JP3511231B2 - α-glucosidase inhibitor - Google Patents

Description

Description: TECHNICAL FIELD [0001] The present invention relates to an α-glucosidase inhibitor effective for preventing diabetes and obesity. [0002] An α-glucosidase inhibitor inhibits a carbohydrate digestive enzyme that decomposes a disaccharide into a monosaccharide in an ingested food or drink, so that the decomposition of the disaccharide into a monosaccharide is slow. In other words, the absorption of monosaccharides from the intestinal tract is delayed, thereby having an effect of suppressing an increase in blood glucose level. Therefore, it is useful for hyperglycemic symptoms and various diseases such as obesity, adiposity, hyperlipidemia, and diabetes caused by hyperglycemia. [0003] Studies on α-glucosidase inhibitors have been carried out for a long time, and many pseudomonosaccharide, oligosaccharide or peptide substances isolated from actinomycetes have been reported. Currently, acarbose and voglibose are carbohydrate digestive enzyme inhibitors used as pharmaceuticals (Progress in Clinical Biochemistry and
Medicine, 77-99, 1988, Journal of the Japanese Society of Agricultural Chemistry, 63, 217, 198
9). Further, recently, a substance obtained from an extract obtained from eel in JP-A-9-2963, an extract of Milchia cephaelocarpe in JP-A-9-48736, JP-A-9-65836
Discloses that a hydrolyzate such as a vegetable protein inhibits α-glucosidase in Japanese Patent Application Laid-Open No. 9-046424. [0004] As described above, many α-glucosidase inhibitors have been developed so far.
However, those derived from plants have not yet been practically used for pharmaceuticals and foods. Therefore, α-glucosidase inhibitors derived from natural products, which are substances contained in food and have excellent processing properties, that is, high safety for living organisms, are edible daily to prevent and improve obesity and diabetes. Is possible. In view of the above problems, the present invention is intended to stably supply a large amount of a highly safe natural product-derived α-glucosidase inhibitor. Therefore, an object of the present invention is to provide an α-mushroom or an edible mushroom extract as an active ingredient.
-To provide a glucosidase inhibitor. Means for Solving the Problems The present inventors effectively inhibit α-glucosidase, one of carbohydrate digestive enzymes, and have no harmful effect on the human body. New α
-Diligent research was conducted to find glucosidase inhibitors. As a result, edible Agaricaceae,
Tricholomataceae, cortinariaceae, hygrophoraceae, Kanoshita family belonging to the Hidanashitake eyes, following one of the mushroom ramaria botrytis family, and phallaceae belonging to phallales
An α-glucosidase inhibitor useful in the prevention and improvement of obesity and diabetes, which is highly safe and effective for the living body, has been found in particular mushrooms , especially in extract extracts, and completed the present invention. [0007] The edible mushrooms that can be used in the present invention are not particularly limited as long as they are edible. Raw
It can be used as a dried product, a dried powder or a solvent extract, and its use form is not limited. Either the mycelium or the fruiting body, or both can be used. For extracting the extract from edible mushrooms, water or an organic solvent such as methanol, ethanol, isopropyl alcohol, butanol, ethyl acetate or acetone, or a mixed solvent thereof may be used as a solvent.
These can be used after appropriately concentrating, purifying, sterilizing, drying and the like. As a method of searching for an α-glucosidase inhibitor using the obtained extract, using sucrose, maltose, and isomaltose as substrates, the extract and the rat small intestine crude enzyme solution are reacted, and the amount of generated glucose is measured using glucose oxidase. A method of quantification by the used enzymatic method can be used. The enzyme solution used was derived from rat small intestine, but may be derived from pigs or cattle. This method is a method generally used as an α-glucosidase inhibitory activity test, but is not limited thereto. However, in the present invention, since a large number of samples cannot be tested by the above-mentioned method, the number of samples is narrowed down in advance by a plate agar medium method in which a large number of samples can be tested at once. Specifically, a rat small intestine crude enzyme solution is spread on a plate agar medium, a hole is formed in the medium, a sample is dropped and reacted, and p-nitrophenyl-α-D-glucose is used as a substrate.
An agar medium containing pyranoside is sown, and enzyme inhibition is determined by the diameter of the inhibition circle generated by the enzyme reaction. [0010] In addition, those having a stronger activity can be selected from those having an effect of suppressing an increase in blood glucose level by an animal test using rats. When 265 kinds of mushrooms were searched by the above screening method,
Agaricales belonging to boletaceae death Ronumeriiguchi, co-cormorant <br/> Jitake, Amorphophallus variabilis, Aishimeji of tricholomataceae, laccaria laccata, of cortinariaceae Tsubafuusentake, Sasatake,
Goat mushrooms of the slime mushroom family, Akayamatake, Shirokanashita of the cynoaceae family belonging to the order Shidanashitake, Scarlet mushrooms of the Asteraceae family, and the extract of Suppontake family of the genus Supponaceae belonging to the order of the order Supponamaceae, in particular, have an α-glucosidase inhibitory activity. . [0011] Furthermore, α-
Glucosidase inhibitors were isolated and identified. As a result of the structural analysis, it was found to be 7-O-β-glucopyranosyl-α-homonojirimycin. It is known that this compound exhibits α-glucosidase inhibitory activity (Rhinehart,
BL: J. Pharmacol.Exp.Therap., 241 , 915-920 (1987); N. As
ano et al: J. Nat. Pro., 60, 98-101 (1997)). It has also been reported that this compound was isolated from a herb, Aglanema treubii (N. Asano et al: J. Nat. Pro., 60 , 98-101).
(1997)). However, this is the first time that the present compound has been isolated and identified from Bombus edulis. [0012] Similarly, an inhibitory substance was similarly isolated from the extracts of Aspergillus niger, and α-homonojirimycin and 7-O
-Β-glucopyranosyl-α-homonojirimycin. It is known that α-homonojirimycin also exhibits α-glucosidase inhibitory activity (Yagi Yamada et al .:
Journal of Pharmaceutical Sciences 47 (1), 47-55 (1993); Geoffrey C. Kite et
al; Tetrahedron Letters 29 (49), 6483-6486 (199
8)), this is the first time that the present compound has been isolated and identified from A. The α-glucosidase inhibitor of the present invention includes, for example, tablets, capsules, soft capsules, powders, injections,
It can be administered in an appropriate dosage form such as a patch. Excipients, solubilizers, binders, stabilizers, flavoring agents, and the like can be used in various formulations using these dosage forms. Specific examples of adjuvants that can be mixed with tablets, capsules and the like are as follows. Binders such as tragacanth gum, gum arabic, corn starch or gelatin,
Excipients such as microcrystalline cellulose, disintegrants such as corn starch, whole gel starch, alginic acid, etc., lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, peppermint, reddish oil When the flavoring agent such as cherry and the unit use form are capsules, a liquid carrier such as fatty oil can be contained in addition to the above-mentioned type of material. Various other materials may be present as coatings or to otherwise alter the physical form of the dosage unit. For example, tablets may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. The sterile composition for injection is water for injection, sesame oil,
By dissolving or suspending the active substance in excipients, for example, naturally occurring vegetable oils such as coconut oil, peanut oil and cottonseed oil, they can be formulated according to conventional pharmaceutical practice. Buffers, preservatives, antioxidants and the like can be mixed as necessary. The drug of the present invention can be administered by various injections such as intravenous injection, subcutaneous injection, intramuscular injection or oral administration, and various methods such as transdermal administration.
Particularly preferred are oral administration and transdermal administration, and the dosage is generally 0.2 to 2 g per day for oral administration,
In the case of intravenous administration, the dose is preferably 0.1 to 1 g per day, which may be administered several times a day. The dose generally varies depending on the type and symptoms of the disease caused by drug resistance, the method of administration, and the like, and the dose can be administered outside the above range. The present invention relates to a substance contained in a plant,
It is a natural α-glucosidase inhibitor that has excellent processing properties and is highly safe for living organisms, and has an inhibitory effect on blood glucose level increase by inhibiting the activity of carbohydrate degrading enzyme, which is a digestive enzyme. It is useful for various diseases such as obesity, adiposity, hyperlipidemia, and diabetes caused by symptoms and hyperglycemia. EXAMPLES Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples. (Example 1) Preparation of edible mushroom extract 100 g of each edible mushroom dry powder shown in Table 1 was ultrasonically extracted with 70% methanol, and each was concentrated under reduced pressure and freeze-dried to obtain 20 g to 40 g of various mushroom extracts. . Using these extracts, an α-glucosidase inhibition test described in Example 3 and below was performed. (Embodiment 2) α-
Isolation and Identification of Glucosidase Inhibitors Butterfly centrifuge (dry weight 308.2 g) was extracted by heating and refluxing three times with 70% ethanol, and the extract was concentrated under reduced pressure to obtain an extract extract. The extract was applied to an Amberlite IR-120B (H ⁺ ) column, eluted with 2N-aqueous ammonia, concentrated, then applied to a Dowex1X4 (AcO-) column, and eluted with 0.3N acetic acid. Further, column chromatography was performed with Dowex50WX4 (Py ⁺ ), and the active fraction was concentrated to dryness to obtain 3.7 g of a mixture. This fraction is further treated with n-propanol-
Silica gel chromatography was repeated with aqueous ammonia to obtain 0.09 g of 7-O-β-glucopyranosyl-α-homonojirimycin. The structure of 7-O-β-glucopyranosyl-α-homonojirimycin obtained above was determined from the following instrumental analysis data. Molecular formula: C ₁₃ H ₂₅ O ₁₀ N FAB-MS m / z: 356 [M + H ⁺ ] ¹ H-nuclear magnetic resonance spectrum [D ₂ O]: δ 3.20 (1H, dd, J
= 8.0, 9.3), 3.24 (1H, dd, J = 9.3, 9.8), 3.33 (1H, ddd, J =
2.2, 5.8, 9.8), 3.36 (1H, t, J = 9.3), 3.39 (1H, ddd, J = 3.
4, 7.4, 10.3), 3.49 (1H, dd, J = 9.0, 10.3), 3.58 (1H, d
d, J = 5.8,12.4), 3.65 (1H, t, J = 9.0), 3.77 (1H, dd, J = 2.
2, 12.4), 3.797 (1H, dd, J = 7.4, 10.3), 3.798 (1H, dd, J =
3.4, 10.3), 3.799 (1H, dd, J = 9.0, 9.4), 3.85 (1H, ddd, J
= 3.4, 8.3, 9.4), 3.96 (1H, dd, J = 8.3, 11.9), 4.22 (1H,
dd, J = 3.4, 11.9), 4.38 (1H, d, J = 8.0) ¹³ C-nuclear magnetic resonance spectrum [D ₂ O]: δ 55.4, 56.4,
57.6, 61.0, 63.8, 68.2, 68.5, 69.9, 73.0, 73.4, 7
6.0, 76.4, 102.8 (Example 3) Isolation and Identification of α-Glucosidase Inhibitor from Kinugasatake Kinugasatake (1.5 kg wet weight) was ultrasonically extracted three times with 70% methanol, and the extract was concentrated under reduced pressure. , An extract was obtained.
The extract was applied to an Amberlite IR-120B (H ⁺ ) column, eluted with 2N aqueous ammonia, and further added to Dowex 1
The mixture was applied to an X8 (OH-) column, and the active fraction eluted with water was concentrated to dryness to obtain 0.75 g of a mixture. This fraction was further subjected to silica gel chromatography with n-propanol, acetic acid, and aqueous ammonia to obtain α-homonojirimycin (0.05 g) and 7-O-β-glucopyranosyl-α-homonojirimycin (0.04 g). Was isolated. The structures of α-homonojirimycin and 7-O-β-glucopyranosyl-α-homonojirimycin isolated from Pseudomonas velutipes were determined by the following analytical data. <Α-Homonojirimycin> Molecular formula: C ₇ H ₁₅ O ₅ N FAB-MS m / z: 194 [M + H ⁺ ] ¹ H-nuclear magnetic resonance spectrum [D ₂ O]: δ 2.89 (1H, ddd ,
J = 2.8, 6.8, 9.8), 3.24 (1H, t, J = 9.8), 3.32 (1H, ddd, J =
5.6, 6.1, 9.8), 3.53 (1H, t, J = 9.8), 3.60 (1H, dd, J = 6.
8, 11.3), 3.77 (1H, dd, J = 6.1, 9.8), 3.81-3.85 (2H,
m), 3.93 (1H, dd, J = 2.8, 11.3) ¹³ C-nuclear magnetic resonance spectrum [D ₂ O]: δ 55.9, 57.9,
58.6, 65.0, 73.8, 75.9, 77.8 <7-O-β-glucopyranosyl-α-homonojirimycin> Molecular formula: C ₁₃ H ₂₅ O ₁₀ N FAB-MS m / z: 356 [M + H ⁺ ] ¹ H -Nuclear magnetic resonance spectrum [D ₂ O]: δ 3.14 (1H, dd, J
= 8.0, 9.1), 3.19 (1H, dd, J = 9.1, 9.8), 3.28 (1H, ddd, J =
2.2, 5.8, 9.8), 3.30 (1H, t, J = 9.1), 3.32 (1H, ddd, J = 3.
3, 7.2, 10.2), 3.42 (1H, dd, J = 8.9, 10.2), 3.53 (1H, d
d, J = 5.8, 12.3), 3.59 (1H, t, J = 8.9), 3.720 (1H, dd, J = 2.
2, 12.3), 3.726 (1H, dd, J = 7.2, 10.2), 3.728 (1H, dd, J =
3.3, 10.2), 3.729 (1H, dd, J = 8.9,9.3), 3.766 (1H, ddd,
J = 3.4, 8.2, 9.3), 3.91 (1H, dd, J = 8.2, 11.8), 4.16 (1
H, dd, J = 3.4, 11.8), 4.33 (1H, d, J = 7.9) ¹³ C-nuclear magnetic resonance spectrum [D ₂ O]: δ 55.4, 56.3,
57.7, 61.0, 63.9, 68.2, 68.6, 69.8, 73.0, 73.3, 7
5.9, 76.4, 102.7 Example 4 α-Glucosidase Inhibition Test This test was carried out by the method of Kurihara et al. (H. Kurihara et al .: Fishheries).
Science 60 (6), 759-761 (1994)). After spreading rat small intestine crude enzyme solution 0.60 mL on 2% plate agar medium,
A hole having a diameter of 8 mm was made in the medium, and 0.10 mL of a sample solution (20 mg of various mushroom extracts dissolved in 1 mL of water) was added dropwise. twenty five
At 150 ° C for 150 minutes, and 5 mM p-nitrophenyl-
Sprinkle 0.5% agar solution containing α-D-glucopyranoside, 25
Reaction was performed at 30 ° C. for 30 minutes. Enzyme inhibition was determined by the diameter of the inhibition circle generated by the enzymatic reaction. The judgment is that the diameter is less than 12 mm:-, the diameter is 12 mm or more
Less than 15 mm: ±, diameter 15 mm or more and less than 18 mm: +, diameter 18 mm or more: ++. Table 1 shows the results of determination of α-glucosidase inhibition of various mushroom extracts. [Table 1] Example 5 Measurement of α-Glucosidase Inhibition Rate (1) This test was carried out by the method of Asano et al. (Toshihiko Asano et al .: Journal of Japan Society of Nutrition and Food Science, 49 (3), 157-162 (1996)) Followed. Using 50 mM sucrose, maltose, and isomaltose as substrates, 0.45 mL of substrate and 0.05 mL of a sample solution of various mushroom extracts (60 mg of various mushroom extracts dissolved in 1 mL of water) and 0.50 mL of rat small intestine crude enzyme solution Was added and reacted at 37 ° C. for 60 minutes. Thereafter, the reaction was stopped, and the amount of glucose produced by the enzyme reaction was quantified by an enzymatic method using glucose oxidase. 10 instead of mushroom extract as control
The reaction was carried out in the same manner using an mM phosphate buffer, and the amount of glucose after the reaction was quantified to determine the inhibition rate (%). Table 2 shows the enzyme inhibition rates of various mushroom extracts. [Table 2] Example 6 Measurement of α-Glucosidase Inhibition Rate (2) 1 ml of each of 7 mg of 7-O-β-glucopyranosyl-α-homonojirimycin and α-homonojirimycin obtained in Examples 2 and 3 The same test as in Example 5 was conducted by using a solution dissolved in water as a sample solution. Table 3 shows the results of the enzyme inhibition rates (%) of 7-O-β-glucopyranosyl-α-homonojirimycin and α-homonojirimycin. [Table 3] (Example 7) Investigation of the inhibitory effect on blood sugar level increase A 6-week-old male SD rat fasted for 20 hours was supplemented with sucrose (2.5 g / Kg) together with an extract (500 mg).
/ Kg) once, before administration, 30 minutes, 60 minutes and 120 minutes
One minute later, blood was collected from the tail vein, and the blood glucose level was measured by the glucose oxidase method. Water was administered to the control group together with sucrose, and the blood glucose level was measured in the same manner as the administration group. Table 4 shows the blood sugar level-suppressing effect of the tsubasa centrifugal extract. [Table 4] (Mean ± SD, mg / dL) * P <0.05 (t-test)

──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Kazuhiro Matsumoto 1-1-21 Midori, Moriya-cho, Kitasoma-gun, Ibaraki Prefecture Asahi Breweries, Ltd. Research and Development Center Basic Research Laboratory (56) References JP-A-9-104624 (JP) , A) Chemical Abs. , 1960, abs. no. 55: 72717: pat. No. Fr 1216259, 1960. Int. J. Peptide Protein Res. , 1995, Vol. 46, no. 6, pp. 508-513 Agric. Biol. Chem. , 1978, Vol. 42, No. 5, pp. 971-980 (58) Fields investigated (Int. Cl. ⁷ , DB name) A61K 35/84 BIOSIS (STN) CA (STN) JICST file (JOIS) MEDLINE (STN)

Claims (1)

(57) Claims: 1. A sheet Ronumeriiguchi, co Ujitake chosen Amorphophallus variabilis, Aishimeji, laccaria laccata, Tsubafuusentake, Sasatake, Yagitake, Hygrocybe conica, hydnum repandum, ramaria flava, Phallus Impudicus, from the group consisting of phallus indusiatus An α-glucosidase inhibitor comprising at least one edible mushroom or its extract as an active ingredient.