JP2009268420A - Functional food composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a functional food composition which contains tea polyphenol compounds and has an inhibiting activity to sugars and/or lipid degradation enzyme, and to provide a method for reinforcing the enzyme-inhibiting activity of the functional food composition. <P>SOLUTION: Provided are the functional food composition comprising 2 to 7 mass% of theaflavin, 2 to 7 mass% of theaflavin-3-gallate, 2 to 10 mass% of theaflavin-3'-gallate, and 2 to 16 mass% of theaflavin-3,3'-digallate; and the method for reinforcing the enzyme-inhibiting activity of the functional food composition, controlling the content of caffeine in the functional food composition containing tea theaflavin compounds to ≤1 mass% to reinforce an α-glucosidase-inhibiting activity and/or a lipase-inhibiting activity. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a functional food composition containing tea theaflavins obtained from tea leaves. More specifically, the present invention relates to a functional food composition having a physiological activity that inhibits enzymes that break down saccharides and lipids by containing tea polyphenols containing tea theaflavins in high concentrations, and a method for enhancing the enzyme inhibitory activity It is.

  The risk of so-called lifestyle-related diseases such as obesity and diabetes is increasing due to excessive intake of fat due to changes in dietary habits and increased intake of carbohydrates, and carbohydrates, especially starch and sucrose (sucrose), There is a demand for a functional food composition that suppresses excessive absorption of blood sugar levels by suppressing digestion and absorption of maltose (malt sugar) and the like, and suppresses insulin secretion to reduce the risk of obesity and diabetes. Therefore, in recent years, there is an interest in natural substances that exist in plants and exhibit physiological activity, in particular, polyphenols. Polyphenols are generally contained in tea, vegetables, fruits, herbs, and the like, and can be expected as physiologically active substances having no side effects and having been ingested for a long time as food or luxury products. In addition, there is a need for a functional food composition having an effect of suppressing an increase in blood glucose level that can be easily and safely ingested while taking a daily meal.

  Tea (Camellia sinensis (L.) O.kuntze) is an evergreen tree belonging to the camellia family, and antibacterial, antiviral, antimutagenic, antioxidant, blood pressure rise suppression, blood cholesterol are added to tea polyphenols, especially tea catechins. It is known to have a wide range of physiological activities such as reduction, anti-caries, anti-allergy, intestinal flora improvement, deodorization, etc. In the past, it has been attracting attention in the fields of pharmacy, phytochemistry, etc. Collecting.

  Tea theaflavins, which are oxidized derivatives of tea catechins, also have a characteristic physiological activity compared to the physiological activity of tea catechins. For example, an antibacterial action against Bordetella pertussis and an action to inhibit dental caries plaque formation are known.

  Tea theaflavins are known to mainly inhibit α-amylase which degrades starch and suppress absorption of sugars in the small intestine (Non-patent Document 1: Hara Y, Honda M., Agric. Biol. Chem). ., 54 (8), 1939-1945, 1990). Quercetin, a kind of flavonoid (Non-patent document 2: Watanabe J, Kawabata J, Kurihara H, Niki R., Biosci. Biotechnol. Biochem., 61 (1), 177-178, 1997), polyphenols of plants of the genus Salacia Components (Non-patent Document 3: Yoshikawa M, Nishida N, Shimoda H, Takada M, Kawahara Y, Matsuda H., Yakugaku Zasshi., 121 (5), 371-378, 2001) inhibit α-glucosidase. It has been known.

  In order to obtain a physiological effect suitable for the purpose by utilizing such a useful physiologically active action, it is necessary to increase the intake amount of the active ingredient. However, when tea catechins are spontaneously fermented, the yield of tea theaflavins produced as oxidized derivatives thereof is extremely low, and conventionally, an enzyme that breaks down sugars is inhibited by containing a high concentration of active ingredients. There is a need for functional food compositions with enhanced physiological activity.

  In order to inhibit not only enzymes that break down starch, but also enzymes that break down sucrose (maltose) and maltose (sucrose), sucrose (sucrose) has been used separately from the enzyme-inhibiting components that break down starch. In addition, it is necessary to use an enzyme-inhibiting component that decomposes maltose (maltose). For example, in JP-A-2004-105157 (Patent Document 1), green tea catechin that inhibits α-amylase and mulberry leaf component that inhibits α-glucosidase are used in combination at a specific ratio.

  Lipids are hydrolyzed by lipase, a lipolytic enzyme secreted from the pancreas, and absorbed into the body. In recent years, dietary high calories, especially an increase in lipid intake, has contributed to obesity. Obesity easily causes hypertension, diabetes, hyperlipidemia, and the like, and causes onset of stroke, heart disease, fatty liver, arteriosclerosis, and the like. In order to suppress lipid absorption, lipase inhibitors that inhibit lipolysis of lipases have been proposed. Examples of tea-derived lipase inhibitors include water extracts of green tea, black tea, and oolong tea (Japanese Patent Laid-Open No. Hei 3-198772: Patent Document 2), and epigallocatechin gallate of a green tea component (Japanese Patent Laid-Open No. Hei 3-228664: Patent Document 3). Have been reported.

  In general, it is known that teas such as green tea and black tea contain caffeine. Caffeine is an irritant, nerve-exciting action, an adverse effect depending on the amount, and may cause hallucinating symptoms if taken excessively. Is preferred. Therefore, the lower the caffeine content in the tea-derived food composition, the less anxiety and the safe continuous intake. Thus, it has been conventionally known to remove caffeine in a tea-derived food composition in order to prevent excessive intake of caffeine, but the presence of caffeine and α-glucosidase activity and lipase activity The relevance was unknown.

JP 2004-105157 A JP-A-3-219872 JP-A-3-228664 Hara Y et al., Agric. Biol. Chem., 54 (8), 1939-1945, 1990. Watanabe J et al., Biosci. Biotechnol. Biochem., 61 (1), 177-178, 1997. Yoshikawa M et al., Yakugaku Zasshi. 121 (5), 371-378, 2001.

A first object of the present invention is a food composition comprising various functionalities, including tea theaflavins, which inhibits enzymes that break down sugars and / or lipids, which are safe, highly effective, and can be continuously ingested on a daily basis. Is in the provision of.
The second object of the present invention is to provide a functional food composition containing tea theaflavins and having enhanced inhibitory activity of enzymes that degrade sugars and / or lipids.
The third object of the present invention is to provide a method for enhancing the α-glucosidase inhibitory activity and / or lipase inhibitory activity of a functional food composition containing tea theaflavins.

  As a result of intensive studies in view of the above problems, the present inventors have found that a functional food composition containing tea theaflavins at a specific component ratio has an effective α-glucosidase inhibitory activity and / or lipase inhibitory activity. The oral administration, particularly continuous oral administration, can enhance the α-glucosidase inhibitory activity, show the effect of improving fatty liver, and the caffeine content in the functional food composition containing tea theaflavins. When the amount is 1% by mass or less, it has been found that both the α-glucosidase inhibitory activity and / or the lipase inhibitory activity can be enhanced, and the present invention has been completed.

That is, the present invention provides the following functional food composition and a method for enhancing the enzyme inhibitory activity thereof.
1. As tea theaflavins, theaflavin 2-7% by mass, theaflavin-3-gallate 2-7% by mass, theaflavin-3′-gallate 2-10% by mass, and theaflavin-3,3′-digallate 2-16% by mass A functional food composition characterized by comprising.
2. As tea theaflavins, theaflavin 4-5% by mass, theaflavin-3-gallate 4-5% by mass, theaflavin-3′-gallate 6-8% by mass, and theaflavin-3,3′-digallate 10-13% by mass 2. The functional food composition as described in 1 above.
3. 3. The functional food composition according to 1 or 2 above, wherein the caffeine content in the functional food composition is 1% by mass or less.
4). 4. The functional food composition according to any one of 1 to 3 above, which has an α-glucosidase inhibitory activity.
5. 5. The functional food composition according to 4 above, which has an α-glucosidase inhibitory activity and an α-amylase inhibitory activity.
6). 6. The functional food composition according to 4 or 5 above, which has a schuclase inhibitory activity.
7). 7. The functional food composition according to any one of 4 to 6, which has a maltase inhibitory activity.
8). 8. The functional food composition according to any one of 1 to 7 above, which is a composition comprising tea polyphenol having α-glucosidase inhibitory activity for oral administration.
9. 9. The functional food composition according to 8 above, which has α-glucosidase inhibitory activity for continuous oral administration.
10. 10. The functional food composition according to any one of 1 to 9, which has a function of improving fatty liver.
11. 11. The functional food composition according to any one of 1 to 10, which has a lipase inhibitory activity.
12 A method for enhancing enzyme inhibitory activity, comprising enhancing α-glucosidase inhibitory activity and / or lipase inhibitory activity by setting the caffeine content in a functional food composition containing tea theaflavins to 1% by mass or less.
13. 13. The method for enhancing enzyme inhibition activity according to 12, wherein the tea theaflavins include theaflavin, theaflavin-3-gallate, theaflavin-3′-gallate, and theaflavin-3,3′-digallate.
14 As tea theaflavins, theaflavin 2-7% by mass, theaflavin-3-gallate 2-7% by mass, theaflavin-3′-gallate 2-10% by mass, and theaflavin-3,3′-digallate 2-16% by mass 14. The method for enhancing enzyme inhibition activity according to 13 above.
15. As tea theaflavins, theaflavin 4-5% by mass, theaflavin-3-gallate 4-5% by mass, theaflavin-3′-gallate 6-8% by mass, and theaflavin-3,3′-digallate 10-13% by mass 15. The method for enhancing enzyme inhibition activity according to 14 above.
16. 16. The enzyme inhibition activity enhancing method according to any one of 12 to 15, wherein the α-glucosidase inhibition activity is enhanced.
17. 17. The method for enhancing enzyme inhibition activity according to any one of the above 12 to 16, which enhances maltase inhibition activity.
18. 18. The method for enhancing enzyme inhibitory activity according to any one of 12 to 17, which enhances lipase inhibitory activity.

ADVANTAGE OF THE INVENTION According to this invention, the food composition which contains tea theaflavins, is safe and highly effective, can inhibit daily consumption of sugars and / or lipids which can be continuously ingested and has various functions. be able to.
ADVANTAGE OF THE INVENTION According to this invention, the functional food composition containing the tea theaflavins and having enhanced the inhibitory activity of the enzyme which decomposes | disassembles saccharide | sugar and / or lipid can be provided.
ADVANTAGE OF THE INVENTION According to this invention, the method of enhancing the alpha-glucosidase inhibitory activity and / or lipase inhibitory activity of the functional food composition containing tea theaflavins can be provided.

ADVANTAGE OF THE INVENTION According to this invention, the functional food composition which has the blood glucose level rise inhibitory effect and / or blood lipid rise inhibitory effect which can be continuously ingested easily and safely, taking a daily meal, and has various functionality Things can be provided.
ADVANTAGE OF THE INVENTION According to this invention, the foodstuff composition which improved the functionality which contains saccharides and / or the active ingredient which has a lipolytic enzyme inhibitory activity in high concentration can be provided.
In order to inhibit enzymes that degrade saccharides and lipids according to the present invention, tea polyphenols containing tea theaflavins in high concentrations obtained by air oxidation of tea leaves, preferably fresh Assam tea leaves, may be used as active ingredients. Well, other components that are used together are not necessarily required. However, such other components may be used in combination as necessary. As a result, tea theaflavins alone can also inhibit saccharides and lipid-degrading enzymes, for example, the effects of inhibiting enzymes that degrade starch contained in food, as well as sucrose (sucrose) and maltose (maltose). ) And enzymes that degrade lipids can be inhibited.

  According to the present invention, a composition containing tea polyphenols containing tea theaflavins in a high concentration obtained by air oxidation of tea leaves, preferably fresh Assam tea leaves, is only used as a component that inhibits α-amylase. In addition, it can be used as a component that inhibits α-glucosidase and lipase, and it is often used with a composition containing tea polyphenols containing tea theaflavins at high concentrations that inhibit enzymes that degrade sugars and lipids. Various kinds of enzyme inhibition effects can be obtained. That is, the functional food composition of the present invention contains tea theaflavins having various enzyme inhibitory activities such as α-amylase inhibitory activity, α-glucosidase inhibitory activity, and lipase inhibitory activity at high concentrations, It is not always necessary to use an α-glucosidase-inhibiting component, and α-amylase, α-glucosidase, and lipase can be inhibited using the multifunctional composition of the present invention that inhibits many kinds of enzymes. In the present invention, α-glucosidase means sucrose and maltase.

According to the present invention, the α-glucosidase inhibitory action can be enhanced by oral administration, particularly continuous oral administration.
According to the present invention, tea containing tea theaflavins at a high concentration, which exhibits various physiological activities including α-amylase inhibitory activity, α-glucosidase inhibitory activity, lipase inhibitory activity, and fatty liver improving effect. The food composition excellent in the functionality containing polyphenol can be provided.

  ADVANTAGE OF THE INVENTION According to this invention, the functional food by which the inhibitory activity of the enzyme which decomposes | disassembles saccharide | sugar and / or lipid was enhanced by making the caffeine content in the functional food composition containing tea theaflavins 1 mass% or less. A composition can be provided. Furthermore, according to the present invention, there is provided a method for enhancing α-glucosidase inhibitory activity and / or lipase inhibitory activity by setting the caffeine content in a functional food composition containing tea theaflavins to 1% by mass or less. be able to. The enhancement of the enzyme inhibitory activity is particularly excellent in that the inhibitory activity of maltase, which is a maltose-degrading enzyme produced from starch, can be enhanced about 3 times.

  The functional food composition of the present invention having saccharide and / or lipolytic enzyme inhibitory activity comprises tea polyphenol containing tea theaflavins at a high concentration as an active ingredient that inhibits α-glucosidase and / or lipase. And The tea theaflavins inhibit α-amylase, α-glucosidase, and / or lipase. In the present invention, tea is not particularly limited, and includes tea leaves produced by Assam-type leaves and powders and tea scraps produced or heated in the production process, and processed by twisting, and fresh tea leaves are particularly preferable.

  The functional food composition preferably contains tea polyphenol containing the tea theaflavins as a main component. The content ratio of the tea theaflavins as the main component in the tea polyphenol is preferably larger than the content ratio of any other components, and the tea theaflavins are contained in the tea polyphenol in an amount of 10% by mass or more, for example, 20% or more. It is preferably contained at a high concentration.

式（１）中、Ｒ¹，Ｒ²は、それぞれ独立して水素原子（Ｈ）またはガロイル基

を表す。 The tea theaflavins are specific dye compounds represented by the general formula (1) and having a benzotropolone ring.

In formula (1), R ¹ and R ² are each independently a hydrogen atom (H) or a galloyl group.

Represents.

式（２）中、Ｒ³，Ｒ⁴は、それぞれ独立して、Ｒ³は水素原子（Ｈ）またはガロイル基

を表し、Ｒ⁴は水素原子（Ｈ）または水酸基（ＯＨ）を表す。 The tea theaflavins represented by the formula (1) are generally produced by oxidative condensation between the B rings of the tea catechins represented by the general formula (2).

In Formula (2), R ³ and R ⁴ are each independently R ³ is a hydrogen atom (H) or a galloyl group

R ⁴ represents a hydrogen atom (H) or a hydroxyl group (OH).

  Tea theaflavins are produced by the action of oxidase in tea leaves during the fermentation process of tea leaves, and are therefore present in high content in oolong tea (semi-fermented tea) and especially black tea (fermented tea). Green tea (non-fermented tea) includes tea catechins. In addition, the content of tea catechins is remarkably low in oolong tea which is semi-fermented tea and black tea which is fermented tea. Usually, tea theaflavins are obtained by natural fermentation of tea catechins, but the yield of tea theaflavins produced as oxidized derivatives thereof is extremely low. For example, black tea contains only about 1%. .

  According to the present invention, tea theaflavins are concentrated at a high concentration by an oxidation reaction that engulfes air using tea leaves, typically fresh tea leaves of Assam, without using oxidases from other plants. Compositions containing tea polyphenols can be produced. That is, when the tea leaves are mixed with water to form a reaction mixture, the tea leaves are preferably homogenized with water to obtain a reaction mixture raw material, and the tea theaflavins with a high concentration when subjected to an oxidation reaction while embracing air therein. Can be obtained. In general, tea leaves, including fresh Assam tea leaves, have an oxidase enzyme in themselves. In the present invention, it is not always necessary to add an oxidase such as polyphenol oxidase to the reaction mixture. The oxidase may be added.

  As the tea leaves, fresh Assam seeds are preferably used. It is preferable to use it by fragmenting before mixing with water. The amount of water for homogenizing the raw tea leaves is usually 2 to 10 parts by mass, preferably 5 parts by mass with respect to 1 part by mass of tea leaves. Preferably, air is entrapped in the reaction mixture that has been homogenized and turned into a mud, and the tea catechins are subjected to an oxidation reaction. The oxidation reaction is usually carried out by oxidative fermentation by vigorously contacting air while stirring at high speed and embracing the air. Stirring may be performed by vibration or the like, but industrially, it is preferable to stir at high speed using a large screw propeller or the like. Although high-speed stirring changes with kinds, shapes, etc. of a reaction kettle and a stirrer, it can be normally implemented as a high speed of 300 rpm-1,000 rpm. In the oxidation reaction, the reaction temperature for incorporating air and reacting is preferably 20 to 50 ° C., and the reaction time is preferably 30 minutes to 6 hours. If the reaction temperature is lower than 20 ° C. or the reaction time is shorter than 30 minutes, tea theaflavins may not be obtained at a high concentration, and the reaction temperature may be higher than 50 ° C. If the time is too longer than 6 hours, the produced tea theaflavins may be decomposed and blackened, resulting in a decrease in yield and inability to obtain tea theaflavins at a high concentration.

  After homogenizing the raw fresh Assam tea leaves in 2 to 10 times the amount of water to make a mud, the reaction mixture was placed in an impeller-type agitator with a baffle-free kettle, or a horseshoe-shaped propeller, Using a lattice type propeller or screw propeller, stirring at high speed and embracing the air, bringing it into contact with air violently, bringing it into contact with the entrapped air and subjecting the tea catechins to an oxidation reaction This is particularly preferable because tea theaflavins can be obtained at a high concentration in a short time of 30 minutes to 1 hour even at a room temperature of 20 to 30 ° C. If there is a baffle (baffle plate) in the reaction kettle, the entrained air will escape, so a kettle without a baffle is preferable, and oxidative fermentation is carried out while enclosing as much air as possible in the reaction mixture. To oxidize tea catechins. As the reaction kettle for the oxidation reaction, it is preferable to use a kettle having a shape that easily generates air suction vortices. The air suction vortex is generated when high-speed stirring is performed in the kettle, and the kettle having a shape in which the air sucking vortex is likely to be generated easily traps air in the reaction mixture. An impeller-type stirrer easily traps air when an agitator with an impeller (stirring blade) is installed at an angle, and a horseshoe propeller, lattice-type propeller, or screw propeller also easily traps air. The kettle, stirrer, or propeller used in the oxidative fermentation reaction of the present invention stirs the reaction mixture at a high speed and makes the tea catechins vigorously contacted with air and oxidatively fermented while embracing the air. Other than the above can be used as long as they can sufficiently oxidize the compounds.

  As described above, the reaction mixture is subjected to an oxidative fermentation reaction that entraps air therein, and includes theaflavin, theaflavin-3-gallate, theaflavin-3′-gallate, and theaflavin-3,3′-digallate. A reaction product containing tea theaflavins can be obtained.

  After the oxidation reaction of the tea catechins, ethanol is added to the reaction product, preferably 0.5 to 3 parts by mass with respect to 1 part by mass of water in the reaction product, and the tea theaflavins are extracted, concentrated and activated. The tea polyphenol containing tea theaflavins at a high concentration can be separated by chromatography on the obtained treatment liquid after the clay treatment.

  Preferably, ethanol is added to the reaction product and stirred, and then the solid (residue) is filtered. The obtained filtrate is concentrated to an aqueous solution, and the resulting precipitate (such as chlorophyll and wax) is added. Unnecessary portion) is filtered off, the filtrate is treated with activated clay, the activated clay adsorbing caffeine in the filtrate is filtered off, and the filtrate is passed through a porous polystyrene resin column. The column adsorbed with a substance is eluted with 10-60% ethanol, and tea theaflavins are recovered at a high concentration from the 30-60% ethanol-eluted fraction.

  The obtained tea polyphenols are theaflavins (TF), theaflavin-3-gallate (TF3G), theaflavin-3′-gallate (TF3′G), and theaflavin-3,3′-digallate as tea theaflavins. (TF3,3′diG) is contained at a high concentration. For example, in the tea polyphenol, as the tea theaflavins, theaflavin 2-7% by mass, theaflavin-3-gallate 2-7% by mass, theaflavin-3′-gallate 2-10% by mass, and theaflavin-3, 2 to 16% by mass of 3′-digallate is included. As tea theaflavins, theaflavin 4-5% by mass, theaflavin-3-gallate 4-5% by mass, theaflavin-3′-gallate 6-8% by mass, and theaflavin-3,3′-digallate 10-13% by mass The inclusion is particularly preferred. Table 1 shows tea polyphenols A and B obtained by the method of the present invention as specific examples of tea polyphenols containing tea theaflavins at a high concentration. Table 1 shows the content of tea theaflavins in the tea polyphenols A and B in comparison with the content of tea theaflavins in tea powder (tea powder).

  As is apparent from Table 1, according to the present invention, tea polyphenols containing a high concentration of tea theaflavins produced by the oxidation reaction of tea catechins of fresh Assam tea leaves are contained, and the caffeine content is 1 mass. % Or less, preferably 0.5% or less, for example, a composition having a content of about 0.3 to 0.5%, having a saccharide and / or lipolytic enzyme inhibitory activity. It is done. While caffeine is involved in the bitter and astringent taste peculiar to tea, it is an irritant and also has a neuro-exciting effect, and depending on the amount, adverse effects appear. Is preferred. Therefore, by using a composition having a caffeine content of 1% by mass or less, it is possible to easily and safely continue to eat while taking a daily meal, and to have an effect of suppressing blood sugar level elevation and / or blood lipid elevation. The functional food composition which has can be provided.

As described above, caffeine is usually contained in teas such as green tea and black tea, and its large intake is not appropriate, and the lower the caffeine content in the tea-derived food composition, the less anxiety is. Since it can be safely and continuously ingested, caffeine was usually removed during the production of tea polyphenols in order to prevent excessive intake of caffeine.
The present inventors paid attention to the relationship between caffeine having a methylxanthine structure and enzyme inhibitory activity, and as a result of intensive studies, the presence of caffeine has an effect on enzyme inhibitory activity, and caffeine removal is α -It was found to enhance glucosidase inhibitory activity and / or lipase inhibitory activity.
That is, according to the present invention, the inhibitory activity of the enzyme that degrades saccharides and / or lipids can be enhanced by setting the caffeine content in the functional food composition containing tea theaflavins to 1% by mass or less. it can. Typically, an enzyme inhibitory activity enhancing method for enhancing α-glucosidase inhibitory activity and / or lipase inhibitory activity by setting the caffeine content in a functional food composition containing tea theaflavins to 1% by mass or less, And a functional food composition with enhanced enzyme inhibitory activity.

  In order to make the caffeine content 1% by mass or less, it is preferable to extract the tea theaflavins from the oxidation reaction product of the tea catechins and then treat the activated tea clay. The activated clay is very excellent as a caffeine removing agent that selectively adsorbs and removes caffeine in tea catechins (Nonaka et al., Journal of Japanese Society of Food Chemistry, Vol. 8 (1), 44-47, 2001). ). However, tea theaflavins are oxidative condensates of two molecules of tea catechins, which have greater molecular weight and steric restrictions than tea catechins, and their specificity as tea theaflavins is the adsorption of tea theaflavins by activated clay. There was a possibility of improving the sex. However, activated clay can selectively adsorb and remove caffeine in tea theaflavins and add it to the tea theaflavin extract so that the caffeine content can be reduced to 1% by mass or less. Was found. Specifically, for example, as described above, after adding ethanol to the oxidation reaction product and stirring, the solid (residue) is filtered, and the obtained filtrate is concentrated to an aqueous solution state, Caffeine content in tea theaflavins is obtained by filtering the generated precipitate (unnecessary matter), treating the filtrate with activated clay, and filtering the activated clay adsorbed caffeine in the filtrate. Can be made 1 mass% or less.

The theaflavins content was analyzed by HPLC under the following conditions.
HPLC conditions (for analysis of theaflavins): Cosmosi 15C ₁₈ -PAQ (4.6 × 250 mm) (Nacalai Tesque);
Solvent: 10-20% (5 min) -34% (40 min) -80% (5 min) CH ₃ CN in 50 mM aq. H ₃ PO ₄ ; 0.8 mL / min; 35 ° C.
5 μL injection; UV absorption at 375 nm,
Theaflavins: t _R 35.7min.
Since the absorption at 375 nm of theaflavin (TF) is not affected by the presence or absence of galloyl groups, a calibration curve was prepared using theaflavin (TF) without galloyl groups as a standard product, and the molar concentration of each theaflavin (TF) was calculated. Based on the content (mg), mass% was calculated.

Caffeine was quantified under the following conditions because it overlapped with other peaks under the analytical conditions that can separate the four theaflavins.
HPLC conditions (for caffeine analysis): Cosmosil 5C ₁₈ -AR-II (4.6 × 250 mm) (Nacalai Tesque);
Solvent: 4-30% (39 min) -75% (15 min) CH ₃ CN in 50 mM aq. H ₃ PO ₄ ; 0.8 mL / min; 35 ° C.
5 μL injection; UV absorption at 270 nm,
Theaflavins: t _R 35.7min.

The analysis result by HPLC of the main components contained in each sample (tea polyphenol A, B, and tea powder) is shown in FIG. 1 and FIG. The numbers attached to the peaks 1 to 13 shown in FIG. 1 and FIG. 2 represent the chemical components shown below.
1: Theaflavin;
2: Theaflavin 3-O-gallate;
3: Theaflavin 3′-O-gallate;
4: Theaflavin 3,3′-di-O-gallate;
5: Caffeine;
6: gallic acid;
7: Gallocatechin;
8: Epigallocatechin;
9: catechin;
10: Theasinensin A;
11: Epicatechin;
12: Epigallocatechin 3-O-gallate;
13: Epicatechin 3-O-gallate

FIG. 1 shows the HPLC chromatogram (conditions for analyzing theaflavins) (Max. Abs.) Of each sample (tea polyphenol A, B, and tea powder).
Sample concentration: ca. 12 mg / 10 mL → 5 μL injection,
HPLC conditions (for analysis of theaflavins): Cosmosi 15C ₁₈ -PAQ (4.6 × 250 mm) (Nacalai Tesque);
Solvent: 10-20% (5 min) -34% (40 min) 80% (5 min) CH ₃ CN in 50 mM aq. H ₃ PO ₄ ; 0.8 mL / min;

FIG. 2 shows an HPLC chromatogram (conditions for caffeine analysis) (Max. Abs.) Of each sample (tea polyphenols A, B, and tea powder).
Sample concentration: Ca. 12 mg / 10 mL → 5 μL injection;
HPLC conditions (for caffeine analysis): Cosmosi 15C ₁₈ -AR-II (4.6 × 250 mm) (Nacalai Tesque);
Solvent: 4-30% (39 min) -75% (15 min) CH ₃ CN in 50 mM aq. H ₃ PO ₄ ; 0.8 mL / min;

  As a result of HPLC analysis of each sample, tea polyphenols A and B are both rich in theaflavins (1-4) and catechins (7-9, 11-13), and caffeine (5) Almost no detection. In tea powder, caffeine (5) and gallic acid (6) were detected, but theaflavins and catechins were not detected at all.

[Α-glucosidase inhibitory activity by theaflavins]
Theaflavins contained in tea leaves inhibit α-amylase and suppress sugar absorption in the small intestine. In order to examine the action of inhibiting α-glucosidase using the tea polyphenol obtained previously as theaflavins, the influence of tea polyphenol on the blood glucose level increasing action by sucrose was examined. Tea polyphenol (300 , 600, 1000 mg / kg) suppressed the blood glucose level-elevating action due to sucrose loading by single oral administration, and it is considered that tea polyphenols inhibit α-glucosidase.

  Next, the effect of continuous administration of tea polyphenols on sucrose loading was examined. Continuous administration to rats was carried out by two methods: free intake from a water bottle and oral administration. In the free intake, the blood glucose level increasing action by sucrose load was suppressed at a concentration of 625 mg / kg / day (1, 2 weeks). Compared with this, in the case of oral administration, the blood glucose level increasing action was suppressed even at a lower concentration than 30,100 mg / kg / 1 day (2 weeks) and a single administration. From this, it is considered that continuous oral administration of tea polyphenol enhances the α-glucosidase inhibitory action.

  Next, the tea polyphenol was continuously administered for 5 to 6 weeks, and the influence on the body weight, motor function, and blood biochemical test value of the rat was examined. Neither free intake from water bottles nor forced oral administration had any effect on body weight, motor function, or blood biochemical test values, and the safety of tea polyphenols was confirmed at the doses and administration periods used. .

  Tea polyphenols were found to inhibit α-glucosidase and suppress sugar absorption in healthy rats. Next, the effects of tea polyphenols on blood glucose levels of pathological model animals were examined using GK / Jcl rats that are type 2 diabetes model animals and Zucker Fatty rats that are obesity model animals.

[Description of disease model animal]
<GK / Jcl>
GK / Jcl rats show no obesity, hyperglycemia after glucose load (insufficient glucose tolerance) due to insulin secretion deficiency, fasting hyperglycemia, and at the same time, plasma insulin during satiation is rather high and mild insulin resistance It is a type 2 diabetes model animal that is also seen in sex.

<Zucker Fatty Rat>
Zucker Fatty rats are well known as obese model animals, and become hyperlipidemia, hypercholesterolemia and hyperinsulinemia, and in addition, fat cell hypertrophy progresses. Obesity is an important environmental factor that causes insulin resistance. In obesity, an increase in the amount of neutral fat that accumulates in fat cells is observed, resulting in enlargement of fat cells. This enlargement is thought to affect adipocytokines (TNF-α, leptin, adiponectin) secreted from adipocytes and free fatty acids, thereby causing insulin resistance.

[Effects of tea polyphenols on blood glucose levels in disease model animals]
In GK / Jcl rats, which are type 2 diabetes model animals, tea polyphenol (30,300 mg / kg) suppressed the blood glucose level-elevating effect due to sucrose loading by single oral administration. 30 mg / kg was an unaffected dose in wistar rats. In GK / Jcl rats, hyperglycemia (abnormal glucose tolerance) after glucose loading is observed, which increases the sensitivity to sugar absorption and is considered to have an effect even at low concentrations (30 mg / kg).

  In continuous administration (free intake), the effects of satiety, fasting, and glucose loading were examined. With respect to the blood sugar level at the time of satiation, hyperglycemia at the time of satiation was significantly reduced on the 10th and 21st days after the administration of tea polyphenol (187 mg / kg). On the 49th and 70th day, a downward trend was observed.

  With respect to the blood glucose level increasing effect due to sucrose loading, the blood glucose level increase due to sucrose (20%) was significantly suppressed in the eighth week after the administration of tea polyphenol (23.8, 187 mg / kg).

  Thus, since tea polyphenol showed an improvement effect on hyperglycemia at the time of satiety and sugar load, it is considered to have an action of suppressing sugar absorption. When fasting, blood sugar is maintained by glycogenolysis and gluconeogenesis in the liver. Since tea polyphenols do not affect blood sugar levels during fasting, it is thought that there is no effect on glycogenolysis and gluconeogenesis in the liver.

  Moreover, about the influence with respect to the blood biochemical test value of tea polyphenol, it did not affect total cholesterol level (T-Cho), HDL cholesterol level (HDL-C), and neutral fat (TG). However, the GOT and GPT values showed a decreasing tendency due to tea polyphenol (23.8 mg / kg, 187 mg / kg). Diabetes is one of the causes of fatty liver and causes liver dysfunction. From this, it is considered that the lowering effect of GOT and GPT values by tea polyphenol indicates that tea polyphenol has an effect of improving fatty liver. To support this, polyphenols suppress the action of reducing fat content in the liver (Fiorini RN, et al., Liver Transpl. 11 (3), 298-308, 2005.) and fatty acid synthase. The action (Wang X et al., Biochem Biophys Res Commun. 288 (5), 1200-6, 2001.) has been reported.

  In addition, changes in body weight, insulin resistance, effects on tissue weight, effects on pancreas and liver tissues were examined, but no effect was observed.

  In Zucker Fatty rats, which are obese model animals, tea polyphenol (300 mg / kg) suppressed the blood glucose level-elevating effect due to sucrose loading by single oral administration.

  In continuous administration (free intake), the effects of satiety, fasting, and glucose loading were examined. As a result, a tendency to suppress the blood glucose level increasing action due to sucrose load was observed in the eighth week after administration of tea polyphenol (187 mg / kg).

  About the influence with respect to the blood biochemical test value of tea polyphenol, it had no influence on total cholesterol level (T-Cho), HDL cholesterol level (HDL-C), and neutral fat (TG). However, the GOT and GPT values were significantly decreased by tea polyphenol (29.6 mg / kg), the GPT value was also significantly decreased by 244 mg / kg, and the GOT value was also decreased. Obesity is also one of the causes of fatty liver and causes liver dysfunction. From this fact, it is considered that the lowering effect of GOT and GPT values in Zucker Fatty rats also indicates that tea polyphenol has an effect of improving fatty liver.

  In addition, changes in body weight, insulin resistance, tissue weight, pancreas and liver tissues were examined, but no effect was observed.

  Tea polyphenols were observed to inhibit sugar absorption even in disease model animals. In particular, the high blood glucose (abnormal glucose tolerance) observed in GK / Jcl rats was observed to be suppressed even by a single administration at a low concentration. Sugar absorption was also significantly suppressed by continuous administration (free intake). Thus, the sensitivity of tea polyphenols to sugar absorption was increased against abnormal hyperglycemia in disease model animals. Moreover, it is considered that tea polyphenol also has an effect of improving liver function, as an effect of lowering GOT and GPT values (liver function index) was observed.

[Effect of maltose and starch on sugar load]
After being ingested, starch, a polysaccharide, is hydrolyzed by the action of α-amylase in the oral cavity and α-amylase secreted from the pancreas in the small intestine, and finally by α-glucosidase present in the intestinal villi of the small intestine. It is broken down to glucose and absorbed. On the other hand, maltose and sucrose, which are disaccharides, are transported as they are to the small intestine, where they are decomposed into glucose and absorbed by the action of α-glucosidase. Then, when the influence of the tea polyphenol with respect to the sugar load by maltose and starch was examined, the inhibitory action with respect to the sugar load by maltose and starch was recognized by the tea polyphenol. Since tea polyphenols were also effective against sucrose sugar loading, tea polyphenols reliably suppressed the blood glucose level-increasing action due to sugar loading, and the mechanism involved α-glucosidase inhibitory action. It became clear. Summarizing the effects of tea polyphenols on sugar load, sucrose was 300 mg / kg, maltose was 30 mg / kg, starch was 300 mg / kg, and glucose was ineffective. It showed a particularly strong inhibitory action. As described above, it has been clarified that tea polyphenols have a sugar absorption inhibitory effect on sugar load caused by sucrose, maltose, and starch, and the α-glucosidase inhibitory action of tea polyphenols is involved in the mechanism. It was.

[Effects on olive oil and corn oil loading]
Tea polyphenols reduced fat absorption by reducing the increase in triglyceride levels caused by loading with olive oil and corn oil by single oral administration. Triglycerides are broken down into free fatty acids and glycerin by pancreatic lipase and absorbed from the intestinal villi. In addition, tea polyphenol having a galloyl group (Theaflavin 3-O-gallate) has been reported to inhibit pancreatic lipase (Kusano R, Andou H, Fujieda M, Tanaka T, Matsuo Y, Kouno I. Chem Pharm Bull (Tokyo). 2008; 56: 266-72). From these facts, it is considered that the tea polyphenol used in this experiment suppresses fat absorption by inhibiting pancreatic lipase and has a preventive effect on hyperlipidemia.

[Effect of caffeine removal on α-glucosidase inhibitory activity]
It was confirmed that the α-glucosidase inhibitory activity was enhanced by setting the caffeine content in the functional food composition containing tea theaflavins to 1% by mass or less.
The amount (IC50) that activates α-glucosidase (maltase schuclase) in 1 mL of solvent in a test tube and inhibits the activity by 50% is shown in the following table. The caffeine-unremoved product has the same amount of theaflavin.

  As is clear from Table 1 (2), maltase and sucrase are removed by removing caffeine from the functional food composition containing tea theaflavins and reducing the caffeine content to 1% by mass or less. ) Each inhibitory activity is enhanced by about 10 to 300%. When the caffeine content in the functional food composition is removed to 1% by mass or less, both the inhibitory activities of both maltase and sucrose are enhanced, and particularly the inhibitory activity of maltase, which is a maltose degrading enzyme produced from sugars such as starch. Was found to be about three times as new. From this, when the tea polyphenol from which the caffeine content in the functional food composition is removed to 1% by mass or less is taken as a food material at the same time as sugars such as starch, the blood glucose level increase suppressing effect is remarkably enhanced. I understand.

[Effect of caffeine removal on lipase inhibitory activity]
It was confirmed that the lipase inhibitory activity was enhanced by setting the caffeine content in the functional food composition containing tea theaflavins to 1% by mass or less.
The amount (IC50) that activates lipase in a test tube and inhibits the activity by 50% is shown in the following table. The caffeine-unremoved product has the same amount of theaflavin.

    As is clear from Table 1 (3), the lipase inhibitory activity can be obtained by removing caffeine from the functional food composition containing tea theaflavins and reducing the caffeine content to 1% by mass or less. About 20% enhancement. It has been a new finding that when the caffeine content in the functional food composition is removed to 1% by mass or less, the inhibitory activity of lipase is enhanced and the inhibitory activity is enhanced by about 20%. From this, it can be seen that when the tea polyphenol from which the caffeine content in the functional food composition is removed to 1% by mass or less is taken as a food material at the same time as the lipid, the blood lipid rise inhibitory effect is enhanced.

  The tea polyphenol has a high concentration of tea theaflavins that exhibit various bioactive effects including an action to inhibit α-amylase, an action to inhibit α-glucosidase, an action to inhibit lipase, and an action to improve fatty liver. Since it contains, the functional food composition containing this is not only excellent in the body active absorption and the physiologically active concentration range that inhibits the enzyme that breaks down saccharides and lipids, but also has various effects including the action of improving fatty liver. It is excellent in that it exhibits a bioactive effect. In addition, according to the functional food composition containing tea polyphenols containing a high concentration of active ingredients exhibiting various bioactive effects, the intake of the active ingredients is increased, so that excellent bioactive effects are obtained. . Moreover, since the functional food composition containing the tea polyphenol which raised the active ingredient purity can be ingested, its usefulness is high.

  Tea theaflavins, which are active ingredients in tea polyphenols contained in the functional food composition according to the present invention, are a kind of plant polyphenols and are also natural ingredients. Tea has been ingested on a daily basis since ancient times, and the safety of tea theaflavins is very high. The functional food composition according to the present invention can be easily and continuously consumed while taking a daily meal.

  The tea polyphenol may be used as it is as the functional food composition according to the present invention, but may be further fractionated and purified as necessary. When the total content of tea theaflavins is 10% by mass or more, for example, 20% or more with respect to the functional food composition according to the present invention, physiological activity that inhibits in vivo absorption and enzymes that degrade saccharides and lipids This is preferable because it is excellent in the action concentration range and the various physiologically active effects are particularly high. In addition, the higher the total content of tea theaflavins in the tea polyphenol contained in the functional food composition according to the present invention, the higher the concentration, the bitterness, astringency, flavor, color, solubility, etc. The tea theaflavins total content is preferably 40% or less because there may be a limit to the blending. Accordingly, the total content of tea theaflavins in the tea polyphenol contained in the functional food composition according to the present invention is usually preferably 20 to 40%, for example, a concentration of around 30% of 25 to 35%. By ingesting the functional food composition according to the present invention, in particular, blood glucose level increase inhibitory effect, blood lipid increase inhibitory effect, and prevention of fatty liver can be expected, improvement effect, prevention of diet or lifestyle-related diseases, An improvement effect can be expected.

  The functional food composition according to the present invention is used, for example, as a powder, granule, or tablet, with tea polyphenol alone or with excipients such as gelatin and sodium alginate, or with tea polyphenol alone or in combination. Can be used, for example, by dissolving it in a solvent such as water or ethyl alcohol and adding it to food. Alternatively, it can be dissolved in water to make a beverage. As the dosage form, for example, an appropriate amount of tea polyphenol is used alone or in a solution obtained by dissolving it in a solvent such as water or ethyl alcohol, or an appropriate excipient or diluent (for example, , Oligosaccharides and carboxymethylcellulose).

  The intake of the functional food composition according to the present invention is usually a tea theaflavin total amount, preferably 80 to 400 mg / kg per day, particularly preferably 120 to 240 mg / kg. Continuous oral intake of the functional food composition is preferable because it enhances the α-glucosidase inhibitory action. In addition, when mix | blending the functional food composition which concerns on this invention with a foodstuff, the compounding quantity should just mix | blend suitably based on said preferable intake.

  The present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.

[Production of tea theaflavins by oxidation reaction of tea catechins]
Use fresh tea leaves (Assam seed) as a raw material, do not use other plant-derived oxidases, charge them in a baffle-free reaction kettle, and use water to make 5 times the amount of tea leaves per kg It was homogenized with 5 L of water at room temperature to obtain a muddy muddy state. To this, air was added at a flow rate of 0.1 L / sec, and a large screw propeller was used to stir at high speed (rotational speed: 300 rpm to 1,000 rpm) to vigorously contact the air while embracing the air. The oxidative fermentation reaction was carried out at 1 ° C. for 1 hour. After adding 10 L of ethanol twice as much as the amount of water in the reaction and stirring, the solid (residue) was filtered and the resulting filtrate was concentrated to an aqueous solution. The generated precipitate (unnecessary components such as chlorophyll and wax) was filtered off, the filtrate was treated with 2 kg of activated clay, and activated clay adsorbed caffeine in the filtrate was filtered off. After passing the filtrate through a column containing 20 L of porous polystyrene resin (Mitsubishi Chemical, Diaion, HP20), tea catechins adsorbed on the column were eluted with 10 to 20% ethanol, and then 30 to 30 Tea theaflavins were eluted with 60% ethanol, and tea polyphenol A (06051) containing a high concentration of tea theaflavins was separated and recovered. Similarly, tea polyphenol B (06112) was separated and collected as a separate lot (yield is typically 10 to 20 g).

(1) Tea polyphenols suppress blood glucose level rise [Experimental method]
1) Glucose and Sucrose increase blood glucose level The experimental animals used were 5-week-old male Wistar rats. After free intake of food and water for 1 week from 5 weeks of age, the animals were fasted for 16 hours from the previous day. Thereafter, Glucose (10, 20%) and Sucrose (10, 20%) were orally administered, and blood glucose levels were measured before administration (pre) and 30, 60, 120 minutes after administration. Blood was collected from the tail vein and measured with a small blood glucose meter (Glutest Ace R, Sanwa Chemical Laboratory Co., Ltd.).

2) Effect of single administration of tea polyphenol on glucose load The experimental animals used were 5 week old male Wistar rats. After free intake of food and water for 1 week from 5 weeks of age, the animals were fasted for 16 hours from the previous day. Thereafter, Glucose (20%) and Sucrose (20%) were orally administered simultaneously with tea polyphenol (polyphenol A or B, the same shall apply hereinafter) (Vehicle, 100, 300, 600, 1000 mg / kg), and before administration (pre ), Blood glucose level was measured 30, 60, 120 minutes after administration. Blood was collected from the tail vein and measured with a small blood glucose meter (Glutest Ace R, Sanwa Chemical Laboratory Co., Ltd.).

3) Effect of continuous administration of tea polyphenols (free intake from water bottles) The experimental animals used were 5-week-old male Wistar rats. After free intake of food and water for 1 week from 5 weeks of age, 5 groups of vehicle administration group (Vehicle), 30 mg / kg administration group, 100 mg / kg administration group, 300 mg / kg administration group, 1000 mg / kg administration group Each group was divided into two groups, and free intake was started by putting each in a water bottle at 6 weeks of age (tea polyphenol (water supply): Vehicle, 30 mg / kg (30 mg / 200 mL), 100 mg / kg (100 mg / 200 mL), 300 mg / kg). (300 mg / 200 mL), 1000 mg / kg (1000 mg / 200 mL)). Body weight is measured every day, glucose tolerance test with Sucrose (20%) is performed in the first, second, and fourth weeks after the start of continuous administration, and motor function measurement and blood biochemical tests are performed in the fifth to sixth weeks. Went to. In the glucose tolerance test, Sucrose (20%) was orally administered, and blood glucose levels were measured before administration (pre) and 30, 60, 120 minutes after administration. Blood was collected from the tail vein and measured with a small blood glucose meter (Glutest Ace R, Sanwa Chemical Laboratory Co., Ltd.). Motor function measurement was performed using the Open-field method and the Rota-rod method. For blood biochemical tests, blood was collected from an inferior vena cava under anesthesia (pentobarbital 50 mg / kg) using a syringe with heparin to prevent blood coagulation. Measurement was performed with an analyzer (Spotchem EZSP-4430, ARKRAY, Inc.).

4) Effect of continuous administration of tea polyphenol (oral administration) Male Wistar rats aged 5 weeks were used as experimental animals. After free intake of food and water from 5 weeks of age to 1 week, it was divided into 3 groups: vehicle administration group, vehicle administration group, 30 mg / kg administration group and 100 mg / kg administration group, and each oral administration from 6 weeks of age. (Tea polyphenol (oral): Vehicle, 30 mg / kg, 100 mg / kg). The body weight was measured every day, the glucose tolerance test with Sucrose (20%) was performed in the first and second weeks after the start of continuous administration, and the motor function measurement and blood biochemical test were performed in the 5th to 6th weeks. In the glucose tolerance test, Sucrose (20%) was orally administered simultaneously with tea polyphenol on each day, and blood glucose levels were measured before administration (pre) and 30, 60, 120 minutes after administration. Blood was collected from the tail vein and measured with a small blood glucose meter (Glutest Ace R, Sanwa Chemical Laboratory Co., Ltd.). Motor function measurement was performed using the Open-field method and the Rota-rod method. For blood biochemistry, blood was collected from an inferior vena cava under anesthesia (pentobarbital 50 mg / kg) using a syringe with heparin to prevent blood coagulation, and the resulting plasma was collected from clinical chemistry. Measurement was performed with an automatic analyzer (Spotchem EZSP-4430, ARKRAY, Inc.).

[Experimental result]
1) Glucose and Sucrose increase blood glucose level
In both Glucose (10, 20%) and Sucrose (10, 20%), blood glucose increased at a peak of 30 minutes, but glucose increased more than sucrose. From these results, it was decided to use a concentration of 20% for both glucose and sucrose (FIGS. 3A and B).

2) Effect of a single administration of tea polyphenol on sugar load 2-1) Glucose load due to Glucose Tea polyphenol had no effect on the increase in blood glucose level due to glucose (20%) (FIG. 4A).

2-2) Sugar load by Sucrose Tea polyphenol suppressed the blood glucose level increase by sucrose (20%) in a dose-dependent manner (FIG. 4B).
In 30 minutes after administration of tea polyphenol and sucrose, it was suppressed almost equally at 300 to 1000 mg / kg (FIG. 4C).

3) Effects of continuous administration of tea polyphenols (free intake from water bottles) 3-1) Water intake from water bottles Assuming that the body weight of a rat is 200g per animal, the daily water intake per animal is from a preliminary test. It was confirmed that the volume was 40 mL. The dose calculated based on these is shown in the left column of Table 2 below. However, tea polyphenol has a bitter taste, and a concentration-dependent decrease in water intake was observed. In the range of 300 to 1000 mg / kg, the water intake decreased by about 10 mL compared to the solvent group. Therefore, the final concentration is as described in the right column of Table 2 below.

3-2) Effects on blood glucose level and body weight
The blood glucose level before loading with Sucrose was not affected at any dose (FIG. 5A). Also, the body weight was not affected (FIG. 5B).

3-3) Sugar loading by Sucrose
None of the doses had an effect on the increase in blood glucose level during the first week after loading with Sucrose (FIG. 6A). However, in the first and second weeks after the administration of tea polyphenol, the highest concentration of 625 mg / kg (set concentration of 1000 mg / kg) significantly suppressed the blood glucose level increasing action (FIG. 6B).
In the 4th week, only the dose that was not affected in the 1st and 2nd weeks (29.5, 90.3, 218 mg / kg) was examined, but none of the doses was affected (FIG. 6C).

3-4) Effect on motor function Administration of tea polyphenol for 5 to 6 weeks did not affect motor function (Table 3).

3-5) Influence on blood biochemical test value The administration of tea polyphenol for 5 to 6 weeks did not affect the blood biochemical test value (Table 4).

4) Effect of continuous administration of tea polyphenol (oral administration) A dose of 30-100 mg / kg that was not affected by a single administration of tea polyphenol was forcibly administered orally.

4-1) Effects on blood glucose level and body weight
The blood glucose level before loading with Sucrose was not affected at any dose (FIG. 7A). Also, the body weight was not affected (FIG. 7B).

4-2) Sugar loading by Sucrose
Tea polyphenol 30,100 mg / kg showed an inhibitory action against the increase in blood glucose level in the first and second weeks when Sucrose was loaded (FIGS. 8A and 8B).

4-3) Influence on motor function Administration of tea polyphenol for 5 to 6 weeks did not affect motor function (Table 3).

4-4) Effect on blood biochemical test values Administration of tea polyphenol for 5 to 6 weeks did not affect blood biochemical test values (Table 4).

(2) Suppression of blood glucose level increase in animal model animals by tea polyphenol [Experimental method]
1) Effect of single administration of tea polyphenol on sugar load by Sucrose Five-week-old male GK / Jcl rats and Zucker Fatty rats were used as experimental animals. After free intake of food and water for 1 week from 5 weeks of age, the animals were fasted for 16 hours from the previous day. Thereafter, Sucrose (20%) was orally administered simultaneously with tea polyphenol (Vehicle, 30, 300 mg / kg), and blood glucose levels were measured before administration (pre) and 30, 60, 120 minutes after administration. Blood was collected from the tail vein and measured with a small blood glucose meter (Glutest Ace R, Sanwa Chemical Laboratory Co., Ltd.).

2) Effect of continuous administration of tea polyphenol (free intake from water bottle) on blood sugar level and sugar load by Sucrose Five-week-old male GK / Jcl rats and Zucker Fatty rats were used as experimental animals. After free intake of food and water for 1 week from 5 weeks of age, it was divided into 3 groups: vehicle administration group (Vehicle), 30 mg / kg administration group, 300 mg / kg administration group (see Table 5), 6 weeks From the age, each was placed in a water bottle, and free intake was started.

  Blood glucose measurement and body weight measurement were performed at 0, 5, 10, 21, 49, and 70 days after administration, glucose tolerance test was performed at 14, 28, and 56 days, insulin resistance test was performed at 75 days, blood biochemical test and Tissue (liver, heart, testicular fat) mass measurements were performed on day 80. Blood collection in blood glucose measurement was performed from the tail vein and measured with a small blood glucose measurement device (Glutest Ace R, Sanwa Chemical Laboratory Co., Ltd.). In the glucose tolerance test, Sucrose (20%) was orally administered after fasting for 16 hours from the previous day, and blood glucose levels were measured before (pre) and 30, 60, and 120 minutes after administration. In the insulin resistance test, after fasting for 4 hours, insulin (0.75 U / kg) was intraperitoneally administered, and blood glucose levels were measured before administration (pre) and 60, 120, and 180 minutes after administration. For blood biochemical tests, blood was collected from an inferior vena cava under anesthesia (pentobarbital 50 mg / kg) using a syringe with heparin to prevent blood coagulation. Measurement was performed with an analyzer (Spotchem EZSP-4430, ARKRAY, Inc.). In histological examination, pancreatic and liver tissues were stained with hematoxylin and eosin (HE) and observed under an inverted microscope (magnification: 50 times).

[Experimental result]
<GK / Jcl rat>
1) Effect of single administration of tea polyphenol on sugar load by Sucrose Tea polyphenol (30,300 mg / kg) showed a dose-dependent inhibitory effect on blood sugar level increase by sucrose (20%) (FIG. 9). .

2) Effects of continuous administration of tea polyphenol (free intake from water bottle) 2-1) Intake from water bottle
The weight of GK / Jcl rats was assumed to be 200 g per animal for 0 to 43 days and 300 g per animal for 44 to 80 days, and it was confirmed from a preliminary test that the daily water intake was 40 mL per animal. The dose calculated based on these is described in the left column of Table 6 below. However, tea polyphenol has a bitter taste, and a concentration-dependent decrease in water intake was observed. At 300 mg / kg, the water intake decreased by about 10 mL compared to the solvent group. Therefore, the final concentration is as described in the right column of Table 6 below.

2-2) Influence on blood glucose level and body weight Tea polyphenol 187 mg / kg (set concentration 300 mg / kg) significantly reduced hyperglycemia at satiation on the 10th and 21st day of administration (FIG. 10A). On days 49 and 70, no significant lowering effect was observed, but this tendency was observed. In addition, tea polyphenols did not affect the blood sugar level at fasting before loading sucrose (FIG. 10B). There was no effect on body weight (FIG. 11).

2-3) Sugar load caused by sucrose Tea polyphenols did not affect the increase in blood glucose level caused by sucrose (20%) at 2 and 4 weeks after administration (FIGS. 12A and 12B). However, in the 8th week, tea polyphenol 23.8 mg / kg (set concentration 30 mg / kg) and 187 mg / kg (set concentration 300 mg / kg) significantly suppressed blood sugar level increase by sucrose (20%) (FIG. 12C). .

2-4) Insulin tolerance test Tea polyphenols had no effect on insulin resistance (Figure 13).

2-5) Effect on tissue mass (liver, heart, testicular fat) Tea polyphenols did not affect the mass of liver, heart, testicular fat (Table 7).

2-6) Influence on blood biochemical test values Total cholesterol level (T-Cho), HDL cholesterol level (HDL-C) and neutral fat (TG) were not affected by tea polyphenols. Moreover, tea polyphenol 23.8 mg / kg (setting concentration 30 mg / kg) and 187 mg / kg (setting concentration 300 mg / kg) showed a decreasing tendency with respect to GOT and GPT values (Table 8).

2-7) Histological examination of pancreas and liver Tea polyphenols did not affect pancreas and liver tissues (Fig. 14).

<Zucker Fatty Rat>
1) Effect of single administration of tea polyphenol on sugar load by Sucrose Tea polyphenol (300 mg / kg) significantly suppressed the increase in blood glucose level by sucrose (20%) (FIG. 15).

2) Effects of continuous administration of tea polyphenols (free intake from picture water bottles) 2-1) Intake from water bottles
The body weight of Zucker Fatty rats was assumed to be 300 g per animal for 0 to 43 days and 500 g per animal for 44 to 80 days, and it was confirmed from a preliminary test that the daily water intake was 40 mL per animal. The dose calculated based on these is described in the left column of Table 9 below. However, tea polyphenol has a bitter taste, and a concentration-dependent decrease in water intake was observed. At 300 mg / kg, the water intake decreased by about 10 mL compared to the solvent group. Therefore, the final concentration is as described in the right column of Table 9 below.

2-2) Effect on blood glucose level and body weight Tea polyphenols did not affect blood glucose level at the time of satiety and fasting (FIGS. 16A and 16B). Also, the body weight was not affected (FIG. 17).

2-3) Glucose load by sucrose Tea polyphenols did not affect the blood glucose level increase by sucrose (20%) at 2, 4, and 8 weeks after administration (FIGS. 18A, B, and C). However, in the 8th week, a tendency of suppression was observed with tea polyphenol 187 mg / kg (set concentration 300 mg / kg) (FIG. 18C).

2-4) Insulin tolerance test Tea polyphenols did not affect insulin resistance (Figure 19).

2-5) Effect on tissue mass (liver, heart, testicular fat) Tea polyphenols did not affect the mass of liver, heart, testicular fat (Table 10).

2-6) Influence on blood biochemical test values Total cholesterol level (T-Cho), HDL cholesterol level (HDL-C) and neutral fat (TG) were not changed by tea polyphenol. In addition, tea polyphenol 29.6 mg / kg (set concentration 30 mg / kg) significantly reduced GOT and GPT values. 244 mg / kg (set concentration 300 mg / kg) also significantly decreased the GPT value and showed a tendency to decrease with respect to the GOT value (Table 11).

2-7) Histological examination of pancreas and liver Tea polyphenols did not affect pancreas and liver tissues (FIG. 20).
Table 12 shows the significance and normal values (persons) of total cholesterol (T-Cho), high-density lipoprotein cholesterol (HDL-C), triglyceride (TG: neutral fat), and transaminase (GOT, GPT). .

(3) Effects of a single dose of tea polyphenol on sugar load from maltose and starch [Experimental method]
As experimental animals, 5-week-old male Wistar rats were used. After free intake of food and water for 1 week from 5 weeks of age, the animals were fasted for 16 hours from the previous day. Thereafter, maltose (20%) and starch (20%) were orally administered simultaneously with tea polyphenol, and blood glucose levels were measured before (pre) and 30, 60, and 120 minutes after administration. Blood was collected from the tail vein and measured with a small blood glucose meter (Glutest Ace R, Sanwa Chemical Laboratory Co., Ltd.).

[Experimental result]
1) Effect on glucose load by maltose Tea polyphenol suppressed the increase in blood glucose level caused by maltose (20%) in a dose-dependent manner (FIG. 21).
2) Effect of starch on sugar load Tea polyphenol suppressed the increase in blood glucose level caused by starch (20%) in a dose-dependent manner (FIG. 22).

(4) Effects of a single dose of tea polyphenol on olive oil and corn oil load [Purpose]
As a pharmacological action of tea polyphenol, lipase inhibition has been reported to improve hyperlipidemia (Maron DJ, Lu GP, Cai NS, Wu ZG, Li YH, Chen H, Zhu JQ, Jin XJ, Wouters BC, Zhao J. Arch Intern Med. 2003; 163: 1448-53). Therefore, in order to examine the effect of tea polyphenols on hyperlipidemia, the effect of tea polyphenols on the blood triglyceride (neutral fat) level increasing effect of olive oil and corn oil was examined.

[experimental method]
As experimental animals, male Wistar rats aged 5 weeks were used. Food and water were freely consumed from 5 weeks of age for 1 week. Olive oil (5mL / kg, Nacalai Tesque) and corn oil (10mL / kg, Sigma) are orally administered simultaneously with tea polyphenols, respectively, with olive oil for 16 hours after fasting, 180 minutes after administration, and for corn oil for 16 hours. The blood triglyceride level was measured 120 minutes after administration after fasting. Laparotomy under anesthesia (pentobarbital 50mg / kg), blood was collected from the inferior vena cava using a syringe with heparin in advance to prevent blood coagulation, and the resulting plasma was collected from an automated clinical chemistry analyzer (Spotchem EZ SP). -4430, ARKRAY Inc.).

[result]
Tea polyphenol (300 mg / kg) significantly suppressed the increase in triglyceride level due to the load of olive oil and corn oil (FIGS. 25 and 26).

(5) Effects of tea polyphenols on α-glucosidase and lipase [Experimental purpose]
It is clear from in vivo experiments that tea polyphenol has a sugar absorption inhibitory action and a fat absorption inhibitory action. In this experiment, the effect of tea polyphenols on α-glucosidase and lipase was examined in an in vitro experiment, and its action was compared with that containing caffeine.

[experimental method]
1) Influence on α-glucosidase activity 50 μL of a tea polyphenol solution was added to 100 μL of a sucrose (74 mM) and maltose (74 mM) solution as a substrate, and pre-warmed at 37 ° C. for 2 to 3 minutes. 50 μL of enzyme solution was added and reacted for 30 minutes, 800 μL of water was added, and the mixture was heated in a boiling water bath for 2 minutes to deactivate the enzyme. Separately, after adding an enzyme solution to each sample, water was immediately added and heated in a boiling water bath for 2 minutes to inactivate the enzyme, and a blank was used. The amount of glucose produced was measured by the glucose oxidase method (glucose CII test Wako). The substrate, enzyme solution, and tea polyphenol were dissolved in 0.1 M maleate buffer (pH 6.0). The enzyme solution was suspended in rat small intestine acetone powder (SIGMA) by adding 10 volumes of 0.1 M maleic acid buffer (pH 6.0), and the supernatant obtained by sonication and centrifugation was used as the enzyme preparation. .

2) Influence on pancreatic lipase activity The lipase activity was measured using a lipase kit S (Dainippon Pharmaceutical Co., Ltd.). Add 50 μL of enzyme solution and 20 μL of esterase inhibitor to 1 mL of coloring solution (2-nitrobenzoic acid), preheat at 30 ° C. for 5 minutes, then add 100 μL of tea polyphenol solution and substrate solution (dimercaprol tributyrate). The reaction was carried out at 30 ° C. for 30 minutes under light shielding. After 30 minutes, 2 mL of the reaction stop solution was added, and the absorbance at 412 nm was measured. Separately, a color developing solution, an enzyme solution, an esterase inhibitor, and a tea polyphenol solution were added and reacted for 30 minutes, and then a reaction stop solution and a substrate solution were sequentially added to make a blank. The enzyme solution was suspended by adding 10 times the amount of distilled water to rat small intestine acetone powder (SIGMA), and the supernatant obtained by sonication and centrifugation was used as the enzyme preparation. In any experiment, the blending ratio of tea polyphenol and caffeine was 1: 0.66.

[Experimental result]
1) Effect on α-glucosidase activity Tea polyphenol (: caffeine content 0.4%), tea polyphenol (: caffeine content 0.4%) + caffeine tea polyphenol (: total caffeine content 40 %) Both inhibited maltase and sucrase in a concentration-dependent manner, but tea polyphenols with a caffeine content of 0.4% were more potent.
Maltase (maltase); tea polyphenols _{IC 50 = 0.51mg / mL, IC} 50 = 1.54mg / ml of tea polyphenol + Caffeine (caffeine), and sucrase (sucrase); tea polyphenols IC ₅₀ = 2.29mg FIG. 23 shows IC ₅₀ = 2.59 mg / mL of / polyester, tea polyphenol + caffeine.

2) Effect on pancreatic lipase activity Tea polyphenol (: caffeine content 0.4%), tea polyphenol (: caffeine content 0.4%) + caffeine tea polyphenol (: total caffeine content 40% ) Both inhibited pancreatic lipase in a concentration-dependent manner, but tea polyphenols with a caffeine content of 0.4% were more potent.
FIG. 24 shows the IC _{50 of} tea polyphenol = 1.73 mg / ml and the IC _{50 of} tea polyphenol + caffeine = 2.1 mg / mL.

It is a HPLC chromatogram (conditions for theaflavins analysis) (Max. Abs.) Of each sample (tea polyphenol A, B, and tea powder). It is a HPLC chromatogram (conditions for caffeine analysis) (Max. Abs.) Of each sample (tea polyphenol A, B, and tea powder). It is a graph showing the time-dependent change of the blood glucose level increase effect by Glucose and Sucrose. It is a graph showing the influence of the tea polyphenol single administration with respect to Sucrose load. It is a graph showing the blood glucose level and weight change by theaflavin continuous administration (free ingestion). It is a graph showing the influence of the Theaflavin continuous administration (free intake) with respect to a glucose load. It is a graph showing the blood glucose level and body weight change by theaflavin continuous administration (oral administration). It is a graph showing the influence of Theaflavin continuous administration (oral administration) with respect to Sucrose load. It is a graph showing the influence of the tea polyphenol single administration with respect to Sucrose load. It is a graph showing the blood glucose level change by tea polyphenol continuous administration (free ingestion). It is a graph showing the body weight change by tea polyphenol continuous administration (free intake). It is a graph showing the influence of the tea polyphenol continuous administration (free intake) with respect to Sucrose load. It is a graph showing the influence of tea polyphenol continuous administration (free intake) in an insulin tolerance test. It is a microscope picture (50 times) showing the influence of the tea polyphenol continuous administration (free ingestion) in the tissue image of a pancreas and a liver. It is a graph showing the influence of the tea polyphenol single administration with respect to Sucrose load. It is a graph showing the blood glucose level change by tea polyphenol continuous administration (free ingestion). It is a graph showing the body weight change by tea polyphenol continuous administration (free intake). It is a graph showing the influence of the tea polyphenol continuous administration (free intake) with respect to Sucrose load. It is a graph showing the influence of tea polyphenol continuous administration (free intake) in an insulin tolerance test. It is a microscope picture (50 times) showing the influence of the tea polyphenol continuous administration (free ingestion) in the tissue image of a pancreas and a liver. It is a graph showing the influence of the tea polyphenol single administration with respect to maltose load. It is a graph showing the influence of the tea polyphenol single administration with respect to starch load. It is a graph showing an in vitro (in vitro) alpha-glucosidase inhibitory activity. It is a graph showing the in vitro (in vitro) pancreatic lipase inhibitory activity. It is a graph showing the influence of the tea polyphenol single administration with respect to olive oil load. It is a graph showing the influence of the tea polyphenol single administration with respect to corn oil load.

Claims (18)

  As tea theaflavins, theaflavin 2-7% by mass, theaflavin-3-gallate 2-7% by mass, theaflavin-3′-gallate 2-10% by mass, and theaflavin-3,3′-digallate 2-16% by mass A functional food composition characterized by comprising.   As tea theaflavins, theaflavin 4-5% by mass, theaflavin-3-gallate 4-5% by mass, theaflavin-3′-gallate 6-8% by mass, and theaflavin-3,3′-digallate 10-13% by mass The functional food composition according to claim 1, comprising:   The functional food composition according to claim 1 or 2, wherein a caffeine content in the functional food composition is 1% by mass or less.   The functional food composition according to claim 1, which has α-glucosidase inhibitory activity.   The functional food composition according to claim 4, which has α-glucosidase inhibitory activity and α-amylase inhibitory activity.   The functional food composition according to claim 4 or 5, which has schuclase inhibitory activity.   The functional food composition according to any one of claims 4 to 6, which has maltase inhibitory activity.   The functional food composition according to any one of claims 1 to 7, which is a composition comprising tea polyphenol having an α-glucosidase inhibitory activity for oral administration.   The functional food composition according to claim 8, which has α-glucosidase inhibitory activity for continuous oral administration.   The functional food composition according to any one of claims 1 to 9, which has a function of improving fatty liver.   The functional food composition according to any one of claims 1 to 10, which has a lipase inhibitory activity.   A method for enhancing enzyme inhibitory activity, comprising enhancing α-glucosidase inhibitory activity and / or lipase inhibitory activity by setting the caffeine content in a functional food composition containing tea theaflavins to 1% by mass or less.   The method for enhancing enzyme inhibitory activity according to claim 12, comprising theaflavin, theaflavin-3-gallate, theaflavin-3'-gallate, and theaflavin-3,3'-digallate as tea theaflavins.   As tea theaflavins, theaflavin 2-7% by mass, theaflavin-3-gallate 2-7% by mass, theaflavin-3′-gallate 2-10% by mass, and theaflavin-3,3′-digallate 2-16% by mass The method for enhancing enzyme inhibition activity according to claim 13.   As tea theaflavins, theaflavin 4-5% by mass, theaflavin-3-gallate 4-5% by mass, theaflavin-3′-gallate 6-8% by mass, and theaflavin-3,3′-digallate 10-13% by mass 15. The method for enhancing enzyme inhibition activity according to claim 14.   The method for enhancing enzyme inhibition activity according to any one of claims 12 to 15, wherein the α-glucosidase inhibition activity is enhanced.   The method for enhancing enzyme inhibitory activity according to any one of claims 12 to 16, wherein maltase inhibitory activity is enhanced.   The method for enhancing enzyme inhibition activity according to any one of claims 12 to 17, wherein the lipase inhibition activity is enhanced.

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