WO2008065796A1 - α-GLUCOSIDASE INHIBITOR - Google Patents

Abstract

It is intended to provide an α-glucosidase inhibitor which is new and has a high activity. Accordingly, after a root or/and aerial part of Salacia reticulata is extracted with water, insoluble components are removed by adding ethanol. Then, separation by fractionation was carried out using a synthetic adsorbent HP-20 (manufactured by Mitsubishi Chemical Corporation) column, an amino column or an ODS column, whereby a compound represented by the chemical formula (Kagaku 1) is obtained. This compound has an excellent α-glucosidase inhibitory activity, and is provided as a new anti-diabetic agent, or a health food capable of exhibiting a sugar absorption inhibitory effect.

Description

 Specification

 a single darcosidase inhibitor

 Technical field

 [0001] The present invention relates to a novel α-darcosidase inhibitor.

 Background art

 [0002] a Darcosidase is an exohydrolase that dissociates gnolecose from the non-reducing terminal side such as α-glucan and short-chain oligosaccharides. This enzyme is involved in starch metabolism in plants, animal digestion and glycogen metabolism, and plays an important role in the carbohydrate metabolism system. Among them, many studies have been conducted on its inhibitors because it controls sugar absorption in the human small intestine, and it is used to prevent and treat diabetes, hyperlipidemia, obesity, etc. caused by hyperglycemic symptoms! It is effective.

 [0003] In fact, α-darcosidase inhibitors, in addition to inhibiting sugar absorption, are not only improved in insulin sensitivity and long-term administration to patients with impaired glucose tolerance (IGT) but also significantly suppressed the onset of type 2 diabetes. It has been shown that the occurrence of disease is significantly suppressed, and further, it has been revealed that it is also significantly suppressed in the development of hypertension (non-patent literature). 1, 2).

 [0004] Since a structure of nojirimycin, which was isolated as a metabolite of Streptomyces sp., Was clarified in 1966 (non-patent document 3), a large number of analogs have been isolated. Synthetic studies on compounds with these motifs have been made (Non-Patent Document 4). These compounds have a common feature in that they are a kind of pseudosaccharide belonging to aza sugar in which the oxygen atom in the monosaccharide bilanose is substituted with nitrogen. In addition to these compounds, compounds substituted with sulfur, phosphorus, carbon atoms, etc. are known as thio-, phospha-, and ruba-sugars, and carbol, voglibose, miglitol, etc., which are already pharmaceuticals, are also these compounds. Belonging to a group.

[0005] Among them, the most studied is azasaccharide, and the above-mentioned miglitol is a drug born from nojirimycin. Nojirimycin was an excellent α- and / 3-darcosidase-inhibiting component, but because it is unstable, it was first reduced with a hydroxyl group at the C1-position. Xinojirimycin was synthesized. As a result, although stability and inhibitory activity were further increased, there was a drawback that the activity decreased in vivo, and further derivatives were synthesized and studied, and miglitol was born. The same is true for voglibose, which has the strongest inhibitory activity. The structure-activity relationship research has been promoted by the discovery of acarbose, and chemical modification using powerful ruba sugar amines such as noridamine, norienamine, and variolamine as lead compounds. Has led to the synthesis.

 [0006] On the other hand, the active site of the dalcosidase enzyme has been studied using the specific binding reaction of conduritol B epoxide (CBE), and the involvement of Asp-carboxyl group has been clearly demonstrated. (Non-Patent Document 5). In addition, it is thought that dexinojirimycin and its isomer isofagogomine act as a selective inhibitory activity, and that they act by ion-pairing between the imino group and the enzyme active site! /.

 [0007] Inhibitory components different from these pseudo-saccharides include attaridone alkaloids (Non-patent Document 6), coumarin glycosides (Non-patent Document 7), flavonoids (Non-patent Documents 8 and 9), tannins ( Non-patent literature 10, 11) Many known plant-derived components are known. These compounds have very low inhibition levels! /, So they are put to practical use! /, N! /.

 [0008] Under such circumstances, in recent years, particularly effective! /, Salacia reticula ta WIGHT has been attracting attention! Kotarahimbutsu is a creeping plant of the Hippocrataceae Salacia, and has been used in Sri Lanka as a medicinal plant effective for rheumatism and skin diseases in addition to rheumatism and skin diseases. (Non-patent Document 12). The site of use is mainly roots and trunks, and when it becomes a thick trunk, it is cut out to make a cup, and if you use hot water or water when eating, it is reported to prevent diabetes. That ’s right.

[0009] With regard to its effectiveness, many studies have been conducted since 1984 when EHKarunanayake et al. At the University of Colombo confirmed the effect of suppressing blood glucose elevation by a glucose tolerance test (Non-Patent Document 13). Have also been clarified for their effects such as anti-obesity and liver protection (Non-Patent Documents 14, 15, 16, 17, 18). Moreover, mutagenicity and acute toxicity tests (Non-Patent Documents 19, 20, 21) have also been studied for safety. Kotara Himubu is a plant that has both excellent efficacy and high safety. If there is, scientific support is obtained coming.

 [0010] Regarding the α-darcosidase inhibitory activity of this kotara himbu, salacinol, a thiosaccharide having a sulfate group in the molecule as an active ingredient (Patent Document 1, Non-Patent Document 22) Nya Kota Ranole (Non-Patent Document 23) and Retikiyuranol (Patent Document 2) has already been disclosed. In addition, since it has a very strong inhibitory activity surpassing that of carbose, which is marketed as a pharmaceutical, isomer synthesis and the like have been actively conducted using this component as a lead compound (Non-patent Documents 24 and 25). .

 [0011] However, on the other hand, considering the contents and activities of the three components that are currently clear, it is said that these three components cannot sufficiently explain the inhibitory activity of Kotarahimbu. There are also reports (Non-Patent Document 26), suggesting the existence of additional active ingredients.

 Patent Document 1: Japanese Patent Application Laid-Open No. 11 29472

 Patent Document 2: JP 2004-323420 A

 Non-patent literature l: Chiasson J. et al., JAMA, .290, p486-494, 2003,

 Non-Patent Document 2: Yuichi Shinoda et al., Metabolism., 55, p935—939, 2006

 Non-Patent Document 3: Inoue S et al., J. Antibiot., 19, p288—292, 1966

 Non-Patent Document 4: Naoki Asano, Glycobiology., 13 (10), p 93-104, 2003

 Non-Patent Document 5: Monique M.P. Hermans et al., J. Biol. Chem., 266 (21), pl3507—135

12, 1991

 Non-Patent Document 6: Jean Duplex Wansi et al., Chem. Pharm. Bull., 54 (3), p292—296, 20 06

 Non-Patent Document 7: Sung Ok Lee et al., Arch. Pharm. Res., 27 (12), pl207-1210, 2004 Non-Patent Document 8: Jun awabata et al., Biosci. Biotechnol. Biochem., 67 (2 ), P445-447, 2003.

 Non-patent document 9: Kiran Iqbal et al., Chem. Pharm. Bull., 52 (7), p785-789, 2004 Non-patent document 10: Miou Toda et al., Biosci. Biotechnol. Biochem., 64 (2) , P294—298,

2000,

Non-patent literature ll: Miou Toda et al., Biosci. Biotechnol. Biochem., 65 (3), p542-547, 2001

 Non-Patent Document 12: Attygalle, Sinhalese Materia Medica., P43, 1917

 Non-patent literature 13: E.H.Karunanayake et al., J Ethonopharmacol., 11, p223—231, 1984 Non-patent literature 14: Osami jimoto et al., Journal of Japan Nutrition's Food Society, 53 (5), pl99—205, 20

00

 Non-patent literature 15: Masayuki Yoshikawa et al., J Nutr., 132, pl819-1824, 2002 Non-patent literature 16: Masayuki Yoshikawa et al., Biol. Pharm. Bull., 25, p72- 76, 2002 Non-patent literature 17: Masayuki Yoshikawa et al., Pharmaceutical Journal, 123 (10), p871—880, 2003, Non-Patent Literature 18: Hidehiko Beppu et al., Journal of Japan Food New Materials Research Society, 8 (2), pl05—117, 2005

 Non-Patent Document 19: Hiroshi Shimoda et al., Edible Journal, 40 (3), pl98-205, 1999

 Non-Patent Document 20: Hiroshi Shimoda et al., Edible Journal, 42 (2), pl44—147, 2001

 Non-patent document 21: Hiroshi Shimoda et al., Medicine and pharmacy, 46 (4), p527-540, 2001 Non-patent document 22: Masayuki Yoshikawa et al., Tetrahedron Letters., 38 (48), p8367-8

370, 1997

 Non-Patent Document 23: Masayuki Yoshikawa et al., Chem. Pharm. Bull ·, 46 (8), pl339— 1340, 1998

 Non-patent document 24: Ahmad Ghavami et al., J. Org. Chem., 66, p2312-2317, 2001 Non-patent document 25: Monica G. Szczepina et al., J. Am. Chem. Soc, 126 (39) , Pl2458—

12469, 2004

 Non-Patent Document 26: Masayuki Yoshikawa et al., Pharmaceutical Journal, 121 (5), p371-378, 2001 Disclosure of the Invention

 Problems to be solved by the invention

[0013] As a result of further research based on the above background art, the present inventors have found a novel α-darcosidase inhibitory active substance from Kotarahimbu and have completed the present invention.

 Means for solving the problem

[0014] The compound of the present invention has an unprecedented force, a novel chemical formula represented by It is a compound and has α-darcosidase inhibitory activity.

 The invention's effect

 [0015] According to the present invention, an α-darcosidase inhibitor having a stronger action than previously known α-darcosidase inhibitors is provided, which causes antihyperglycemia such as antidiabetic drugs and antihyperlipidemic drugs. It is expected to be applied to the specific health foods that have been clinically applied to each symptom.

 Brief Description of Drawings

 FIG. 1 is an HPLC chart of a 70% acetonitrile nitrile eluate obtained by fractionating HP-20 fraction of Kotarahimbu water extract with an amino column.

FIG. 2 is a ¹ H-NMR spectrum of the final fraction of Kotara Himbu extract.

FIG. 3 is a ¹³ C-NMR spectrum of a final fraction of Kotarahimbu extract.

 [Fig. 4] FAB-MS spectrum of the final fraction of Kotara Himbu extract.

 FIG. 5 is an estimated plan structure diagram of the final fraction of Kotara Himbu extract.

 FIG. 6 is a graph showing efficacy evaluation by a sugar tolerance test (sucrose).

 FIG. 7 is a graph showing efficacy evaluation by a glucose tolerance test (maltose).

 BEST MODE FOR CARRYING OUT THE INVENTION

[0017] The novel compound according to the present invention has a structure represented by the following chemical formula (Chemical Formula 1), and is abundantly contained in the roots and aerial parts of the plant of the genus Sarchinae. This compound has a very excellent α-darcosidase inhibitory activity. This compound is mainly obtained from the solvent extract of the root and aerial parts of the genus Salacia plant, for example, Salacia reticulata, Salacia prinoides (Salacia reticulata). ), Sarachia oblonga and Sarachia chinensis. However, it goes without saying that the compound of the present invention may be extracted from other sites or plants other than the plant.

[Chemical 1]

[0018] The purification method is not particularly limited, and can be obtained, for example, by combining various known methods. For example, it can be obtained by adding extraction solvent to the roots of the genus Sarakia or the cut parts of the above-ground parts, extracting the filtrate, collecting the filtrate, concentrating, and then purifying it by fractionation using a solvent or column. It is done.

 [0019] To give a more specific example, about 2 to 20 times the amount of extraction solvent is added by weight to the roots and / or the aerial parts of the genus Sarakia. The extraction solvent may be selected from water, alcohols, or a mixed solvent of water and a hydrophilic solvent such as alcohols or acetone, with water being particularly preferred. A supercritical extraction method can also be used.

 [0020] The extraction time is an appropriately determined force, and is preferably refluxed overnight or longer. Then remove the residue by centrifugation or filtration. The filtrate obtained is concentrated under reduced pressure and then lyophilized. By the above operation, Kotarahimbu extract powder as a starting material is obtained. Note that the solvent may be added again to the residue, and the same extraction operation may be repeated.

[0021] An aqueous solution is prepared using this Kotarahimbu extract powder, and an ethanol precipitation treatment is performed by adding an equivalent amount of ethanol. In addition, methanol, isopropanol, acetonitrile, etc. may be used as the precipitation solvent. Thereafter, the precipitate is removed by centrifugation or filtration. Then, column chromatography using the organic solvent removed by vacuum concentration It is possible to obtain the novel α-darcosidase inhibitor of the present invention by the separation force S.

 [0022] The α-darcosidase inhibitor thus obtained can be used as a single component as it is. It can be used as an additive in various foods and beverages such as tea, suppresses the increase in blood sugar after meals, It is provided as a food for specified health use that can be used for the indications such as suppressing absorption. In addition, it may be formulated in the form of tablets, capsules, soft capsules, powders, granules, and other internal preparations and injections together with appropriate excipients, preservatives, fragrances and the like. It can be used not only for pharmaceuticals but also for health foods similar to so-called pharmaceutical forms.

 That is, the pharmaceutical composition of the present invention comprises an α-darcosidase inhibitor (compound) represented by the chemical formula (Formula 1) and a pharmacologically acceptable carrier. Here, the pharmacologically acceptable carrier means an excipient, a binder, a disintegrant, a lubricant, a ρΗ adjuster, a solvent and the like that are necessary for formulation. The excipients are lactose, sucrose, starch, bound cellulose, mannit, talc, etc., the binders are syrup, gum arabic, dextrin, etc. The disintegrants are polyethylene glycol, propylene glycol, D-mannitol, hydroxy Propyl cellulose, hydroxypropyl methylcellulose, etc. S, lubricants include magnesium stearate, talc, etc., pH adjusters include hydrochloric acid, citrate, phosphoric acid, sodium hydrogen phosphate, sodium hydroxide, etc. Examples thereof include water and ethanol. The form of the pharmaceutical composition is not limited. Examples of the form of the pharmaceutical composition include internal preparations such as tablets, capsules, soft capsules, powders, granules, and injections as described above. In addition, the pharmaceutical composition of the present invention includes a pharmaceutical composition containing other active ingredients than just a pharmaceutical composition containing only an α-darcosidase inhibitor (compound) represented by the chemical formula (Chemical Formula 1). Is included.

[0024] The food composition of the present invention comprises an α-darcosidase inhibitor (compound) represented by the chemical formula (Chemical Formula 1) and a nutritionally acceptable carrier. Here, the food composition refers to confectionery such as gum, candy, jelly, rice cakes such as udon and soba, dairy products such as yogurt and cheese, seasonings such as miso and soy sauce, sauces, soups, juices, For general foods such as coffee, tea, and nutritional drinks, and pharmaceuticals such as tablets and capsules It has a similar form! /, And refers to health foods (including foods for specified health use that are publicly approved to claim their effects). Nutritionally acceptable carriers refer to raw materials for food preparation necessary for the preparation of these foods, including pigments such as food red, antioxidants such as vitamin E and vitamin C, vitamin A and vitamin B1, etc. These are used to include various additives such as vitamins, preservatives, and preservatives that are added as needed. However, according to the present invention, an extract derived from a natural product adjusted to contain an α-gnorecosidase inhibitor (compound) represented by the chemical formula (Chemical Formula 1), that is, a known Kotarahimbu extract Food compositions containing are excluded. However, adding the α-darcosidase of the present invention to the food composition containing the extract to increase its effect is not excluded from the present invention.

 [0025] In order to effectively exhibit the α-darcosidase inhibitory activity in these food compositions and pharmaceutical compositions, the component of the present invention is at least 0.0001%, more preferably 0, based on the total weight. 005-0. About 1% is required. However, this amount can be appropriately set depending on the symptoms, age, intake form, etc. of the patient or animal to be administered.

 [0026] Hereinafter, the present invention will be described in more detail based on examples. The present invention is not limited to the following examples! Needless to say,

 Example 1

 [0027] [Coarse fraction of Kotarahimbu extract]

Ten times the amount of water was added to approximately 1 kg of dried stems of Salacia reticulata, and hot water extraction was performed overnight (14 to 15 hours). Insoluble matter in the obtained extract was removed by centrifugation (8,000 rpm / 30 min) and filtration. By these operations, 100 g of Kotarahimbu hot water extract powder was obtained. Water was added thereto to prepare a 500 ml aqueous solution. Thereafter, an equivalent amount of ethanol was added with stirring, and the resulting precipitate was removed by centrifugation (8,000 rpm / 30 min). The obtained supernatant was ethanol-removed by concentration under reduced pressure (yield 69.0 g). A 500 ml aqueous solution was prepared again using this, and then treated with an HP-20 column (carrier used: 1 kg of a synthetic adsorbent HP-20 manufactured by Mitsubishi Chemical Corporation, column: 8 × 35 cm). Elution was carried out using water (5 U, 20 vol%, 40 vol%, 60 vol%, and 80 vol% methanol aqueous solution (2.5 U each, stepwise elution). W ”fraction; Yield 34. lg), 20 vol% methanol fraction (“: HP-20-20 ”fraction; Yield 5.89 g), 40 vol% methanol fraction (“: HP-20-20 40 Fraction; Yield 9. 48 g), 60% methanol fraction (“HP-20-60” fraction; Yield 4.85 g), 80% methanol fraction (“: HP-20-60” fraction) Min; yield 0.667 g) (total recovery 86.0%). Each of the obtained fractions was subjected to an α-darcosidase inhibitory activity test. The inhibitory activity test was performed according to the following operation method. The results are shown in Table 1.

 [0028] [a Measurement of darcosidase inhibitory activity (in vitro)]

 (Preparation of crude enzyme solution)

 Reagent 1: Rat small intestine acetone powder (manufactured by SIGMA), reagent 2: l OOmM potassium phosphate buffer (5 mM EDTA, pH 7.0), reagent 3: l OOmM potassium phosphate buffer (0.1% Triton , pH 7.0), Reagent 4: 10 mM potassium phosphate buffer (pH 7.0) is used.

 [0029] First, 50 ml of Reagent 2 was added to 5 g of Reagent 1, and homogenized (1 minute x 3 times) with ice cooling. The resulting solution was centrifuged (15,000 X g / 60 min or 21,000 X g / 6 Omin), and the supernatant was removed. Thereafter, 50 mL of Reagent 3 was added to the precipitate, solubilized by stirring, and left at 4 ° C. for 60 minutes. This was further centrifuged (15,000 X g / 90 min or 110,000 X g / 90 min) to obtain a supernatant. The resulting supernatant was dialyzed with reagent 4 for 24 hours. The internal solution obtained after dialysis was used for the test as a crude enzyme solution.

 [0030] (a Gnolecosidase inhibitory activity test)

 As test reagents, reagent 1: crude enzyme solution, reagent 2: DMSO, reagent 3: l OOmM sucrose aqueous solution (25 mM maltose aqueous solution), reagent 4: 2MTris_HCl buffer (pH 7.0), reagent 5: glucose CII test A coloring solution (manufactured by Wako Pure Chemical Industries) was prepared. The test sample solution was prepared by dissolving the test substance (l Omg) in DMSO (10mU (diluted appropriately according to the strength of the inhibitory activity of the sample). Also, measured at n = 3 per sample. Then, each sample was blanked, and then the inhibitory activity was measured by the following procedure.

[0031] 10 L of sample solution and 25 μL of reagent 1 were added to a 96-well plate, and preincubated for 5 minutes in a thermostatic bath. At this time, before adding reagent 1, add 50 L of reagent 4 to the blank so that the reaction does not proceed. Then add 20 L of Reagent 3 to start the reaction. [0032] 30 minutes after the start of the reaction, 50 μL of Reagent 4 was added to the reaction solution other than the blank to stop the reaction. Then, 150 L of Reagent 5 was added to each reaction solution to develop color for 10 minutes, and then the absorbance at 492 nm was measured using a microplate reader. From the obtained measurement result, the inhibition rate was calculated by the following formula 1.

 Inhibition rate (%) = {(GlcO-Glcx) / GlcO} X 100 · · · · Equation 1

 However, GlcO: Control (DMS O in the same amount as the sample) is obtained by subtracting the average absorbance (n = 3) of the corresponding blank from the average absorbance (n = 3).

 Glcx: Average absorbance of the sample (n = 3) minus the corresponding blank absorbance (n = 3)

[0033] [Table 1]

Yield after HP-20 treatment and α-darcosidase inhibitory activity (IC ₅₀ )

 [0034] As can be seen from Table 1, most of the inhibitory active ingredients in the extract were collected in the water-eluted fraction, and the inhibitory activity decreased with increasing methanol concentration. Inhibitory activity was not observed at a concentration of 60% by volume or more. Therefore, it was decided to further separate the HP-20W fraction that showed remarkable activity.

 [0035] [Fractionation of Kotarahimbubu crude fraction 'purification]

 The fraction was made into a 200 mL aqueous solution, an equivalent amount of ethanol was added and stirred, and the insoluble portion was removed by centrifugation (7, 500 rpm / 30 min). The resulting supernatant was freed from ethanol by concentration under reduced pressure. This was made into a 40 mL aqueous solution and applied to an amino column (a carrier used: a column packed with 950 g of N H-DM 1020SG manufactured by Fuji Silicon Chemical Co., Ltd .: 8 × 49 cm).

[0036] 80% by volume (6L), 70% by volume (9L), 60% by volume, 40% by volume, 20% by volume acetonitrile aqueous solution (6L each), water (12U is used for stepwise elution. 80% capacity Acetonitrile-eluted fraction (“80% CN” fraction; yield 4.43 g), 70% -acetonitrile-eluted fraction (“70% CN” fraction; yield 12.10 g), 60% -acetonitrile-eluted fraction (“60% CN ”fraction: Yield 2 · 78 g), 40% acetonitrile-eluted fraction (“ 40% CN ”fraction; Yield 6.75 g), 20% acetonitrile-eluted fraction (“ 20% CN ”fraction; Yield 1. 90 g), a water-eluted fraction (“W” fraction; 4.20 g) was obtained (total recovery 94.3%). Each of the obtained fractions was subjected to an α-darcosidase inhibitory activity test by the above method. The results are shown in Table 2.

[Table 2] α-Dalcosidase inhibitory activity (IC ₅₀ ) of amino column treated fractions

[0038] From the test results, it was found that almost no inhibitory activity was observed in the elution part other than the 70% CN fraction, and the main inhibitory active component was present in the 70% CN fraction. In addition, when the inhibitory activity test was carried out on the precipitate formed during the pretreatment, no activity was observed.

[0039] Next, HPLC fractionation was performed using this fraction. The preparative conditions were as follows: column used: YMC pack polyamine-11 (20 250111111), mobile phase: 65% by volume acetonitrile, flow rate: 4.5 mL / min, column temperature: 30 ° C, detector: RI. The sample for separation was dissolved in a mobile phase solvent. However, since a large amount of white precipitate was formed at this time, the supernatant portion obtained after centrifugation was used (the white precipitate was used, and when the inhibitory activity test was performed, the activity was not recognized. , Tsuta). From the HPLC eluates obtained at this time, Frl (7—15 min / yield 433. Omg), Fr2 (25—35 min / yield 1744.0 mg), Fr3 (35—40 min / yield 526.7 mg), Fr4 ( Fr5 (53-57min / yield 33.6mg) and 5 fractions were obtained. Figure 1 shows the HPLC chart at this time. Furthermore, in order to recover the column adsorbed components, the column was washed with 20% by volume acetonitrile solution. Purification was performed (total recovery rate 88.2%). The obtained fraction was subjected to a <<-darcosidase inhibitory activity test by the above method. The results are shown in Table 3.

 [Table 3]

Α-Dalcosidase inhibitory activity (IC ₅₀ ) of HPLC fractions

[0041] As will be understood from Table 3, inhibitory activity was observed for Frl and Fr3～5, in Fr ²及beauty 20% Asetonitoriru recovery unit was not observed at all inhibitory activity. The force with a relatively strong inhibitory activity in Fr3-5, in particular, Frl showed a strong inhibitory activity. Frl can also be separated by using 50 to 70% by volume aqueous acetonitrile as the mobile phase. In this case, the retention time varies, but it can be separated from other active ingredient fractions (Fr3-5) by collecting the peak within 10-25 min.

 [0042] With the YMC pack polyamine-II column, as shown in the spectrum shown in Fig. 1, it is very difficult to further separate the peaks because they become broad. Changed and further fractionated.

 [0043] Preparative conditions are as follows: Column used: DAISOPAK SP-120-5-ODS_BP (20 X 250mm), mobile phase: water, flow rate: 5. OmL / min, column temperature: 30 ° C, detector: RI Carried out. From the obtained spectrum, ODSFrl (10—10 · 7min / yield 230 · 4mg), ODSFr2 (10 · 7—11 • 2min / 5.8mg), ODSFr3 (11.2-13min / 70.8mg) Three fractions were obtained. These fractions were then tested for inhibitory activity. The results are shown in Table 4.

[0044] [Table 4] Cc-glucosidase inhibitory activity (ICso) of HPLC fractions

[0045] [Fractionation · Structure determination of purified product]

The ODSFr2 fraction was repeated several times in the same manner to obtain almost a single substance. Various NMR analyzes to clarify the structure of this substance ^ H, ¹³ C, 'H-' HCOS Y, ^ 1 ^ (3 and ^ 1 ^ [8)) and? Eight: 6 — ^ [3 analysis was performed, and structural analysis was performed using the obtained spectrum data. NMR analysis (1D— and 2D—) was performed by analyzer: JN M AL-400 (manufactured by JEOL, 400 MHz), solvent: DO (acetone as internal standard reagent)

 2

The analysis temperature was 25 ° C. FAB—MS analysis was performed with an analyzer: 7000QQ type (manufactured by JEOL Ltd.), ionization method: fast atom bombardment method (FAB), acceleration voltage: 6 kV, matrix: glycerin, measurement mass range: ~ m / zl000. Fig. 2 shows the ¹ H-NMR spectrum (inD O / 400MHz), Fig. 3 shows the ¹³ C-NMR spectrum (inD O / 400MHz), and Fig. 4

 twenty two

 Shows the FAB-MS spectrum.

 [0046] As a result of analysis based on the above results, it was suggested that the component obtained from ODSFr2 is cyclitol having sulfoxide as shown in FIG. However, since the presence of sulfoxide cannot be confirmed from the obtained spectrum data, the elemental composition was further clarified by HR-MS analysis. Analytical apparatus: JMS—T1 00LC (manufactured by JEOL Ltd.), ionization method: APCI, measurement mass range: to m / z 1000. From this analysis, the results shown in Table 5 were obtained.

以上のことより、図 5に示した構造との整合性が得られたことから、 ODSFr2で得ら れた成分は、次の化学式 (化 1)に示すようにスルフォキシドを有する 13員環シクリト ール構造であることが明らかとなった。 [0047] [Table 5] APCI-MS analysis results of the components of the present invention

Based on the above, consistency with the structure shown in FIG. 5 was obtained, so the component obtained with ODSFr2 was a 13-membered cyclic cyclite having a sulfoxide as shown in the following chemical formula (Chemical Formula 1). It was clarified that it was a rugged structure.

[0049] [Chemical 1]

[0050] Further IR analysis was performed to confirm the functional group. Analysis apparatus: JASCO FT / IR-460 plus infrared spectrometer, analysis method: The analysis was performed under the analysis conditions of the KBr method. As a result of measurement, an absorption band of 1071CHT ¹ indicating sulfoxide was detected. As a result, it was confirmed that sulfoxide was contained in the molecule.

 Example 2

 [0051] [Activity evaluation by sugar tolerance test (sucrose tolerance test)]

 Breeding environment is clear cycle (17:00-19:00 lighting 12 hours cycle), temperature 23-25 ° C

Under the condition of humidity 60-70%, Wistar rats (male) 5 weeks old were used for pre-feeding for 1 week before the test. During the preliminary breeding period, both feed and drinking water were freely consumed.

[0052] The groups were divided into 4 groups, 6 animals each, based on body weight and blood glucose level. The administration groups were the control group (purified water) and the α-darcosidase inhibitor administration group (0.15, 0.30 and 0.4), respectively.

5 mg / kg).

 [0053] In the glucose tolerance test, sucrose (2.5 g / kg) was orally administered to each group, and before glucose tolerance (after 0 minutes),

Blood was collected from the tail vein 60 and 120 minutes later. Α-Dalcosidase inhibitor is It was dissolved in a loin solution and co-administered. The blood glucose level in the obtained serum was measured using a glucose CII test KOKO (manufactured by Wako Pure Chemical Industries). Efficacy evaluation was performed by Dunnett multiple comparison test using blood glucose in the control group as a control. The results are shown in Table 6 and FIG. The values in Table 6 are the average soil standard deviation. Table 6 and Fig. 6 show that “*” indicates a significant difference at p 0.05 compared to the control group, and “* * indicates a significant difference at p 0.01. "It is shown.

 [0054] As a result, the 0.15 mg / kg inhibitor administration group showed no difference in blood glucose level 60 minutes after glucose load compared to the control group. Administration of 0.30 and 0.45 mg / kg The group showed significantly low values at a risk rate of 5% and 1%, confirming the sugar absorption inhibitory effect.

 [0055] [Table 6]

 Glucose tolerance test (sucrose) results

 Blood glucose level (mgZdL): Average value

 *: p <0.05; **: p <0.01 vs control group Example 3

 [0056] [Evaluation of efficacy by sugar tolerance test (maltose tolerance test)]

 The test method was performed under the same conditions as in Example 2. The administration groups were a control group (purified water) and an α-darcosidase administration group (0 · 30, 0.60 and 0.90 mg / kg), respectively.

 [0057] In the glucose tolerance test, maltose (2.5 g / kg) was orally administered to each group, before the glucose load (after 0 minutes),

Blood was collected from the tail vein 60 and 120 minutes later, and the blood glucose level was measured using the obtained serum. The α-darcosidase inhibitor was dissolved in a maltose solution and coadministered. The blood glucose level in the obtained serum was measured using a glucose CII test KOKO (manufactured by Wako Pure Chemical Industries). Efficacy was evaluated by Dunnett's multiple comparison test using blood glucose in the control group as a control. The results are shown in Table 7 and FIG. The values in the table are the average soil standard deviation It is. Table 7 and Figure 7 show that “a” indicates a significant difference at p 0.1 compared to the control group, and “*” indicates a significant difference at p <0.05. When there is a significant difference between p and 0.01, "* *" is indicated.

 [0058] As a result, in the 0.30 mg / kg group, the blood glucose level showed a tendency to decrease after 60 minutes (p 0.1) compared to the control group. In the kg-administered group, the risk rate was 1%, showing a significantly low value, confirming the sugar absorption inhibitory effect. Thus, it was revealed that the anti-diabetic action was also effective for rats.

[0059] [Table 7]

 Glucose tolerance test (maltose) results

 Blood glucose level (mgZdL): Average value

 a: p <0.1; *: p <0.01; **: p <0.01 vs control group

[0060] [Fractionation comparison of purified fraction activity]

 The activity of the α-darcosidase inhibitor whose structure was determined was compared with previously reported components obtained from Kotarahimbu (cited from Non-Patent Document 26) and vodaribose, which is approved for sale as a pharmaceutical product. The results are shown in Table 8. As can be seen from this table, the activity intensity is almost the same as that of voglibose on the market, and it is about 7;-103 times higher for maltase inhibition and about 10 times lower for sucrase inhibition compared to previously reported components obtained from the same plant The inhibitory activity intensity was 19 to 34 times. In addition, in the glucose tolerance test, it showed a sugar absorption inhibitory effect at a dose lower than the reported components. Thus, the α-darcosidase inhibitor according to the present invention is clearly a component exhibiting inhibitory activity far exceeding the previously reported components, and is considered to be the main active component in the Kotarahimbu extract.

[0061] [Table 8] Comparison of α-darcosidase inhibitory activity with the components of the present invention (IC ₅₀ )

 1) Quoted from Pharmaceutical Journal 121 (5), ρ371-378 (2001).

 2) Tested at our company using Basin OD Tablet 0.2 (manufactured by Takeda Pharmaceutical Company Limited).

 The molar concentration in parentheses is calculated based on molecular weight.

[0062] As described above, it was confirmed that the compound represented by the chemical formula (Formula 1) has excellent α-darcosidase inhibitory activity. The α-darcosidase inhibitory activity of this compound is considerably higher than that of salacinol and kotalanol, which have been reported so far, in the kotarahimbu extract. The contribution ratio of such compounds is considered to be high.

 Example 4

 [0063] Next, a formulation example using this component is shown.

 [Production Example 1: Tablet]

 Α-Dalcosidase inhibitor of the present invention 0.07 g

 Maltosyl cyclodextrin 600g

 Sucrose fatty acid ester 50g

 Calcium carbonate 15g

 Reduced maltose syrup 75g

 Lactic anhydride 1 10g

 Crystalline cellulose 100g

 Milk calcium 50g

 According to the above-mentioned prescription, according to a conventional method !, 5000 tablets were produced.

 [Production Example 2: Candy]

 Α-Dalcosidase inhibitor 1.0 g of the present invention

 120g sugar

Mizuame 100g i 4g

 Fragrance 0.4 g

 According to the above formulation, 50 candies were produced according to a conventional method.

[0065] [Production Example 3: Soft drink (500mU)]

 Chenic acid 0.2 g

 Fructose dextrose liquid sugar 25g

 Perfume appropriate amount

 Α-Dalcosidase inhibitor 0.5 mg of the present invention

 Appropriate amount of water

 According to the above formula, 500 mL of soft drink was produced according to the conventional method!

[0066] [Production Example ⁴ : Soft capsule]

 (Contents)

 Safflower oil 88 · 0% by mass

 Emulsifier 1 1. 8% by mass

Of the present invention alpha - Darukoshidaze inhibitor 0.2 wt ^_0/0

 Soft capsules were produced in accordance with a conventional method using the contents according to the above formulation and a coating consisting mainly of gelatin.

 Industrial applicability

[0067] The compound of the present invention has a novel and highly active α-darcosidase inhibitory action, whereby anti-diabetic drugs that are more effective for anti-diabetic patients and people in the pre-diabetes group are used for specific health care. Foods are provided.

Claims

A compound represented by the following chemical formula (Chemical Formula 1):  [Chemical 1]

[3] A pharmaceutical composition comprising the α-darcosidase inhibitor according to claim 2 and a pharmacologically acceptable carrier.  [4] A food composition comprising the α-darcosidase inhibitor according to claim 2 and a nutritionally acceptable carrier (provided to contain the α-darcosidase inhibitor according to claim 2) Except food compositions containing extracts derived from natural products).  [5] Use of a compound represented by the following chemical formula (Formula 1) as an α-darcosidase inhibitor or an antidiabetic agent.  [Chemical 1]