HK1103643B - Lipase inhibitor - Google Patents

Description

Lipase inhibitors

Technical Field

The present invention provides a lipase inhibitor containing flavan-3-ol monomers derived from tea or acylates thereof.

Background

In recent years, the intake of high-fat foods has been increasing with the westernization of the lifestyle of japanese. According to the report of national nutrition survey in 11 average years, although the intake of energy decreases year by year, the ratio of lipid energy exceeds 25% of the suitable ratio, and the proportion of people with high neutral fat value and cholesterol value accounts for 5 to 6 or more years [ the national fat labour province is the summary clinical nutrition 2001 of the national nutrition survey result in 11 average years; 98(5): 577 + 588], laid-open jequirity , laid-open granted Chinese Eikes , outline bed Eikes 2001, 98(5), (577 + 588).

Obesity is one of the most important diseases in today's society, and the main cause thereof is excess fat intake. In addition, excessive fat intake not only causes obesity, but also causes diabetes, hyperlipidemia, hypertension, arteriosclerosis, and the like. The only therapeutic drug for obesity recognized in japan is the appetite suppressant, Mazindol (japanese registered trademark "マジンド - ル"), but it has been reported that it has side effects such as thirst, constipation, stomach discomfort, nausea, vomiting, etc. [ clinical evaluation 1985; 13(2): 419-459, clinical evaluation 1985; 13(2): 461-. Further, Xenike (Xenica) (Japanese registered trademark "ゼニカル"), which has an effect of inhibiting the absorption of fat in the intestinal tract by inhibiting lipase activity in overseas, is also marketed as an obesity-ameliorating agent, but is still considered to be safe because of the side effects of fatty stools, increased frequency of defecation, soft stools, diarrhea, abdominal pain, and the like (Lancet 1998; 352: 67-172).

Although calorie intake reduction control is an effective means for preventing obesity, strict nutritional guidelines must be accepted, and most of them are difficult to be practiced in daily life. Therefore, it is a realistic and effective method for safely and healthily inhibiting the absorption of fat from meals into the body and treating obesity and diseases related thereto or improving health.

In this context, the development of specified health foods which have proven to be both safe and effective for the human body is very compelling. Heretofore, as a food material for suppressing the increase in the serum neutral fat value after meal, there has been sold a globin hydrolysate for suppressing fat absorption by suppressing pancreatic lipase [ j.nutr.1988; 128: 56-60, journal of Japan society of clinical and food and diet 1999; 52(2): 71-77 health and nutritional food research 2002; 5(3): 131. and144 ] (J.Nutr.1988; 128: 56-60, original name in Japanese, bed-food society, 1999, 52(2) 71-77, original name in Japanese, health food , research 2002, 5(3) 131. 144), and diglyceride [ J.Am.Coll.Nutr.2000 ] having digestion and absorption characteristics different from triglyceride; 19(6): 789-; 11(2): 109-117], eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and the like extracted from fish oil.

Recently, substances inhibiting lipase activity derived from plants have also been drawing attention, and particularly regarding polyphenols inhibiting lipase activity, tannins derived from plant bark (Japanese examined patent publication No. Sho 60-11912); tannins, flavonoids and their glycosides contained in Cassia occidentalis (Japanese patent application laid-open No. 8-259557); a food for inhibiting lipid absorption, which contains epigallocatechin gallate and epicatechin gallate as main ingredients of green tea (Japanese patent application laid-open No. Hei 3-228664); lipase inhibitor containing water extracts of green pepper, Tricholoma matsutake, fructus Cucurbitae Moschatae, Grifola frondosa, Cyrtymenia Sparsa, green tea, and oolong tea (Japanese patent application laid-open No. 3-219872); flavones and flavonols (Japanese patent application laid-open No. Hei 7-61927) and hydroxybenzoic acids (gallic acid) (Japanese patent application laid-open No. Hei 1-102022); triterpenes and their derivatives (Japanese patent application laid-open No. Hei 9-40689); an anti-obesity agent containing procyanidin as effective component of fructus Tamarindi Indicae (Japanese patent application laid-open No. 9-291039). And lipase inhibition of grape seed extract is also known [ Nutrition 2003; 19(10): 876-879 ]; lipase inhibition of Salacia-derived polyphenols and anti-obesity action in rats (J.Nutr.2002; 132: 1819-1824); the anti-obesity effect of oolong tea extract on mice (int.J.Obes.1999; 23: 98-105), etc.

However, the lipase inhibitors from plants which have been reported as shown above cannot be said to be effective sufficiently. For example, even if some plant extracts are effective, it is difficult to maintain a stable lipase inhibitory activity because the amount of active ingredients contained therein is unknown and the extracts are derived from natural substances. Further, when a plant-derived inhibitor having low preference is used as a food or drink, there is a problem that the flavor is affected. For example, in a report showing the lipid-improving effect of oolong tea, a result of a significant decrease in the neutral fat value in blood after drinking commercially available oolong tea for 6 weeks at 1330ml per day was reported [ journal of japan nutrition and diet society 1991; 44(4): 251 < 259 >, (original Japanese Eiken < food 1991; 44 < 4 >, < 251 >); also, when oolong tea (2g × 4/day) was orally ingested for 6 weeks for a male female 102 who had simple obesity, 67% of subjects showed a weight reduction of 1kg or more, and subjects with high blood neutral fat values showed a significant improvement effect after ingesting oolong tea (journal of japanese clinical nutrition institute 1998; 20(1): 83-90 (original Japanese name; Eiken Eyeso. , Eyeso.35468; 20 (1): 83-90). Such a large amount of oolong tea, though effective, is difficult to sustain in daily life. Furthermore, even if a concentrated oolong tea is provided, the bitterness and astringency are too strong, and the amount of caffeine increases, which is not practical.

[ 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 ]

Outline of "Pingyuan 11 year national nutrition investigation result" in the province of thicknessing labour in China (Japanese original name; thicknessing Eyren investigation result in the year 11 year)

[ non-patent document 2 ]

Clinical nutrition 2001; 98(5): 577 Eyre 588 (original Japanese name; Eyre 2001; 98 (5): 577 Eyre 588)

[ non-patent document 3 ]

Clinical evaluation 1985; 13(2): 419-459, clinical evaluation 1985; 13(2): 461-515 (Japanese original name; bed value 1985; 13 (2): 419-459, Japanese original name; bed value 1985; 13 (2): 461-515)

[ non-patent document 4]

Lancet 1998；352：67-172

[ non-patent document 5]

J.Nutr.1988；128：56-60，1988

[ non-patent document 6 ]

Japanese clinical food society will note [ japanese original name; japanese society of bed and food 1999; 52(2): 71-77]

[ non-patent document 7]

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

[ Japanese original name; espresso food product study 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 2003；19(10)：876-879

[ non-patent document 11 ]

J.Nutr.2002；132：1819-1824

[ non-patent document 12 ]

Int.J.Obes.1999；23：98-105

[ non-patent document 13 ]

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

[ Japanese original name; eikeka , food being food 1991; 44(4): 251-259]

[ non-patent document 14 ]

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

[ Japanese original name; japanese Eikei , via 35/1998; 20(1): 83-90]

[ non-patent document 15]

Chem.Pharm.Bull 1987；35(2)：611-616

[ non-patent document 16 ]

Chem.Pharm.Bull 1989；37(12)：3255-3563

Disclosure of Invention

The present invention focuses on components contained in tea having high palatability, and provides a lipase inhibitor containing at least one of flavan-3-ol monomers derived from tea and acylates thereof.

The present invention also provides a health food or beverage containing the lipase inhibitor, which is highly palatable and capable of reducing neutral fat in blood and promoting health.

The present invention further provides a pharmaceutical composition containing the lipase inhibitor, which inhibits absorption of dietary fat and inhibits an increase in neutral fat in blood.

As a means for solving the above problems, a component of pancreatic lipase necessary for inhibiting fat absorption was sought from tea, and the lipase inhibitory activity of various polyphenols present therein was evaluated, and it was confirmed that flavan-3-ol monomers or acylated products thereof have a strong lipase inhibitory activity.

More specifically, the lipase inhibitor of the present invention is characterized by comprising a compound represented by the formula:

[ chemical formula 1 ]

(in the formula, R₁And R₃Each independently is H or OH, R₂Is H, R₄Is H, G, 3MeG or p-cou, R₅Is H or G, R₄' and R₅' independently of one another, are H or G, R₄"is H or G, and G, 3MeG and p-Cou are each of the formula:

[ chemical formula 2 ]

Group (2)

At least one of flavan-3-ol monomers represented by (a) or acylates thereof.

Preferred flavan-3-ol monomers or acylates thereof are selected from the group consisting of (-) -epigallocatechin 3-O-gallate ((-) -epigallocatechin 3-O-gallate) (Compound 1), (-) -epicatechin 3-O-gallate ((-) -epi-catechin 3-O-gallate) (Compound 3), (-) -epicatechin 3-O- (3 '-O-methyl) -gallate ((-) -epicatechin 3-O- (3' -O-methyl) -gallate) (Compound 4), (-) -epigallocatechin 3-O-gallate ((-) -epigallocatechin 3-O-gallate) (Compound 6), (-) -epigallocatechin3, 5-di-O-gallate ((-) -epigallocatechin3, 5-di-O-gallate) (Compound 7), (-) -epigallocatechin 3-O-p-gallate ((-) -epigallocatechin 3-O-p-gallate) (Compound 8), (-) -catechine 3-O-gallate ((-) -catechine 3-O-gallate) (Compound 10), (-) -gallocatechin 3-O-gallate ((-) -gallocatechin 3-O-gallate) (Compound 12), gallocatechin3, 5-di-O-gallate (11 ocatechin3, 5-di-O-gallate) (Compound 13), 8-C-ascorbic acid- (-) -epigallocatechin (8-C-ascorbyl- (-) -epigalloatechin) (Compound 14), and 8-C-ascorbic acid- (-) -epigallocatechin 3-O-gallate (8-C-ascorbyl- (-) -epigalloatechin 3-O-gallate) (Compound 15). (-) -epigalloatechin 3, 5-di-O-gate (Compound 7) is particularly preferable in terms of the intensity of the lipase inhibitory activity.

The flavan-3-ol monomers or acylates thereof of the present invention are commercially available, and may be extracted from commercially available natural materials such as green tea, black tea, and oolong tea. For example, chem. pharm. bull1987; 35(2): 611-616, chem.pharm.Bull 1989; 37(12): 3255-3563, the flavan-3-ol component was purified from oolong tea leaves, and the presence of catechin and its acylated product was confirmed. Gallocatechin3, 5-di-O-gate can be obtained according to the method described in example 2 of this specification.

Lipase inhibitors

The flavan-3-ol monomers or acylates thereof of the present invention may be used alone as a lipase inhibitor without containing other components, or may be used together with a solvent or a solid carrier as a lipase inhibitor. In view of the fact that the solvent or carrier is used as a food or drink and/or a pharmaceutical as described below, it is preferable that the solvent or carrier be used safely as a food or pharmaceutical. The lipase inhibitor of the present invention has various uses, for example, in experimental studies, and is useful as an active ingredient for foods and medicines for preventing accumulation of neutral fat.

Method for measuring lipase inhibitory activity

The lipase inhibitor of the invention has strong inhibition effect on lipase, especially pancreatic lipase. The inhibitory activity can be measured by the method specifically described in example 1.

Beverage and food containing lipase inhibitor

When a lipase activity inhibitor containing the flavan-3-ol monomer or its acylate of the present invention is added to a food or drink as a component for inhibiting lipase activity, it is possible to prevent an undesirable increase in neutral fat in blood accompanying the intake of a fat component at meal time and/or to reduce the neutral fat in the blood which has risen. Preferred examples of the food and drink are food and drink products for daily intake, such as green tea, barley tea, oolong tea, black tea, coffee, sports drinks, drinking water, seasonings, and sauces. However, the beverage or food is not limited to a usual one, and may be a refreshing beverage, a cocktail, beer, whisky, distilled liquor, wine, sake, seasoning, sauce, seasoned rice, processed food, convenience food, soft canned food, chocolate, whipped cream, western-style pastry, dairy products, health food, nutritional supplement food, or the like.

The amount of the lipase activity inhibitor of the present invention added to the food or beverage is 0.1mg to 10g per meal of the flavan-3-ol monomer or the acylated product thereof. However, since the flavan-3-ol monomers or the acylates thereof of the present invention are derived from foods and are very safe, the amount of the flavan-3-ol monomers or the acylates thereof to be added to food and drink is not limited to a practical amount.

Pharmaceutical containing lipase activity inhibitor

The lipase inhibitor containing the flavan-3-ol monomer or the acylase thereof of the present invention can inhibit the absorption of fat from the diet and is useful as an active ingredient of a medicament for preventing and/or reducing undesirable increase in neutral fat in blood. Preferred drugs are orally administered drugs such as health drinks, tablets, capsules, granules, powders, candies, dropping pills and the like. The dosage of the compound of the invention contained in the medicament is O.1mg-10 g for 1 dose.

Since the lipase activity inhibitory component of the pharmaceutical composition of the present invention is highly safe, it is safe to be taken for a long period of time. Therefore, it can be daily administered to prevent or eliminate obesity caused by lifestyle-related diseases.

The present invention provides a health food and beverage which can reduce neutral fat and promote health without spoiling flavor and having high palatability by adding a lipase inhibitor containing at least one of flavan-3-ol monomers derived from tea leaves and acylates thereof. In order to suppress the absorption of edible fat, it is preferable to ingest the food at the same time, so that the beverage enriched with the effective ingredient obtained from tea is of great significance. Particularly, by enhancing these ingredients, neutral fat-reducing tea can be developed.

Drawings

FIG. 1 shows the chemical structural formula of the compound for evaluating the lipase inhibitory activity in example 3.

Examples

Example 1Measurement of Lipase inhibitory Activity

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

For the assay, the buffer used contained 150mM NaCl, 1.36mM CaCl₂13mM Tris-HCl (pH 8.0). Substrate 4-UMO (manufactured by Sigma Co., Ltd.) which was a product obtained by diluting a 0.1M DMS0 solution prepared 1000 times with the above-mentioned buffer solution, and lipase which was a product obtained by preparing a 400U/ml solution of porcine pancreatic lipase (manufactured by Sigma Co., Ltd.) with the above-mentioned buffer solution were used for enzyme assay.

Enzyme reaction at 25 ℃ in 96 hole enzyme plate adding, mixing 50 u l 4-UMO buffer solution and 25 u l distilled water (or sample water solution), by adding 25 u l lipase buffer solution to the reaction start. After the reaction was carried out for 30 minutes, 100. mu.l of 0.1M citric acid buffer (pH 4.2) was added to stop the reaction, and the fluorescence (excitation wavelength of 355nm, emission wavelength of 460nm) of 4-methylumbelliferone produced by the reaction was measured with a fluorescence analyzer (Floroskan asset CF, manufactured by Labsystems).

The inhibitory activity of the test sample was determined as IC by taking the amount of the sample inhibited by 50% relative to the activity of the control (distilled water)₅₀(. mu.M) was determined.

Assay sample

(-) -epictecthin (Compound 2), (-) -epictecthin 3-O-gate (Compound 3), (-) -epicyclotatechin (Compound 5), (-) -epicyclotatechin 3-O-gate (Compound 6), (+) -catechin (Compound 9), (-) -catechin 3-O-gate (Compound 10), (+) -gallocatechin (Compound 11), (-) -gallocatechin 3-O-gate (Compound 12) was purchased from Wako pure pharmaceutical industries.

(-) -epiczelechin 3-O-gate (Compound 1), (-) -epicocchien 3-O- (3' -O-methyl) -gate (Compound 4), (-) -epicoccatechin 3, 5-di-O-gate (Compound 7), (-) -epicoccatechin 3-O-p-coumarate (Compound 8), according to chem.phase.Bull 35(2), 611-door 616(1987) and 8-C-ascorbyl- (-) -epicoccatechin (Compound 14), 8-C-ascorbyl- (-) -epicocchien 3-O-gate (Compound 15) according to chem.phase.Bull 37(12), 3255-door 3563 (1989). Gallocatechin3, 5-di-O-gate (Compound No. 13) was purified by the method of example 2.

Example 2

Mixing cocoa tea (Camellia ptilophylla)100g of the leaf (dried product) of (1) was extracted with 2000ml of hot water (90 ℃ C.) for 4 minutes, and the following purification was performed using the above lyophilized product. A1% aqueous solution of the lyophilized powder was adsorbed to Sep-Pak C18Cartridge (5g, manufactured by Waters), washed with water, and eluted with acetonitrileThe components of (a) are freeze-dried. The fraction (250 mg) was injected into Develosil C30-UG-5(20 mm. times.250 mm, manufactured by Nomura chemical Co., Ltd.), and the fraction was separated while monitoring the absorption of A280nm by eluting with a linear gradient (5ml/min, 180min) of 5-30% acetonitrile in the presence of 0.05% TFA. The resulting fraction was further injected into YMC-Pak ODS (20X 250mm, manufactured by Y M C Co., Ltd.), and eluted and purified with a linear gradient (6ml/mim, 60min) of 20-25% acetonitrile in the presence of 0.1% TFA to obtain gallocatechin3, 5-di-O-gallate (13).

Example 3Lipase inhibitory Activity of Catechins

The lipase inhibitory activities of catechins and acylated catechins are shown in table 1. FIG. 1 shows the chemical structural formula of the compound used for evaluation.

TABLE 1

Lipase inhibiting activity of various polyphenols

Among the major catechins (8 out of 2, 3, 5, 6, 9, 10, 11, 12) present in tea, 4 out of 3, 6, 10, 12, and flavan-3-ol species in which gallic acid forms an ester bond, showed lipase inhibitory activity. Particularly, (-) -epigalloatechin 3-O-gate (EGCG: 6), which is the most abundant in tea leaves, showed the strongest activity among these catechins. Another 1 molecule of gallic acid-conjugated (-) -epigalloatechin 3, 5-di-O-gate (7) in EGCG showed about 3.5 times activity of EGCG, and 1 molecule of gallic acid-conjugated galloatechin 3, 5-di-O-gate (13) showed about 2 times activity of GCG. The fact that a substance having no gallic acid ester group such as (+) -catechin in the molecule does not exhibit lipase inhibitory activity, and the more gallic acid ester groups, the stronger the activity, indicates that the presence of gallic acid ester groups in the molecule is essential for the expression of the activity.

Claims (5)

1. Use of (-) -epigallocatechin3, 5-di-O-gallate for the preparation of a beverage food or medicament for inhibiting the absorption of fat from a meal and/or for inhibiting the elevation of neutral fat in the blood.
2. 2. The use according to claim 1, wherein the food and drink product contains the compound in a total amount of from 0.1mg to 10g per meal.
3. 3. The use according to claim 1, wherein the medicament contains the compound in a total amount of 0.1mg to 10g per dose.
4. 4. The use according to claim 1 or 2, wherein the beverage food is selected from the group consisting of tea beverages, refreshing beverages, and health foods.
5. Use of (-) -epigallocatechin3, 5-di-O-gallate in the preparation of a beverage food or medicament for preventing or treating obesity.