JP2012136445A - Novel compound derived from sargassum patens and its use as glycolytic enzyme inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a component having useful functions from Sargassum patens, and to provide useful applications of Sargassum patens or a processed product thereof.SOLUTION: An ethanol extract of Sargassum patens contains a novel compound 2-(4-(3,5-dihydroxy-phenoxy)-3,5-dihydroxy-phenoxy)benzene-1,3,5-triol. The compound and the ethanol extract of Sargassum patens have excellent glycolytic enzyme inhibitory effect and hypoglycemic effect.

Description

  The present invention relates to a novel compound derived from Yatsuma Tamoku, a composition containing the compound, and uses of the compound as a glycolytic enzyme inhibitor and a blood glucose level increase inhibitor.

Yatsuma Tamoku (Scientific name = Sargassum patens C. Agardh) is a seaweed belonging to the genus Honda. Yatsuma Tamoku lives in various parts of Japan, and in some areas, the tip of the alga body may be edible. In the Noto region of Ishikawa Prefecture, it is used as a scaffold for mozuku in the cultivation of silk mozuku, but it is not common for yamtamamok itself to be edible.

  On the other hand, it is known that some seaweed extracts inhibit glycolytic enzymes such as α-glucosidase (Patent Documents 1 and 2, etc.). However, little is known about the functionality of the ingredients contained in Yatsuma Tamoku. Non-patent document 1 suggests that long-term administration of algal body powder of Yamagatamok to rats may reduce the blood lipid concentration, but there is no report on the usefulness of Yamatamamok.

Japanese Patent Laid-Open No. 2002-212095 JP 2006-104100 A

Fisheries Science, 60, 8.-88 (1994)

  An object of this invention is to acquire the component which has a useful function from Yamagatamok. Another object of the present invention is to provide a useful application of Yatsuma Tamoku or a processed product thereof.

  As a result of intensive studies, the present inventors have found that the α-amylase inhibitory action of the ethanol extract of Yamagatamok is significantly higher than that of other seaweed ethanol extracts, and the ingestion of components derived from the ethanol extract of Yamamatsumok Was found to suppress an increase in blood glucose level. Furthermore, the present inventors have found that the compound responsible for these actions is a novel fluorotannin compound having a structure represented by the following formula I ′. The present invention includes the following inventions.

［式中、R₁、R₂、R₃、R₄、R₅、R₆及びR₇は、それぞれ独立に、水素、又は -C(=O)-R’で表されるアシル基であり、該アシル基中、-R’は直鎖状又は分岐鎖状の飽和又は不飽和の炭化水素基であり、該炭化水素基中の水素は1つ以上の置換基により置換されていてもよく、-R’全体の炭素数は1〜20である］
で表される化合物又は該化合物の塩。
(2) 上記式I’で表される化合物又は該化合物の塩を、固形分あたり0.3重量％以上の濃度で含有する組成物。
(3) 経口摂取用組成物である、(2)の組成物。
(4) エタノール又はエタノール含有溶液によるヤツマタモクの抽出物。
(5) (1)の化合物又は塩を有効成分として含む糖分解酵素の阻害剤。
(6) 糖分解酵素がα-アミラーゼ又はα-グルコシダーゼである、(5)の阻害剤。
(7) (1)の化合物又は塩を有効成分として含む血糖値上昇抑制剤。 (1) Formula I ':

[Wherein R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and R ₇ are each independently hydrogen or an acyl group represented by —C (═O) —R ′. In the acyl group, —R ′ is a linear or branched saturated or unsaturated hydrocarbon group, and the hydrogen in the hydrocarbon group may be substituted with one or more substituents , -R ′ has 1 to 20 carbon atoms in total]
Or a salt of the compound.
(2) A composition comprising the compound represented by the formula I ′ or a salt of the compound at a concentration of 0.3% by weight or more per solid content.
(3) The composition according to (2), which is a composition for ingestion.
(4) An extract of Yamamamok by ethanol or ethanol-containing solution.
(5) A glycolytic enzyme inhibitor comprising the compound or salt of (1) as an active ingredient.
(6) The inhibitor according to (5), wherein the glycolytic enzyme is α-amylase or α-glucosidase.
(7) A blood glucose level increase inhibitor comprising the compound or salt of (1) as an active ingredient.

  ADVANTAGE OF THE INVENTION According to this invention, the novel compound which has a glycolytic enzyme inhibitory effect and a blood glucose level raise inhibitory effect, and the composition containing this compound are provided.

The preparation procedure of the extract from seaweed is shown. The inhibition rate of α-amylase activity by various extracts is shown. The isolation | separation procedure of the active ingredient by the solvent extraction from a Yatsuma Tamoku ethanol extract is shown. The HPLC chromatogram of the ethyl acetate fraction obtained from the Yatsuma Tamoku ethanol extract is shown. The relationship between the substrate (amylopectin) concentration ([Amylopectin]) and the reaction product (maltose) production rate (M / min) used in the reaction rate analysis of the α-amylase inhibitory action of HPLC_01 is shown. The reciprocal plot of the substrate (amylopectin) concentration ([Amylopectin]) and the reaction product (maltose) production rate (M / min) used for the kinetic analysis of the α-amylase inhibitory action of HPLC_01 is shown. The delayed effect of HPLC_01 of amylopectin digestion by human salivary α-amylase is shown. The delayed effect of HPLC_01 digestion of amylopectin by human pancreatic α-amylase is shown. 1 shows the effect of suppressing an increase in blood glucose level by oral administration of an ethyl acetate fraction to a mouse. ¹ shows assignments in chemical formulas of chemical shift values of ¹ H-NMR and ¹³ C-NMR. The inhibition rate of α-amylase activity by 10% ethanol extract of Yatsuma Tamoku is shown.

The compound of the present invention has a structure represented by the above general formula I ′.
R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and R ₇ are each independently hydrogen or an acyl group represented by —C (═O) —R ′.

  -R 'in the acyl group is a linear or branched saturated or unsaturated hydrocarbon group, and the hydrogen in the hydrocarbon group may be substituted with one or more substituents, The total number of carbon atoms in -R '(including the number of carbon atoms in the substituent group if any) is 1 to 20. Examples of the hydrocarbon group include methyl group, ethyl group, propyl group, butyl group, pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, phenyl group, benzyl group, t-butyl group and the like. . Examples of the substituent include a hydroxyl group, an amino group, and a carboxy group. The number of substituents is not particularly limited, but is usually 3 or less, preferably 2 or less, more preferably 1 or 0. The total number of carbon atoms in -R '(including the number of carbon atoms of the substituent, if any) is more preferably 1-10.

で表される化合物は、2-(4-(3,5-ジヒドロキシフェノキシ)-3,5-ジヒドロキシフェノキシ)ベンゼン-1,3,5-トリオールと命名されるフロロタンニン化合物である。該化合物は、下記実験2.7に示す物性値を示す化合物である。 R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and R ₇ are all hydrogen and have the following formula I:

Is a fluorotannin compound named 2- (4- (3,5-dihydroxyphenoxy) -3,5-dihydroxyphenoxy) benzene-1,3,5-triol. The compound is a compound having physical property values shown in Experiment 2.7 below.

  The compound represented by Formula I can be produced by isolating or increasing the concentration from Yatsuma Tamoku. As a method for isolating the compound represented by the formula I from Yatsuma Tamoku, an extract of the dried alga body of Yatsuma Tamoku with ethanol or an ethanol-containing solvent is prepared, and the solvent is distilled off from the extract to obtain a residue. After adding and mixing aqueous methanol solution and n-hexane, the upper layer (n-hexane layer) and the lower layer (aqueous layer) were separated to obtain the lower layer (aqueous layer), and chloroform was added to the lower layer (aqueous layer) and mixed. Separate the upper layer (aqueous layer) and the lower layer (chloroform layer) to obtain the upper layer (aqueous layer), add ethyl acetate to the upper layer (aqueous layer), mix, and then mix the upper layer (ethyl acetate layer) and the lower layer (aqueous layer). Examples of the method include a step of separating and obtaining an upper layer (ethyl acetate layer). A solvent is appropriately removed from the upper layer (ethyl acetate layer), and a fraction containing the compound represented by formula I can be obtained by column chromatography as necessary.

  By appropriately derivatizing the compound represented by Formula I, one or more of the hydrogens of the seven hydroxyl groups can be substituted with an acyl group represented by -C (= O) -R '. The method for derivatization is not particularly limited, and can be performed by a usual method for protecting a hydroxyl group with an acyl group.

  Any compound of formula I or I 'may be provided in the form of a salt as appropriate. The salt is not particularly limited as long as it is a salt that can be taken orally as a component of food and drink, pharmaceuticals and the like. Examples of salts that can be taken orally include sodium salts and potassium salts.

  The compound represented by the formula I or I 'or a salt thereof is not necessarily provided as a pure compound, and may be provided as a crude product. For example, a crude product containing a compound represented by the formula I or I ′ or a salt thereof at a concentration of 60% by weight or more, more preferably 80% by weight or more, more preferably 90% by weight or more per solid content is also represented by “Formula I 'Compound represented by', 'compound represented by formula I', 'salt of compound represented by formula I', 'salt of compound represented by formula I' or formula I or I 'described later In the range of the composition containing the compound represented by these, or its salt.

  The compound represented by the formula I or I ′ or a salt thereof may exist in the form of a hydrate with water or a solvate with a lower alcohol or the like. The hydrate or solvate is not particularly limited as long as it is a hydrate or solvate that is acceptable for uses such as pharmaceuticals and foods and drinks. That is, in the present invention, “a compound represented by formula I ′”, “a compound represented by formula I”, “a salt of a compound represented by formula I ′”, “a salt of a compound represented by formula I” These include hydrate or solvate forms as subordinate concepts.

  It is confirmed in the following examples that a composition containing a compound represented by the formula I or I ′ or a salt thereof at a concentration higher than that in natural yamatamamok also has an advantageous action such as an α-amylase inhibitory action. Has been. As such a composition, the compound represented by the formula I or I ′ or a salt thereof is preferably at a concentration of 0.3% by weight or more, more preferably 0.35% by weight or more, particularly preferably the total solid content of the composition. A composition containing 1.0% by weight or more, most preferably 2.0% by weight or more. The upper limit of the concentration of the compound represented by the formula I or I ′ in the composition or a salt thereof is not particularly limited, and can be a value up to 100% by weight based on the total solid content of the composition. Is less than 60% by weight.

  The present invention or an extract of Yatsuma Tamoku by ethanol or an ethanol-containing solution is provided. The ethanol-containing solution contains ethanol at a concentration of 10 (v / v)% or more, more preferably 50 (v / v)% or more with respect to the total amount of the solvent, and the balance is one or more types compatible with ethanol. An ethanol-containing solution that is another solvent may be mentioned. The “one or more other solvents compatible with ethanol” is preferably water. The extract of Yatsuma Tamoku with ethanol contains about 2.1% by weight of the compound represented by Formula I per total solid content, and the extract of Yatsuma Tamoku with 10 (v / v)% ethanol aqueous solution contains About 0.35% by weight of the compound of formula I is included. The Yatsuma Tamoku extract of the present invention may be used in a form in which the extraction solvent is appropriately removed, or may be used in a form combined with other components. The extract of Yatsuma Tamoku of the present invention is useful as an active ingredient of a degrading enzyme inhibitor and a blood sugar level increase inhibitor.

  It is preferable that the said composition and extract are compositions for oral consumption. Examples of the composition for ingestion include a food and drink composition and a pharmaceutical composition for oral administration. The composition for oral ingestion contains the compound represented by the formula I or I 'or a salt thereof, or an extract of Yamatamamok, and other components that can be orally ingested as food or drink or pharmaceuticals.

  The present invention also provides a glycolytic enzyme inhibitor comprising a compound represented by the formula I or I 'or a salt thereof as an active ingredient. Examples of the glycolytic enzyme include α-amylase and α-glucosidase (maltase activity, sucrase activity, etc.). The glycolytic enzyme inhibitor of the present invention is ingested by mammals such as humans and inhibits the degradation of sugar by a glycolytic enzyme in vivo. Therefore, the glycolytic enzyme inhibitor of the present invention can be used for the purpose of preventing or treating a disease or symptom that is improved by inhibiting a glycolytic enzyme. Such diseases or symptoms include diabetes and an increase in blood glucose level. The glycolytic enzyme inhibitor of the present invention may be used as a reagent for inhibiting a glycolytic enzyme in vitro.

  The present invention also relates to a blood glucose level increase inhibitor comprising a compound represented by the formula I or I 'or a salt thereof as an active ingredient.

  The inhibitor of glycolytic enzyme and the inhibitor of increase in blood glucose level of the present invention may be in the form of a pharmaceutical product or food or drink. The pharmaceutical may be formulated into a form suitable for various administration routes, and forms such as oral preparations such as liquids, tablets and granules, and parenteral preparations such as injections. These pharmaceutical preparations comprise a compound represented by the formula I or I ′ or a salt thereof, or a composition or extract containing the compound or the salt, and other pharmaceutically acceptable components such as excipients. Including. Examples of the food and drink include beverages, tablets, granules, and other ordinary food and drink. These foods and drinks contain a compound represented by the formula I or I 'or a salt thereof, or a composition or extract containing the compound or the salt, and other ingredients acceptable as food.

Experiment 1: Screening distilled water (DW), ethanol or dimethyl sulfoxide (DMSO) was used as an extraction solvent to prepare extracts from seaweeds, and the α-amylase inhibitory activity of each seaweed extract was measured. Extraction with distilled water was performed at 25 ° C or 85 ° C. Extraction with ethanol and DMSO was performed at 25 ° C.

  The seaweed used was Sugimok, Yanagimok, Togemoku, Yoremoku, Umitorano, Yatsuma Tamoku, Aonori, Habanori, Mozuku, Kurome, Mekabu, Gimbasa, Wakame, Kajime, Kinumozuku.

  The extract was prepared by the following procedure. An overview is shown in FIG. Specifically, the collected seaweed was gently washed with running water and stored frozen at -80 ° C. Subsequently, it was freeze-dried, the dried product was pulverized with a blender, and passed through a 0.5 mm mesh sieve to obtain a powder sample. The powder sample (weight 1.0 g) and the extraction solvent (about 40 mL) were mixed, and the mixture was pulverized with a homogenizer. However, when the extraction solvent was distilled water at 85 ° C., the mixture was kept warm at 85 ° C. for 1 hour before pulverization with a homogenizer. The pulverized product by the homogenizer was shaken at 25 ° C. for 1 hour and then centrifuged. The extraction solvent was added again to the precipitate, and the extraction operation was repeated. After the supernatants by centrifugation were combined into one, the extraction solvent was added to make 100 mL, and 100 mL of seaweed extract was obtained from 1 g of the powder sample.

  For comparison, extracts were prepared in the same manner for green tea, guava tea, and bay leaves known to have α-amylase inhibitory activity.

The α-amylase inhibitory activity was measured by an iodine starch reaction method using human saliva α-amylase. Specifically, for each extraction solvent, seaweed extract, 100 nM human saliva α-amylase (α-Amylase From Saliva, manufactured by Sigma), 20 mM Tris-HCl buffer (pH 7) .2) and starch (soluble starch, manufactured by Nacalai Tesque) are added to the sample solution so that the volume of the sample solution after starch addition is 50 μL and the starch concentration is 0.1% by weight. The enzymatic reaction was allowed to proceed. Moreover, each solution was added as positive control as No. 2 row | line | column of the following table, and as blank No. 3 row | line | column of the following table as a blank, and reaction was advanced at room temperature. Ten minutes after starch addition, 150 μL of an iodine solution (an aqueous solution containing 0.1 (w / v)% iodine and 1 (w / v)% potassium iodide, both manufactured by Nacalai Tesque) was added. The absorbance at 595 nm (A ₅₉₅ ) of the sample after addition of iodine was measured using a plate reader.

The inhibitory activity (%) of α-amylase was measured by the following formula.
(Inhibition activity of α-amylase) = (No. 1 A ₅₉₅ -No. 3 A ₅₉₅ ) / (No. 2 A ₅₉₅ -No. 3 A ₅₉₅ ) × 100

  The inhibition rate is shown in FIG. Of Yanagimoku, Yoremoku, Yatsumata Moku, Kurome, Kajime, which have particularly high inhibitory activity, distilled water extract, 85 ° C water extract and ethanol extract for Yanagimoku and Yoremok, and only ethanol extract for Yatsumata Moku, Kurome and Kajime As for, the distilled water extract and the 85 ° C. water extract each showed high α-amylase inhibitory action. Among these seaweeds, dietary habits are known for Yatsuma Tamoku, Kurome and Kajime.

The 50% inhibitory concentration (IC ₅₀ ) of human salivary α-amylase activity was determined using extracts of willow moku, yoremoku, yasumamamok, kurome, and kajime. The 50% inhibitory concentration indicates the concentration of the extract necessary for inhibiting human salivary α-amylase activity by 50% by the concentration (dry-mg / mL) converted to the weight of dried seaweed. It was confirmed that the α-amylase inhibitory action of these seaweeds was remarkably higher than that of the ethanol extract of bay leaves, which is known to have an inhibitory action on α-amylase.

Experiment 2: Isolation of α-amylase inhibitory component from Yasumata Moku
Experiment 2.1: Isolation of an active ingredient with α-amylase inhibitory activity from the ethanol extract of Tatsuma Tamoku, which was confirmed in Experiment 1 as having solvent fractionation and HPLC purification dietary habits, and having α-amylase inhibitory activity did.

  An outline of the isolation procedure of the active ingredient by solvent extraction is shown in FIG. 100 mL of an ethanol extract (containing 1 g in terms of dried seaweed body) was distilled off under reduced pressure to obtain 113.3 mg of residue. To the residue, 30 mL of 50% aqueous methanol solution and 50 mL of n-hexane were mixed and allowed to stand, and the upper layer (n-hexane layer) and the lower layer (aqueous layer) were separated. The upper layer contains 6.1 mg solids. To the lower layer (aqueous layer), 50 mL of chloroform was added and mixed, and allowed to stand to separate the upper layer (aqueous layer) and the lower layer (chloroform layer). The lower layer contains 13.8 mg solids. 50 mL of ethyl acetate was added to and mixed with the upper layer (aqueous layer) and allowed to stand to separate the upper layer (ethyl acetate layer) and the lower layer (aqueous layer). The upper layer (ethyl acetate layer) contains 9.0 mg of solids and the lower layer (aqueous layer) contains 98.0 mgs of solids.

  When human salivary α-amylase activity was confirmed for the fractions obtained from the n-hexane layer, chloroform layer, ethyl acetate layer, and aqueous layer, activity was found only in the fraction of the ethyl acetate layer.

  Ethyl acetate layer fraction was subjected to high performance liquid chromatography (Condition = Intersil Ph-3 column [manufactured by GL Sciences Inc.], acetonitrile 10 (v / v)%-(10min) -10 (v / v)%-50 ( (v / v)%-(15 minutes) -50 (v / v)%-100 (v / v)%-(10 minutes) -100 (v / v)% (stepwise), flow rate: 1 ml / min) In the chromatogram shown in FIG. 4, a fraction containing a peak with a retention time of 15.5-17.6 minutes was obtained. This fraction is hereinafter referred to as “HPLC_01”. HPLC_01 (dry weight) of 2.4 mg was obtained from 1.0 g of the dried Yamamotomok.

Experiment 2.2: Reaction kinetic analysis
The α-amylase inhibitory action of HPLC_01 was analyzed kinetically. The initial reaction rate of human salivary α-amylase was measured by the potassium ferricyanide method for the amount of maltose produced using amylopectin as a substrate. Specifically, according to the table below, amylopectin (manufactured by Nacalai Tesque) dissolved in 300 μL of 10 (v / v)% ethanol containing 1 μg of HPLC_01, 50 μL of 10 mM calcium chloride and 100 mM sodium phosphate buffer (pH 7.0) 140 μL of the solution was mixed and incubated at 37 ° C. for 5 minutes, and then 10 μL of 1 μM human saliva α-amylase (Sigma, α-Amylase From Saliva) dissolved in 100 mM sodium phosphate buffer (pH 7.0) was added. The reaction was started and the reaction was allowed to proceed at 37 ° C. The amylopectin solution was prepared such that the concentration after enzyme addition was 0.07, 0.08, 0.098, 0.14, 0.28, 0.56, 0.84, 1.12 (w / v)%. In addition, 300 μL of 10 (v / v)% ethanol without HPLC_01 as a control, 10 μL of 100 mM sodium phosphate buffer (pH 7.0) without human salivary α-amylase as a sample blank, and HPLC_01 as a control blank 10 (v / v)% ethanol (300 μL) and human saliva α-amylase-free 100 mM sodium phosphate buffer (pH 7.0) (10 μL) were used instead of each solution, and the reaction proceeded at 37 ° C. I let you. Five minutes after the addition of the enzyme, 50 μL was collected, and 500 μL of potassium ferricyanide solution (2.0 (w / v)% sodium carbonate solution containing 10 mM potassium ferricyanide, both manufactured by Nacalai Tesque) was added. After heating for 10 minutes in a heat block at 100 ° C., the mixture was centrifuged, 50 μL of the supernatant was mixed with 200 μL of distilled water, and the absorbance of the sample at 415 nm was measured using a plate reader. To prepare a calibration curve, 0, 0.83, 1.7, 2.5, 3.3, 4.2, 5.0, 5.8, 6.7, 7.5, 8.3 mM maltose (manufactured by Nacalai Tesque) solution was added to 50 μL of potassium ferricyanide solution (2.0 containing 10 mM potassium ferricyanide). (w / v)% sodium carbonate solution (both manufactured by Nacalai Tesque) Add 500 μL, heat for 10 minutes in a heat block at 100 ° C., centrifuge, mix 50 μL of the supernatant with 200 μL of distilled water, and sample The absorbance at 415 nm was measured using a plate reader. The maltose molar concentration ([maltose]) in each sample was determined using a maltose calibration curve, and the reaction product formation rate was calculated using the following equation.
Reactant (maltose) production rate (M / min)
= ([Maltose] of S− [maltose] of SB) / ([maltose] of C− [maltose] of CB) / 5

FIG. 5 shows the relationship between the substrate (amylopectin) concentration ([Amylopectin]) and the reaction product (maltose) production rate (M / min). The Michaelis-Menten constant (Km) and maximum velocity (Vmax) are as shown in the following table. From the reciprocal plot (FIG. 6), the antagonistic inhibition constant (Ki) was calculated to be 1.8 × 10 ⁻⁴ (%). The inhibition constant (Ki) of acarbose, known as a non-competitive inhibitor of α-amylase, is 1.8 × 10 ⁻³ (%). It was confirmed that HPLC_01 of the present invention has an excellent α-amylase inhibitory action.

Experiment 2.3: Digestive delay effect (human salivary α-amylase)
HPLC_01 was added at various concentrations to the digestion reaction system of amylopectin by human saliva α-amylase (manufactured by Sigma), and the relationship between reaction time and amylopectin digestibility was examined. The initial reaction rate of human salivary α-amylase was measured by the potassium ferricyanide method for the amount of maltose produced using amylopectin as a substrate. Specifically, amylopectin (Nacalai Tesque) dissolved in 300 μL of 10 (v / v)% ethanol containing 0, 5, 0.5 μg of HPLC_01, 50 μL of 10 mM calcium chloride and 100 mM sodium phosphate buffer (pH 7.0) After mixing 140 μL of solution and incubating at 37 ° C. for 5 minutes, 10 μL of 1 μM human saliva α-amylase (α-Amylase From Saliva, Sigma) dissolved in 100 mM sodium phosphate buffer (pH 7.0) was added. The reaction was started by addition, and the reaction was allowed to proceed at 37 ° C. The amylopectin solution was prepared so that the concentration after enzyme addition was 0.28 (w / v)%. This concentration corresponds to a maltose concentration of 8.2 mM. 50 μL was collected at 2, 5, 10, 15, 20, 30, 40, 50, and 60 minutes after addition of the enzyme. As a sample blank, 10 μL of 100 mM sodium phosphate buffer (pH 7.0) was added instead of human saliva α-amylase, and the same operation was performed. The molar concentration ([maltose]) of glucose produced by the potassium ferricyanide method was determined in the same manner as in Experiment 2.2, and the amylopectin digestibility was calculated using the following formula.
Amylopectin digestibility = ([maltose] of HPLC_01− [maltose] of sample blank) / (8.2 × 10 ⁻³ ) × 100
The results are shown in FIG.

Experiment 2.4: Digestive delay effect (human pancreatic α-amylase)
HPLC_01 was added at various concentrations to the digestion reaction system of amylopectin by human pancreatic α-amylase (manufactured by Sigma), and the relationship between reaction time and amylopectin digestibility was examined. The initial reaction rate of human pancreatic α-amylase was measured by the potassium ferricyanide method for the amount of maltose produced using amylopectin as a substrate. Specifically, amylopectin (Nacalai Tesque) dissolved in 300 μL of 10 (v / v)% ethanol containing 0, 5, 0.5 μg of HPLC_01, 50 μL of 10 mM calcium chloride and 100 mM sodium phosphate buffer (pH 7.0) After mixing 140 μL of solution and incubating at 37 ° C. for 5 minutes, 10 μL of 1 μM human pancreatic α-amylase (Sigma, α-Amylase From Saliva) dissolved in 100 mM sodium phosphate buffer (pH 7.0) was added. The reaction was started by addition, and the reaction was allowed to proceed at 37 ° C. The amylopectin solution was prepared so that the concentration after enzyme addition was 0.28 (w / v)%. This concentration corresponds to a maltose concentration of 8.2 mM. 50 μL was collected at 2, 5, 10, 15, 20, 30, 40, 50, and 60 minutes after addition of the enzyme. The same operation was performed using 10 μL of 100 mM sodium phosphate buffer (pH 7.0) instead of human pancreatic α-amylase as a sample blank. Similar to the method of Experiment 2.2, the molar concentration of maltose ([maltose]) in each sample was determined by the potassium ferricyanide method, and the amylopectin digestibility was calculated using the following formula.
Amylopectin digestibility = ([maltose] of HPLC_01− [maltose] of sample blank) / (8.2 × 10 ⁻³ ) × 100
The results are shown in FIG.

Experiment 2.5: α-amylase activity 50% inhibitory concentration
The 50% inhibitory concentration (IC ₅₀ ) of α-amylase activity by HPLC_01 and conventionally known α-amylase inhibitors was determined. As a conventionally known α-amylase inhibitor, acarbose (C ₂₅ H ₄₃ NO ₁₈ , molecular weight 645.60, manufactured by LKT Laboratories, Inc.), quercetagetin (quercetagetin, C ₁₅ H ₁₀ O ₈ , molecular weight 318.25, EXTRASYNTHESE SA), commercially available wheat-derived α-amylase inhibitor (α-Amylase Inhibitor from Triticumaestivum (wheat seed), Sigma, protein with a molecular weight of about 21,000, literature: Biochimica et Biophysica Acta, 422 (1976) 159-169 ) Was used.

Regarding the IC ₅₀ values of polyphenols (phlorotannins), laurel leaves extract, and guava leaves extract, the anti-diabetic effect of seaweed polyphenols (phlorotannins) (2) <Nagase Sangyo November 2007, http http://www.nagase.co.jp/assetfiles/news/20071121.pdf>, amylase inhibitor “α-amylase inhibitor” <Nippon Flour Milling Patent Nos. 1919036, 2032272>, “Guba leaf hot water extract Cited from the anti-diabetic effect in db / db mice and the inhibitory effect on postprandial blood glucose level increase by human drinking test "<Japan Agricultural Chemical Society, Vol.72, No.8, pp923-931, 1998>.

IC ₅₀ is _half of the digestibility of the amylopectin digestion reaction system with human saliva α-amylase (manufactured by Sigma) compared to the digestion rate when the inhibitor is added at various concentrations and the inhibitor is not contained (control). It was calculated as the inhibitor concentration giving the digestibility. Specifically, the inhibitor solution (solvent is 100 mM sodium phosphate buffer (pH 7.0) for wheat-derived α-amylase inhibitor, 10 (v / v)% ethanol for other inhibitors) in 300 μL, 10 mM chloride 50 μL of calcium and 140 μL of 1.0 (w / v)% amylopectin (Nacalai Tesque) solution dissolved in 100 mM sodium phosphate buffer (pH 7.0) were mixed and incubated at 37 ° C. for 5 minutes, and then 100 mM sodium phosphate The reaction was started by adding 10 μL of 1 μM human saliva α-amylase (α-Amylase From Saliva, Sigma) dissolved in buffer (pH 7.0), and the reaction was allowed to proceed at 37 ° C. The same operation was performed by adding 10 μL of 100 mM sodium phosphate buffer (pH 7.0) instead of human saliva α-amylase as a sample blank for each inhibitor concentration. As a control, the same reaction was performed by adding 300 μL of the solvent of the inhibitor instead of the inhibitor. As a control blank, 300 μL of an inhibitor solvent was used instead of the inhibitor, and 10 μL of 100 mM sodium phosphate buffer (pH 7.0) was added instead of human saliva α-amylase, and the same operation was performed. Five minutes after the addition of the enzyme, 50 μL was sampled, and the maltose molar concentration ([maltose]) in each sample was determined by the potassium ferricyanide method in the same manner as in Experiment 2.2. Calculating the inhibition rate using the following equation, and the inhibitor concentration that gives inhibition of 50% and IC _50.
Inhibition rate = {(inhibitor [maltose] −sample blank [maltose]) / (control [maltose] −control blank [maltose])} × 100
The results are shown in Table 5. It was revealed that HPLC_01 has a superior inhibitory action than existing α-amylase inhibitors.

Experiment 2.6: Suppressing blood glucose level in mice
Six 10-week-old male DDY mice (35-42 g body weight) were used. The blood glucose level of the mouse was measured by collecting blood from the tail vein using a blood glucose level measurement kit and a care fast meter (both manufactured by Nipro Corporation). First, a control experiment was conducted. The mice were fasted overnight (with free collection of water) and then blood glucose levels were measured. In either case, 2 g / kg body weight starch was administered 30 minutes after administration of 100 μL of physiological saline using a sonde. The blood glucose level was measured 30 minutes, 60 minutes, 90 minutes, 120 minutes, and 180 minutes after physiological saline administration. After breeding the same mice for 1 week, an ethyl acetate fraction administration experiment was conducted. After fasting overnight (free collection of water), blood glucose levels were measured. The ethyl acetate fraction (see Experiment 2.1) was suspended in 110 μL of physiological saline so that 100 mg / kg body weight was administered for each mouse body weight, and 30 g later, 2 g / kg body weight starch was administered. did. A sonde was used for both administrations. The blood glucose level was measured 30 minutes, 60 minutes, 90 minutes, 120 minutes and 180 minutes after starch administration.

  The results are shown in FIG. It was confirmed that an increase in blood glucose level was suppressed by single administration of the ethyl acetate fraction to mice.

Experiment 2.7: Determination of the structure of HPLC_01 by instrumental analysis
HPLC_01 was subjected to ¹ H-NMR, ¹³ C-NMR, mass spectrometry, and elemental analysis and found to exhibit the following physical property values.
¹ H-NMR (CD ₃ OD): δ 5.90 (1H, bs), 5.92 (1H, bs), 5.94 (1H, bs), 6.00 (1H, bs), 6.04 (1H, bs), 6.15 (1H, bs), 6.29 (1H, bs).
¹³ C-NMR (CD ₃ OD): δ 95.8, 97.1 (x4), 98.8 (x2), 125.8, 126.8, 153.0 (x2), 153.2, 153.4, 154.8 (x2), 157.1 (x2), 158.9.
MS (m / z): 375.2 (MH ⁺ )
MS calculated (C ₁₈ H ₁₄ O ₉ ): 374.0638 (100.0%)
Elemental analysis: C, 57.76; H, 3.77; O, 38.47

により表される化合物であると推定された。¹H-NMR及び¹³C-NMRの化学シフト値は図10に示すように帰属される。当該化合物は、2-(4-(3,5-ジヒドロキシフェノキシ)-3,5-ジヒドロキシフェノキシ)ベンゼン-1,3,5-トリオールと命名されるフロロタンニン化合物である。 From these analysis results, HPLC_01 has a molecular formula of C ₁₈ H ₁₄ O ₉ and has the following structural formula:

It was estimated that it was a compound represented by. Chemical shift values of ¹ H-NMR and ¹³ C-NMR are assigned as shown in FIG. The compound is a fluorotannin compound named 2- (4- (3,5-dihydroxyphenoxy) -3,5-dihydroxyphenoxy) benzene-1,3,5-triol.

Experiment 3: In extraction experiment 1 with a 10% aqueous ethanol solution , extraction was carried out using ethanol from a dried powder of Yamamotomok, and the α-amylase inhibitory activity of the resulting extract was measured.

  In this experiment, an extract was obtained in the same procedure as in Experiment 1 using a 10 (v / v)% aqueous ethanol solution instead of ethanol, and α-amylase inhibitory activity was evaluated. The inhibition rate of α-amylase was determined by the same procedure as in Experiment 1 using the extract itself (stock solution), the extract diluted 2 times (1/2 solution), and the extract 5 times diluted (1/5 solution).

  The results are shown in FIG. When an aqueous ethanol solution was used as an extraction solvent, it was confirmed that an active ingredient having an α-amylase inhibitory action could be extracted, although the extraction efficiency was lower than when pure ethanol was used.

Experiment 4: α-Glucosidase Inhibitory Action α-Glucosidase was extracted from rat small intestine acetone powder (Intestinal acetone powders from rat, Sigma), and the inhibitory action of HPLC_01 on maltase activity and sucrase activity was examined.

Rat small intestine acetone powder 0.5 g was added with 10 mL of 100 mM potassium phosphate buffer (pH 6.8) and 30 mL of distilled water, homogenized, and the centrifuged supernatant was used as an α-glucosidase extract. Maltose and sucrose (both manufactured by Nacalai Tesque) were used as substrates. HPLC_01 used an ethanol solution. Each solution was mixed according to the following table, and the enzyme reaction was allowed to proceed at 37 ° C. for 30 minutes. After heating in a 100 ° C. heat block for 3 minutes, the mixture was centrifuged and 50 μL of the supernatant was collected. In order to quantify glucose, 100 μL of glucose C-II test (manufactured by Wako Pure Chemical Industries, Ltd.) was added and reacted for 5 minutes, and the absorbance at 505 nm (A ₅₀₅ ) was measured using a spectrophotometer. Inhibitory activity was calculated as a percentage of the amount of glucose produced in the absence of HPLC_01 using the following formula.
Maltase inhibitory activity (%) = (M A ₅₀₅ −MB A ₅₀₅ ) / (MC A ₅₀₅ −MCB A ₅₀₅ ) × 100
Sucrase inhibitory activity (%) = (S A ₅₀₅ -SB A ₅₀₅ ) / (SC A ₅₀₅ -SCB A ₅₀₅ ) × 100

  The maltase inhibitory activity was 61.6%, and the sucrase inhibitory activity was 94.7%.

Experiment 5: α-Glucosidase 50% inhibitory concentration
The 50% inhibitory concentration (IC ₅₀ ) for α-glucosidase of HPLC_01 was determined for maltase activity and sucrase activity. HPLC_01 using at various concentrations to obtain the inhibitory activity in the same manner as in Experiment 4 was the inhibitor concentration that gives inhibition of 50% and IC _50.

The IC _{50 for} maltase inhibition was 114 μg / mL and the IC ₅₀ for sucrase inhibition was 25.4 μg / mL. Therefore, HPLC_01 was shown to have an inhibitory effect on α-glucosidase.

Claims (7)

Formula I ':

[Wherein R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and R ₇ are each independently hydrogen or an acyl group represented by —C (═O) —R ′. In the acyl group, —R ′ is a linear or branched saturated or unsaturated hydrocarbon group, and the hydrogen in the hydrocarbon group may be substituted with one or more substituents , -R ′ has 1 to 20 carbon atoms in total]
Or a salt of the compound. Formula I ':

[Wherein R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and R ₇ are each independently hydrogen or an acyl group represented by —C (═O) —R ′. In the acyl group, —R ′ is a linear or branched saturated or unsaturated hydrocarbon group, and the hydrogen in the hydrocarbon group may be substituted with one or more substituents , -R ′ has 1 to 20 carbon atoms in total]
Or a salt of the compound at a concentration of 0.3% by weight or more per solid content.   3. The composition of claim 2, which is a composition for oral consumption.   Extract of Yatsuma Tamoku with ethanol or ethanol-containing solution.   An inhibitor of a glycolytic enzyme comprising the compound or salt of claim 1 as an active ingredient.   6. The inhibitor according to claim 5, wherein the glycolytic enzyme is α-amylase or α-glucosidase.   2. A blood sugar level increase inhibitor comprising the compound or salt of claim 1 as an active ingredient.

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