JP4815421B2 - Postprandial blood neutral fat concentration inhibitor and food and drink - Google Patents

Description

  The present invention relates to a postprandial blood neutral fat concentration increase inhibitor and a food or drink containing the postprandial blood neutral fat concentration increase inhibitor.

  In recent years, the Japanese lifestyle has changed and life expectancy has increased rapidly. However, dietary habits have increased in calories and fat and are accompanied by lack of exercise. Obesity, hyperlipidemia, hypertension Lifestyle-related diseases such as diabetes and diabetes are increasing. According to the summary of the 2005 National Health and Nutrition Survey, the average energy intake for both men and women has gradually decreased, but the lipid energy ratio is less than 25%, the appropriate ratio. Both men and women are gradually decreasing, and the ratio of those over 30% is increasing gradually for both men and women. The percentage of those with exercise habits is the lowest in the working generation, with 15.6% in the 40s for men and 14.0% in the 30s for women. Based on the above lifestyle habits, it is considered that one in two men and one in five women are those who are strongly suspected of metabolic syndrome (visceral fat syndrome) or a reserve group when they are 40-74 years old. In order to improve or prevent these lifestyle-related diseases, reducing calorie intake through dietary restrictions is an effective means, but it requires careful nutritional guidance and is difficult to implement in daily life. There are many cases. In addition, although regular exercise is effective, it is often difficult for those who have not had exercise habits until now.

  On the other hand, efforts are being made to search for organisms useful for improving human disease or promoting health from around the world, but overfishing is not desirable from the viewpoint of resource conservation and the Convention on Biological Diversity. In addition, so-called health foods that use raw materials that have insufficient traceability such as unknown origin and cultivation history, or that cannot ensure a stable supply, are on the market, causing serious side effects and social disruption.

  Tea (Camellia sinensis) is a perennial woody evergreen tree belonging to the family Theaceae, and has the longest history of beverages.In 2005, 100,000 tons were produced in Japan and 3,201,000 tons worldwide. It is safe and reliable to use as a raw material.

Various functions have already been clarified in green tea. The center of its bioactivity is often epigallocatechin 3-O-gallate, a type of tea catechin (Liao A. et al. Vitamins and Hormons 62, 1-94, 2001). in use. However, in such foods and beverages, the action of epigallocatechin 3-O-gallate is insufficient, so it is necessary to use a high concentration of catechin, and as a result, the bitterness is strong and the original flavor of tea is often impaired. For these reasons, when using a high concentration of catechin, a special method for masking bitterness is required. In addition, foods and beverages (such as Japanese Patent Application Laid-Open No. 2006-1909) in which a polymerized polyphenol produced by fermentation of tea inhibits fat absorption have been put on the market, such as epigallocatechin 3-O-gallate. The tea catechins are significantly reduced and the original flavor of tea is lost.
Liao A. et al Vitamins and Hormons 62, 1-94, 2001 JP 2006-1909 A

  Therefore, in order to improve lifestyle-related diseases, it has been a challenge to maintain a proper balance between the flavor and the tea catechin and the polyphenol produced by fermentation. Therefore, a tea-derived polymerized catechin that is effective at a lower concentration than epigallocatechin 3-O-gallate has been desired.

  Therefore, an object of the present invention is to find a new substance that replaces epigallocatechin 3-O-gallate, which is suitable for improving lifestyle-related diseases, and particularly has an excellent neutral fat absorption inhibitory effect. The object is to provide a blood neutral fat level increase inhibitor.

  Furthermore, the objective of this invention is providing the food / beverage products which have the neutral fat absorption inhibitory effect containing the said novel postprandial blood neutral fat concentration raise inhibitor.

  As a result of intensive studies to solve the above problems, the present inventors have found that laccase-treated green tea extract and epitea fragalin 3-O-gallate contained in black tea are excellent in postprandial blood at low concentrations. It has been found that it has an effect of suppressing the increase in sex fat concentration, and the present invention has been completed.

The present invention is as follows.
[1] A pancreatic lipase inhibitor containing epitheaflavalin 3-O-gallate as an active ingredient.
[2] The pancreatic lipase inhibitor according to [1], which is used as a postprandial blood neutral fat concentration increase inhibitor added to food and drink.
[3] The pancreatic lipase inhibitor according to [1] or [2], which is used for obesity prevention and / or obesity treatment.
[4] Epiteaalagalin-3-O-gallate is obtained by adding gallic acid to a tea extract containing epigallocatechin gallate and allowing polyphenol oxidase to act. The pancreatic lipase inhibitor according to any one of [1] to [3], which is epitheaflavalin 3-O-gallate contained in a mixture containing.

  ADVANTAGE OF THE INVENTION According to this invention, the food-drinks containing the postprandial blood neutral fat concentration increase inhibitor which has the outstanding postprandial blood neutral fat concentration increase inhibitor, and this postprandial blood neutral fat concentration increase inhibitor can be provided. .

Epithea fragalin 3-O-gallate

  Epitea fragalin-3-O-gallate is a black tea-derived substance represented by the above chemical formula, but the amount contained in green tea and black tea is very small, and the extract of ordinary black tea with the highest content The amount is generally 0.1% or less (Itoh N. et al., Teterahedron, 63, 9488-9492, 2007). Therefore, it was virtually impossible to ingest epiteafragalin-3-O-gallate from a normal tea extract. On the other hand, the present inventors have studied to establish a new production method of epitheafragalin-3-O-gallate, and epitheaflagalin-3-O-gallate is converted to epigallocatechin gallate. It has been found that it can be produced by the action of polyphenol oxidase in the presence of gallic acid to convert it to epitheaflavalin-3-O-gallate, and a patent application has already been filed (Japanese Patent Application No. 2006-156488). ing.

  Epitheaflavalin-3-O-gallate used in the present invention can be produced as a food material in large quantities for the first time by the above-mentioned method, and as a result, the inventors of the present invention for the first time reveal its efficacy. Thus, the present invention was completed.

  Epigallocatechin gallate, which is the raw material of the above method, is contained in the tea extract. A mixture containing epitheafragalin-3-O-gallate by converting at least a portion of epigallocatechin gallate contained in the tea extract into epiteafragalin-3-O-gallate Can also be manufactured. Examples of the tea extract include green tea extract, oolong tea extract, and black tea extract.

Green tea extract Green tea is an extract of the leaves of Camellia plants, which are mainly dried from Camellia sinensis and Camellia assamica sprouts. Examples of the green tea extract include SD Green Tea Extract Powder No. 16714 (San-Eigen FFI Co., Ltd.), Sunphenon BG (Taiyo Chemical Co., Ltd.), and the like. As an extraction method, there is a method in which the raw material is extracted with hot water, hydrous alcohol, glycerin aqueous solution, ethyl acetate or the like, purified and concentrated, and spray-dried or freeze-dried.

Oolong tea extract Oolong tea is obtained by fermenting tea leaves similar to green tea extract for a certain period of time and then heating to stop fermentation (semi-fermented tea). As the oolong tea extract, for example, FD oolong tea extract powder No. 16297 (San-Eigen FFI Co., Ltd.). As an extraction method, there is a method in which the raw material is extracted with hot water, hydrous alcohol, glycerin aqueous solution, ethyl acetate or the like, purified and concentrated, and spray-dried or freeze-dried.

Black tea extract Black tea is obtained by strongly fermenting tea leaves similar to the green tea extract and then heating to stop fermentation (strongly fermented tea). Examples of the black tea extract include SD black tea extract powder No. 16691 (Saneigen FFI Co., Ltd.). As an extraction method, there is a method in which the raw material is extracted with hot water, hydrous alcohol, glycerin aqueous solution, ethyl acetate or the like, purified and concentrated, and spray-dried or freeze-dried.

  Examples of tea extract are shown in Table 1 below. However, these tea extracts are examples, and are not limited to these.

  In said method, it is suitable to add 1-10 gallic acid by molar ratio with respect to the total amount of epigallocatechin gallate contained in a tea extract. Preferably, gallic acid having a molar ratio of 2 to 5 is added to the total amount of epigallocatechin gallate.

  The polyphenol oxidase is not particularly limited as long as it is an enzyme capable of converting epigallocatechin gallate to epitheafragalin-3-O-gallate. Examples of such polyphenol oxidase include laccase (EC1.10.3.2), tyrosinase (EC 1.14.18.1), bilirubin oxidase (EC1.3.3.5), phenol and polyphenol oxidase (EC1.10.3.1). Mention may be made of at least one enzyme selected from the group. Tyrosinase (EC 1.14.18.1) is also partly classified as 1.10.3.1, but the enzyme classified as 1.10.3.1 is also a part of tyrosinase (EC 1.14.18.1) in the present invention.

  The polyphenol oxidase can be a free enzyme or an immobilized enzyme. When polyphenol oxidase is a free enzyme, a predetermined amount of polyphenol oxidase is added to a tea extract to which gallic acid is added, and a conversion reaction is performed at a predetermined temperature for a predetermined time. The predetermined amount of polyphenol oxidase is, for example, in the range of 10 to 200 U with respect to 1 ml of a solution containing 0.5 (w / v)% to 15 (w / v)% of tea extract.

  Note that how much epigallocatechin gallate is contained in the tea extract varies depending on the tea extract. For example, SD green tea extract powder contains about 13 (w / w)% of epigallocatechin gallate. Thus, for example, a solution containing 1% tea extract has an epigallocatechin gallate of about 1.3 mg / ml.

  The predetermined time for the conversion reaction is, for example, 10 minutes to 15 hours, and the predetermined temperature is in the range of 20 to 60 ° C. The method further comprises heating the tea extract to inactivate the enzyme after the conversion reaction with polyphenol oxidase. The heating condition is suitably 2 to 10 minutes at 70 to 90 ° C.

  Polyphenol oxidase can also be an immobilized enzyme. When using an immobilized enzyme, the conversion reaction with polyphenol oxidase can be performed by adding the immobilized enzyme to a tea extract to which gallic acid has been added. The time and temperature of the conversion reaction are, for example, 10 to 240 minutes, and the predetermined temperature is in the range of 20 to 60 ° C. After the conversion reaction, the immobilized enzyme is removed from the tea extract. The removal can be performed, for example, by filtering the immobilized enzyme.

  When polyphenol oxidase is an immobilized enzyme, the conversion reaction by polyphenol oxidase can also be performed by passing a tea extract to which gallic acid has been added into a container filled with the immobilized enzyme. The circulation conditions of the tea extract can be performed by setting the temperature in the range of 20 to 60 ° C. and the contact time with the immobilized enzyme in the range of 10 to 240 minutes, for example.

  The conversion reaction with polyphenol oxidase can be performed while aeration of oxygen or air through the tea extract. The conversion reaction can be promoted by ventilating oxygen or air. The ventilation condition of oxygen or air can be, for example, 0.2 to 10 L / L / min. Furthermore, in order to enhance the aeration effect, aeration can be performed while stirring the reaction solution.

  The conversion reaction with polyphenol oxidase is carried out so as to convert at least a portion of epigallocatechin gallate contained in the tea extract into epitheaflavalin-3-O-gallate. Depending on the purpose, the amount of gallic acid added and the reaction conditions can be selected so that the total amount of epigallocatechin gallate is converted to epitheaflavalin-3-O-gallate.

  The conversion reaction with polyphenol oxidase is performed by adding gallic acid to the tea extract. However, in consideration of the pH dependence of the polyphenol oxidase activity, a buffer may be added to the tea extract for the purpose of pH adjustment. it can. However, considering that the conversion reaction product is used as a beverage as it is, it is preferable to use, for example, phosphoric acid and its salts, citric acid and its salts, etc. as the buffer. Of course, the conversion reaction by polyphenol oxidase can also be performed without adding a buffer.

  In addition to food / beverage products, the epithea fragalin-3-O-gallate or the epithea fragalin-3-O-gallate-containing mixture obtained by the above production method can be used as the food / beverage product of the present invention. The food and drink of the present invention is specifically a beverage, and more specifically a tea beverage. Examples of the tea beverage include green tea beverage, green tea beverage, oolong tea beverage, oolong tea beverage, black tea beverage, and black tea beverage. The beverage of the present invention may contain, for example, 0.01 to 0.5% by mass of epiteafragalin-3-O-gallate.

  Furthermore, epithea flagaline-3-O-gallate or epitea fragalin-3-O-gallate-containing mixture obtained by the above production method can be used as it is by adding it to foods and drinks. The extract powder can also be prepared by sizing the liquid obtained by treating the tea extract with polyphenol oxidase under the following conditions, extracting, purifying or concentrating, spray drying or freeze drying. Such concentrated solutions or extract powders can also be used as raw materials for various forms of food and health care products.

  Examples of foods to which the concentrated solution or extract powder can be applied include gums, confectionery, candy, and supplements. Examples of the health care product to which the concentrated solution or the extract powder can be applied include an oral cleaning liquid and a toothpaste.

  In the present invention, in order to obtain a desired postprandial blood triglyceride concentration-inhibiting effect, based on the data of the examples described later, about 20 mg / kg or less as a guideline is used as epiteaflavalin-3-O-gallate. As long as it is taken after meals. However, the intake amount of epitheaflavalin-3-O-gallate can be changed as appropriate according to the amount of fat taken from the meal and individual differences in fat absorption capacity. For example, if 200 ml (equivalent to 0.2 to 1 g) of a beverage containing 0.1 to 0.5% by mass of the above-described epitheaflavalin-3-O-gallate according to the present invention is ingested, an effect of suppressing postprandial blood neutral fat concentration can be obtained. .

<Reference Example 1> Preparation of laccase-treated green tea extract and epitheafragalin 3-O-gallate ・ Laccase-treated green tea extract 1 g of green tea extract (Camelia extract 40R (Taiyo Kagaku)) and gallic acid monohydrate (pure chemistry) 0.48 g was taken and dissolved in 100 mL of water. Laccase Daiwa Y120 (Amano Enzyme, 108,000 U / g) was added in 0.0093 g to 0.093 g, and the reaction was carried out at a reaction temperature of 45 to 50 ° C. for 1 to 4 hours. After the reaction, the reaction solution was heated, allowed to cool, and freeze-dried. The resulting dry powder was used as a laccase-treated green tea extract (Japanese Patent Application No. 2006-156488). The extract contained 0.2 to 1.5% (w / w) of epitheafragalin 3-O-gallate.

Epithea fragalin 3-O-gallate 4 g of epigallocatechin 3-O-gallate was added to 300 mL of water and stirred (ice cooling). Potassium ferricyanate 10 g, sodium hydrogen carbonate 6 g / water 60 mL, pyrogallol 2.8 g / water 60 mL were added dropwise. The reaction solution was extracted with ethyl acetate (100 mL × 3), evaporated under reduced pressure, and purified by column chromatography [polyamide C-200, 50 g (column diameter: 3 cm), eluted with water / ethanol mixture], epitheaflavalin 3- The O-gallate fraction was collected and recrystallized from water / methanol to obtain 840 mg of orange crystals.

<Example 1> Inhibitory effect on pancreatic lipase Pancreatic lipase is an essential enzyme for degrading and digesting triglyceride, which is an oil and fat component in diet, into fatty acid and glycerol and absorbing it in the small intestine. Therefore, if pancreatic lipase can be inhibited, fat accumulation can be directly suppressed, and anti-obesity can be expected. Therefore, pancreatic lipase inhibition by tea-derived polymerized catechin was measured.

  The lipase activity was calculated by measuring the amount of oleic acid released from triolein. A uniform suspension was obtained by sonicating 80 mg of triolein, 10 mg of lecithin, and 5 mg of bile acid in 9 mL of 0.1 M Tris buffer, and this was used as a substrate solution. As an experimental operation, 0.05 mL of a porcine pancreatic lipase solution and 0.1 mL of a sample solution were added to 0.1 mL of a substrate solution, reacted at 37 ° C. for 30 minutes, and the liberated fatty acid was quantified by a copper reagent method. The activity value of each sample was calculated with the value of no sample added as 100%. As a positive control, commercially available pancreatic lipase inhibitor Xenical (Orlistat) was used.

As shown in FIG. 1, the laccase-treated green tea extract (containing 3 mg / g extract of epithea fragalin 3-O-gallate) and epithea fragalin 3-O-gallate obtained in Reference Example 1 have pancreatic lipase activity. Was inhibited. As shown in FIG. 2, they inhibited pancreatic lipase activity in a dose-dependent manner, and the concentrations showing 50% inhibition (IC ₅₀ ) were 1.4 and 1.2 mg / mL, respectively. On the other hand, both components of epigallocatechin 3-O-gallate (Sanphenon EGCG, manufactured by Taiyo Kagaku Co., Ltd.) and epitheaflagalin used as controls did not show pancreatic lipase inhibitory action. Xenical inhibited pancreatic lipase activity by about 96% at a final concentration of 0.04 mg / mL.

Moreover, it measured also by the method of measuring a fluorescence intensity using 4-methylumbelliferyl oleate (MUO) which is a synthetic substrate using bovine origin pancreatic lipase. Specifically, 25 μl of lipase (derived from bovine pancreatic juice, 50 U / ml) dissolved in 13 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl and 1.3 mM CaCl ₂ , 50 μl of 0.1 mM 4-MUO solution and 25 μl of test solution were mixed. And reacted at 25 ° C. for 30 minutes. After stopping the reaction by adding 100 μl of 0.1 M citrate buffer (pH 4.2), the fluorescence intensity (Ex: 360, Em: 450) was measured. As a result, the laccase-treated green tea extract obtained in Reference Example 1 and epitheaflavalin 3-O-gallate showed stronger lipase inhibition than epigallocatechin 3-O-gallate used as a control, and olein from triolein. Data consistent with lipase inhibition by acid release was obtained (Table 2).

* IC ₅₀ (μg / ml) In parentheses, inhibition rate at 100 μM action

Laccase treatment green tea extract (epi tare Fraga phosphate 3-O-gallate containing), was found to exhibit inhibition of epi tare Fraga phosphate 3-O-gallate is a strong pancreatic lipase.

<Example 2> Postprandial neutral fat increase inhibitory effect in rats Rats fasted overnight (around 226 g, 5 each, 9 controls) were given lipid emulsion and each sample (laccase-treated green tea extract [Epitea Fragalin 3- 3 mg / g extract of O-gallate], epitheafragalin 3-O-gallate, and a lipid emulsion containing distilled water instead of the test solution as a control) Administered. Blood was collected from the tail vein over time up to 60, 120, 180, 240, and 300 minutes after administration, and the amount of neutral fat in the blood was measured with a triglyceride-E-test kit (manufactured by Wako Pure Chemical Industries). . The lipid emulsion was prepared by mixing 6 ml of corn oil, 6 ml of physiological saline, 100 mg of bile acid and 2 g of cholesterol oleate, and prepared by sonication to obtain a lipid emulsion.

  The results are expressed as mean values ± standard error (SE) in FIG. Super ANOVA software was used for the significant difference test between groups, and the test was performed by Fisher's Protected LSD test, and p <0.05 was regarded as significant. This point is indicated by * mark. As a result, the green tea extract treated with laccase showed a significant inhibitory effect on postprandial neutral fat elevation. On the other hand, when 20 mg / kg of epitheaflavalin 3-O-gallate was administered, a clear and significant postprandial neutral fat increase inhibitory effect was shown.

  The present invention provides a postprandial blood neutral fat concentration increase inhibitor, and is useful, for example, in the field of food and drink such as health foods.

Inhibitory action on pancreatic lipase Inhibitory effect on pancreatic lipase (dose-dependent) Inhibition of postprandial neutral fat elevation

Claims (4)

A pancreatic lipase inhibitor comprising epiteafragalin 3-O-gallate as an active ingredient. 2. The pancreatic lipase inhibitor according to claim 1, wherein the pancreatic lipase inhibitor is used as a post-meal blood neutral fat concentration increase inhibitor added to a food or drink. 3. The pancreatic lipase inhibitor according to claim 1 or 2, which is used for obesity prevention and / or obesity treatment. Epithea fragalin 3-O-gallate contains epitea fragalin-3-O-gallate obtained by adding gallic acid to a tea extract containing epigallocatechin gallate and allowing polyphenol oxidase to act. 4. The pancreatic lipase inhibitor according to claim 1, wherein the pancreatic lipase inhibitor is epitheaflavalin 3-O-gallate contained in the mixture.