0.068. mu.M), and IC of monomeric EGCG₅₀0.16. mu.g/ml (0.349. mu.M), indicating a very strong activity compared to the monomer.

Claims (4)

1. Use of oolong theanine-3' -O-gallate represented by the following formula in preparing foods, beverages or medicines for inhibiting postprandial fat absorption and/or inhibiting blood neutral fat increase and/or preventing or eliminating obesity,

2. the use according to claim 1, wherein the food, drink, or pharmaceutical product contains the oolong theanine-3' -O-gallate in an amount of 0.1mg to 1000mg per meal or 0.1mg to 1000mg per meal.

3. The use according to claim 2, wherein the use is for preparing a food or drink selected from the group consisting of tea beverages, refreshing beverages, and health foods.

4. Use of oolong theanine-3' -O-gallate ester represented by the following formula in preparing foods, beverages or medicines for inhibiting lipase activity,