JP5061282B2 - Naringenin derivative, glucose uptake promoter and blood sugar level increase inhibitor containing the same - Google Patents

Description

The present invention relates to a novel naringenin derivative, a glucose uptake promoter and a blood glucose level increase inhibitor containing the same.

As the diet becomes richer, "obesity" has become one of the most serious concerns of modern people. Obesity is not only cosmetically unfavorable, but is also known to cause various diseases such as diabetes, arteriosclerosis, hypertriacylglycerolemia, hypercholesterolemia, thrombosis and the like.

Obesity is caused by adipocyte differentiation / hypertrophy or an increase in the number of fat cells itself, and in either case, “glucose uptake” is deeply involved. Since glucose is a polar substance, a glucose transporter (GLUT) is required for glucose to be taken into each cell from the blood. Currently, 9 or more types of GLUTs have been cloned, and among them, GLUT1 and GLUT4 are mainly expressed in adipocytes that are greatly involved in sugar and lipid metabolism in vivo. Among them, GLUT4 is known to play a major role in glucose uptake activity on the adipocyte membrane.

GLUT4 is called insulin-sensitive GLUT and is usually present in intracellular vesicles in adipocytes and muscle cells. When stimulated by insulin, GLUT4 moves (translocates) onto the cell membrane and makes glucose available. The translocation of GLUT4 is triggered by the binding of insulin to the receptor and the phosphorylation of the β subunit of the receptor, followed by phosphorylation of the insulin receptor substrate (IRS), phosphatidylinositol 3 kinase ( The phosphoinositide 3-kinase (PI3K) activation and Akt / Protein Kinase B activation pathway complete the translocation from the intracellular endoplasmic reticulum to the cell membrane. In addition, GLUT4 works to move more glucose into muscle cells, and it has been reported that athletes have more GLUT4 than ordinary people ("Latest carbohydrate loading knowledge" Keio University Sports) Medical Research Center Bulletin 1996).

Reflecting the increase in obesity, the number of diabetic patients tends to increase. Diabetes is a troublesome disease that not only is difficult to cure once it develops, but is also likely to develop complications.

As complications that specifically develop in diabetic patients, there are diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, etc., which are also called the three major complications of diabetes. These complications are thought to be due to microangiopathy based on fine vascular lesions. Specifically, the function of an enzyme such as protein kinase C is abnormally enhanced and cell function is decreased, or glucose is chemically bound to a protein such as enzyme due to the persistence of hyperglycemia, and the enzyme function is decreased. It is thought that sorbitol (sugar alcohol) accumulates in cells due to metabolic disorders due to blood sugar and causes cell damage, which causes abnormalities in small blood vessels and blood cells, causing complications .

  For example, Patent Document 1 describes a lipid metabolism improving agent containing naringenin as an active ingredient, and further treating or preventing hyperlipidemia, treating or preventing hypercholesterolemia containing naringenin as an active ingredient, arterial A sclerosis treatment or prevention agent and a myocardial infarction treatment or prevention agent are described. Patent Document 2 describes a food effective for improving lipid metabolism, which contains nariningen.

Furthermore, Non-Patent Document 1 shows that naringenin has an inhibitory action on phosphatidylinositol 3-kinase (PI3K), a main regulator of GLUT4 translocation, and that grapefruit-derived naringenin is dose-dependent. Describes inhibition of glucose uptake activity in 3T3-L1 adipocytes. Non-Patent Document 2 (Breast
Cancer Research and Treatment, 85, 2, 103-110, 2004) states that naringenin inhibits mitogen-activated protein kinase (MAPK) phosphorylation and is deeply involved in inhibiting glucose uptake inhibitory activity in breast cancer cells. Seem.

  However, it has not been known so far that naringenin having a specific glycoside promotes glucose uptake activity into muscle cells, such naringenin has an effect of suppressing blood sugar increase, and has an increase in fat increase. .

JP-A-8-283154 JP-A-8-280358 Biochenical and Biophysical Research Communications 35, 2, 229-234, 2003 Breast Cancer Research and Treatment, 85,2, 103-110, 2004

An object of the present invention is to provide a therapeutic or prophylactic agent having an effect of suppressing an increase in blood sugar and an effect of suppressing an increase in fat by promoting sugar uptake activity into muscle cells, and a food and drink containing such a therapeutic agent.

  As a result of diligent research, the present inventors have found that naringenin represented by the following chemical formula [1] has an action of taking up glucose into muscle cells and an action of suppressing an increase in blood sugar, and completed the present invention.

（式中、Ｒ１は６単糖−ｎ分子数（ｎ≠０）と５単糖−ｍ分子数（ｍ≠０）を示し、Ｒ２は水素原子を示す。＊は異性体であることを示す。）で表されるナリンゲニン誘導体、
（２） ５単糖の分子数が１である（ｍ＝１）ことを特徴とする上記（１）記載のナリンゲニン誘導体、
（３） ６単糖の分子数が３である（ｎ＝３）ことを特徴とする上記（１）又は（２）記載のナリンゲニン誘導体。
（４） 発酵茶由来であることを特徴とする上記（１）〜（３）のいずれか記載のナリンゲン誘導体。
（５） 上記（１）〜（３）のいずれか記載のナリンゲン誘導体を含有する植物由来抽出物。
（６） 抽出物が熱水抽出物又は水溶性抽出物であることを特徴とする上記（５）記載の植物由来抽出物。
（７） 発酵茶由来であることを特徴とする上記（５）又は（６）記載の植物由来抽出物。
（８） 上記（１）〜（４）のいずれか記載のナリンゲン誘導体及び／又は上記（５）〜（７）のいずれか記載の植物由来抽出物を有効成分として含有するグルコース取込み促進剤。
（９） 上記（１）〜（４）のいずれか記載のナリンゲン誘導体及び／又は上記（５）〜（７）のいずれか記載の植物由来抽出物を有効成分として含有する血糖値上昇抑制剤に関する。 That is, the present invention
(1) The following general formula [I]

(In the formula, R1 represents 6 monosaccharides-n molecules (n ≠ 0) and 5 monosaccharides-m molecules (m ≠ 0), R2 represents a hydrogen atom, and * represents an isomer. .) Naringenin derivatives represented by
(2) The naringenin derivative according to (1) above, wherein the number of molecules of five monosaccharides is 1 (m = 1),
(3) The naringenin derivative according to (1) or (2) above, wherein the number of molecules of 6 monosaccharides is 3 (n = 3).
(4) The naringen derivative according to any one of (1) to (3) above, which is derived from fermented tea.
(5) A plant-derived extract containing the naringen derivative according to any one of (1) to (3) above.
(6) The plant-derived extract as described in (5) above, wherein the extract is a hot water extract or a water-soluble extract.
(7) The plant-derived extract according to (5) or (6) above, which is derived from fermented tea.
(8) A glucose uptake promoter containing the naringen derivative according to any one of (1) to (4) and / or the plant-derived extract according to any one of (5) to (7) as an active ingredient.
(9) The present invention relates to a blood sugar level increase inhibitor comprising as an active ingredient the naringen derivative according to any one of (1) to (4) and / or the plant-derived extract according to any one of (5) to (7). .

ADVANTAGE OF THE INVENTION According to this invention, a glucose uptake | capture active compound, a glucose uptake activation promoter, a blood glucose rise inhibitor, and food / beverage products containing these can be provided.

Naringenin is generally 4 ', 5,7-trihydroxyflavanone (4', 5,
7-trihydroxyflavanone), a typical compound of flavanones. The narinning derivative in the present invention has a characteristic structure in the 7-position sugar chain, and the sugar species involved are composed of 6 monosaccharides of glucose and rhamnose and 5 monosaccharides such as xylose. Furthermore, it is also known that an isomer is formed at the 2-position, and is included in the narinning derivative of the present invention. The structural formula of the naringenin derivative of the present invention is shown below.

The naringenin derivatives in the present invention include those having a structure in which 5 monosaccharides are composed of 2 molecules of glucose and 1 molecule of rhamnose. Also in the naringenin derivative having such a configuration, an isomer is observed at the 2-position (part of *). (Hereinafter, it may be called the compounds 1 and 2.) The chemical structure of the compound 1 is shown below.

In addition, the naringenin derivatives in the present invention include those in which the sugar chain at the 7-position of the mother nucleus is composed of 2 molecules of 6 monosaccharides and 1 molecule of 5 monosaccharides. Similar to the above compound, a difference in partial structure is observed at the 2-position. (Hereinafter, it may be called the compounds 3 and 4.) The chemical structure of the compound 2 is shown below.

  It is known that some nariningen derivatives are composed of 2 molecules of 6 monosaccharides and 1 molecule of glucose and 1 molecule of rhamnose and do not have 5 monosaccharides. Similar to the above compound, a difference in partial structure is observed at the 2-position. (Hereinafter, it may be called the compounds 5 and 6.) As described in Examples, the glucose uptake activity of the compound 6 was weak compared with the glucose uptake activities of the compounds 1 and 2. . The chemical structure of Compound 6 is shown below.

  Incidentally, it is well known that the same black tea extract, theaflavin, has an action of promoting glucose uptake into muscle cells, an action of inhibiting glucose uptake into fat cells, an action of suppressing an increase in blood glucose level, and the like (Japanese Patent Laid-Open No. 2006-1929). However, as a result of analyzing the structure, the present inventors have clarified that the compound involved in the action and effect of the present invention is not a theaflavin but a novel naringenin derivative. For reference, the structure of theaflavins is shown below. Theaflavins have (1) a 7-membered ring moiety, (2) a compound in which gallate is present, and (3) no sugar chain. This clearly differs structurally from the naringenin derivative in the present invention.

Theaflavins easily migrate to the ethyl acetate layer, and the compound of the present invention was quite polar because the aqueous phase after distribution was treated with a synthetic adsorption resin and isolated, but this was due to the number of OH groups. It seems to be. That is, it can be said that theaflavins and the naringenin derivative of the present invention have different polarities.

  The naringenin derivative in the present invention has an activity of taking up glucose into muscle cells. As described above, it is known that naringenin has an activity of inhibiting glucose uptake into fat cells and an activity of inhibiting glucose uptake into cancer cells, and thus naringenin has an anti-obesity effect and an anti-cancer cell growth inhibitory activity. However, it has not been known that a specific naringenin derivative has an activity of promoting glucose uptake into muscle cells. The naringenin derivative of the present invention has effects on enhancing vitality, improving endurance, nourishing tonic, anti-fatigue, and suppressing increase in blood glucose level.

  The naringenin derivative in the present invention may be obtained by any method of natural origin, biosynthesis, and chemical synthesis, but is preferably obtained by extraction from a plant in consideration of availability and industrial cost. The target plant is not particularly limited as long as it can extract the naringenin derivative of the present invention, but tea, particularly fermented tea such as black tea, is preferable.

The method for extracting the naringenin derivative of the present invention is not limited as long as it can extract the naringenin derivative of the present invention, but hot water extraction, alcohol extraction such as ethanol and the like are preferable from the viewpoint of simplicity. The naringenin derivative can be used as a high-purity product that has undergone one or more purification steps after extraction, but contains the naringenin derivative as long as the activity in the present invention is not lost. Extracts and fractions to be used can be used instead.

  The naringenin derivative of the present invention or an extract or fraction containing the derivative can be blended in a drug or food or drink containing these as active ingredients.

  The total content of the naringenin derivative in the present invention in the food varies depending on the food to be applied and individual differences (individual differences, racial differences, animal species differences, etc.), but 0.001 to 99.9 w / w% is appropriate. , Preferably 0.01 w / w%, more preferably 0.1 to 5 w / w%. If the amount is less than 0.001 w / w%, sufficient activity in the present invention may not be expected.

  The food and drink in the present invention are mixed in accordance with a conventional method by adding the naringenin derivative itself, an extract or a fraction, or a mixture containing these at an appropriate stage during the process of producing the food and drink. It can be manufactured by a normal manufacturing method. Moreover, it can also manufacture by adding the mixture containing a naringenin derivative to the food / beverage products once manufactured, and mixing by a conventional method.

  The form of the naringenin derivative to be added may be a crystal or powder of the naringenin derivative, or may be a mixture of an appropriate excipient or a part of the raw material in food and drink with the naringenin derivative. A naringenin derivative may be dissolved in ethanol or an aqueous solution thereof. Suitable excipients include starch, lactose, cellulose, dextrin, sugar alcohol, thickening polysaccharide and the like.

  The administration target of food and drink in the present invention is not particularly limited. In addition to food and drink consumed by humans, it also includes feed for mammals other than humans such as dogs, cats, rabbits, rats, and mice. In addition, food and drink additives include luxury items. Specifically, processed wheat products such as bread, noodles, biscuits, confectionery such as biscuits, cakes, candy, jelly, chocolate, fermented foods such as miso, marine products such as kamaboko and chikuwa, ham , Livestock foods such as sausages, cheese, seasoning foods such as sauce, dressing, sauce, coffee drinks, tea drinks, grain extract drinks (eg barley tea, bean tea, corn tea), vegetable drinks, fruit drinks, lactic acid bacteria drinks , Carbonated beverages, natural waters, sports beverages, various functional beverages, health foods such as supplements, etc. can be mentioned, but blending into various beverages is particularly preferred from the viewpoint of ease of ingestion. Moreover, since it became clear that the naringenin derivative in this invention is contained in teas, especially fermented tea, especially black tea, the functionality can also be expected from the naturally derived naringenin derivative.

  The naringenin derivative of the present invention can be produced after the naringenin is produced by an ordinary production method. Naringenin extracts fermented tea leaves using hot water, phosphate solution or the like, and after standing, separates the precipitated portion. If necessary, it can be recrystallized with water or glacial acetic acid. It can be produced by hydrolyzing it with vinegar or an enzyme and separating and purifying it by a conventional method.

  In each of the above methods, if necessary, decolorization, filtration, and recrystallization can be performed by a conventional method. The above hydrolysis conditions are not absolute, and can be appropriately changed depending on the heating temperature, time, concentration and amount of reagent used, and the like.

  Since the naringenin derivative of the present invention has an action of activating glucose uptake into muscle cells and an action of suppressing the increase in blood sugar, it has effects on enhancing vitality, improving endurance, nutrition and toughness, anti-fatigue, and suppressing an increase in blood sugar level. Moreover, since the naringenin derivative of the present invention can be extracted from, for example, tea leaves, it is excellent in safety. Furthermore, by adding the naringenin derivative in the present invention to foods and drinks, regular intake is facilitated over a long period of time.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited to an Example at all.

Example 1: Effect of enhancing sugar uptake of black tea hot water extract and its water-soluble fraction It was examined whether black tea contains an active ingredient that enhances uptake of glucose (sugar) into muscle cells. Glucose uptake activity was measured using black tea hot water extract and its water-soluble fraction as test substances.

Rat skeletal muscle-derived L6 myotube cells were cultured in MEM medium containing 10% fetal bovine serum (FBS) at 37 ° C., 5% CO ₂ -95% air. After L6 cells were grown to a subconfluent state in a 24-well plate, the medium was changed every 2 days in a MEM medium containing 2% FBS, and differentiated into myotube cells by culturing for 10 days. Differentiated L6 myotube cells were desensitized with MEM medium containing 0.2% BSA for 18 hours, and then the medium was removed and Krebs-Ringer HEPES buffer (KRH; 50 mM HEPES, pH 7.4, 137). (Mm NaCl, 4.8 mM KCl, 1.85 mM CaCl 2, 1.3 mM MgSO 4) 300 μl. After treatment with insulin to a final concentration of 100 nM in KRH or a test substance to 50 μg / ml for 15 minutes, [ ³ H] -labeled 2-deoxyglucose (2-DG) is finally It was allowed to act for 5 minutes so that the concentration was 6.5 mM (18.5 μCi). Thereafter, the cells were washed four times with ice-cooled KRH to remove 2-DG that was not taken up into the cells.

After complete removal of KRH, cells solubilized with 250 μl of 0.05 N NaOH were collected. Further, each well of the plate was washed twice with 200 μl of KRH, and this washing solution was also collected. Using the liquid scintillation system LSC-5000 series (manufactured by Aloka), the [ ³ H] radioactivity of the recovered solubilized cells was measured to determine the uptake activity of 2-DG incorporated into the cells. The non-specific uptake amount was obtained by treating cytochalasin B, an inhibitor of glucose transport, with 20 μM for 15 minutes and measuring 2-DG uptake activity in the same manner as described above.

As a result, although the hot water extract only had a certain effect on the 2-DG uptake activity at a concentration of 50 μg / ml, the water-soluble fraction significantly increased uptake activity at the same concentration, It showed the same effect as a certain 100 nM insulin (FIG. 1). From this, it was found that there is a compound that increases the glucose uptake activity in the black tea water-soluble fraction.

Example 2: Dependence of concentration on sugar uptake activity of black tea water-soluble fraction For the water-soluble fraction found to increase 2-DG uptake activity in Example 1, uptake activity varies depending on the concentration of action. I investigated. In the same manner as in Example 1, when 2-DG uptake activity was measured after allowing a water-soluble fraction as a test substance to act on differentiated L6 myotube cells at various concentrations, the water-soluble fraction was The uptake activity was increased significantly and concentration-dependently at 0.5 μg / ml, and the activity was maximized at 5 μg / ml (FIG. 2).

Example 3: Effect of enhancing the sugar uptake of compounds in the black tea water-soluble fraction In Examples 1 and 2, it was suggested that the black tea water-soluble fraction contains an active ingredient. The separated compounds 1 to 6 were examined for effects on sugar uptake activity. When the test substance was allowed to act to 50 μg / ml as in Example 1, the 2-DG uptake activity was measured. As a result, compounds 1 and 2 were significantly increased to a level equivalent to 100 nM insulin as a positive control. Was found to increase the uptake activity (FIG. 3).

Example 4: Concentration dependence on sugar uptake activity of compounds 1 and 2 in black tea water-soluble fractions Concentration dependence of compounds 1 and 2 confirmed to increase sugar uptake activity in Example 3 It confirmed by the method similar to Example 2. FIG. As a result, it was revealed that compounds 1 and 2 significantly increased the uptake activity in a concentration-dependent manner at 5 μg / ml or more (FIG. 4).

It is a figure which shows the result of having investigated the uptake | capture effect of the sugar intake of black tea hot water extract and its water-soluble fraction. The differentiated L6 myotube cells were allowed to act for 15 minutes with hot black water extract or water-soluble fraction at 50 μg / ml, and then [ ³ H] -labeled 2-DG was added to a final concentration of 6.5 mM ( 18.5 Ci) was allowed to act for 5 minutes. The cells were solubilized with NaOH and collected, and the radioactivity of [ ³ H] was measured to determine 2-DG uptake activity. The uptake amount when 100 nM insulin (dotted line in the figure) was used as a positive control, the same amount of solvent (solid line in the figure) as a negative control, and 20 μM cytochalasin B as a nonspecific uptake amount was measured. The figure shows the ratio when the uptake activity of the negative control is 1 as an average value ± standard error, and the asterisk has a risk rate of less than 5% and is significantly different from the negative control. It is a figure which shows the result of having investigated the density | concentration dependence with respect to the sugar uptake | capture activity of the black tea water-soluble fraction. After allowing the water-soluble fraction to act on the differentiated L6 myotube cells for 15 minutes to be 0.5, 2, 5, 50 μg / ml, the 2-DG uptake activity was measured in the same manner as in FIG. 100 nM insulin (broken line in the figure) was used as a positive control, and the same amount of solvent (solid line in the figure) was applied as a negative control. The figure shows the ratio when the uptake activity of the negative control is 1 as an average value ± standard error, and indicates that the asterisk is less than 5% risk and has a significant difference compared to the negative control. It is a figure which shows the result of having investigated the sugar uptake | capture enhancement effect of the compound in a water-soluble fraction. The compound 1 to 6 contained in the water-soluble fraction was allowed to act on the differentiated L6 myotube cells for 15 minutes to 50 μg / ml, and then 2-DG uptake activity was measured in the same manner as in FIG. 100 nM insulin (broken line in the figure) was used as a positive control, and the same amount of solvent (solid line in the figure) was applied as a negative control. The figure shows the ratio when the uptake activity of the negative control is 1 as an average value ± standard error, and indicates that the asterisk is less than 5% risk and has a significant difference compared to the negative control. It is a figure which shows the result of having investigated the density | concentration dependence with respect to the sugar uptake | capture activity of the compounds 1 and 2 in the black tea water-soluble fraction. After allowing compound 1 and 2 in the water-soluble fraction to act on differentiated L6 myotube cells for 15 minutes to be 0.5, 5, and 50 μg / ml, 2-DG uptake activity as in FIG. Was measured. 100 nM insulin (broken line in the figure) was used as a positive control, and the same amount of solvent (solid line in the figure) was applied as a negative control. The figure shows the ratio when the uptake activity of the negative control is 1 as an average value ± standard error, and indicates that the asterisk is less than 5% risk and has a significant difference compared to the negative control.

Claims (4)

Naringenin derivative represented by the following formula [I] or formula [II].

Or

The naringen derivative according to claim 1, wherein the naringen derivative is derived from fermented tea. A glucose uptake promoter containing the naringen derivative according to claim 1 as an active ingredient. A blood sugar level increase inhibitor comprising the naringen derivative according to claim 1 as an active ingredient.

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