JPH0672884A - Alpha-amylase inhibitor and medicine using the same - Google Patents

Abstract

PURPOSE:To obtain the subject medicine useful for preventing and treating super blood sugar disease such as corpulence and diabetes, free from side effects, showing strongly inhibitory action on alpha-amylase, especially alpha-amylase of pancreatic juice, comprising a specific deoxymaltooligosaccharide. CONSTITUTION:The objective medicine comprises a 6-deoxymaltooligosaccharide of the formula ((n) is 1-5). 6<3>-deoxy-D-maltotriose, 6<4>-deoxy-D-maltotetrose and 6<5>-deoxy-D-maltopentaose have high activity and are preferable as the compound of the formula. The compound of the formula, for example, is obtained by treating mono-6-deoxycyclodextrin with cyclodextrinase, simultaneously with an exo type saccharifying enzyme and purifying by active carbon column chromatography. A dose is preferably 0.5-10g calculated as the active ingredient of the medicine per adult daily.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an α-amylase inhibitor containing 6-deoxymalto-oligosaccharide as an active ingredient and a method for preventing or treating hyperglycemia such as obesity and diabetes using the same. It relates to medicine.

[0002]

2. Description of the Related Art Carbohydrates ingested by mammals are partially digested (hydrolyzed) by saliva α-amylase in the oral cavity and stomach.
Then, it is fully digested by pancreatic juice α-amylase in the duodenum and jejunum to become oligosaccharides and disaccharides,
These are further hydrolyzed by glucoside hydrolases such as glucoamylase and maltase to finally become monosaccharides such as glucose, which are absorbed from the cilia on the mesentery.

[0003] After ingesting carbohydrates, the absorbed glucose causes a temporary increase in blood glucose level, so-called hyperglycemia, which usually occurs due to the glucose homeostasis system in the living body. It recovers by adjusting to a certain range.

However, if the dietary hyperglycemia is sustained for a long time or the blood glucose level becomes abnormally high, a disease called hyperglycemia is caused, which causes cases such as obesity and diabetes.

This obesity is caused by hyperglycemia caused by overeating.
It promotes high levels of insulin secretion, which results in increased lipogenesis and decreased lipolysis, which results from the accumulation of fat in the body.

[0006] In addition, obesity is easily associated with adult diseases such as diabetes, hypertension, arteriosclerosis, cardiovascular disease, and fatty liver, has a high mortality rate, and is said to have a short life.

Further, diabetes causes many serious complications such as neuropathy, infertility, visual impairment, blindness, arteriosclerosis, and autonomic neuropathy. Especially, adult-type diabetes is predominant in obese people. , It is known to relieve by reducing the intake of calories and losing weight.

[0008] As such prophylactic or therapeutic agents for hyperglycemia, an appetite suppressant, an intestinal absorption suppressant, a digestive enzyme inhibitor, etc. have been proposed ("Medical History", No. 141). Vol., Pp. 249-251).

On the other hand, an α-amylase inhibitor listed as one of digestive enzyme inhibitors is said to be useful as a preventive or therapeutic agent for hyperglycemia such as obesity and diabetes. (Acarbose)
[Chapman and Hall, "Dictionary of Organic Compounds (Dictionary)
of Organic Compounds) "No. 1
Vol., 5th edition, page 7, (1982)] and similar amino sugar derivatives (JP-A-52-122342).
It has been known.

The above-mentioned acarbose is 4 ³ -amino-6 ³ , 4 ³ -consisting of a cyclitol moiety and an amino sugar and two glucoses by a reductive C--N bond cleavage reaction.
It is a compound having a structure that produces dideoxymaltotriose (trisaccharide), but the trisaccharide produced at this time has no inhibitory effect on porcine pancreatic α-amylase [Bradbeck ed., "Enzyme Inhibitors". Proceedings of a Meeting Held in Basel (EnzymeInhib
itors Proceedings of a me
etingheld in Basel) ", 1980
Year, March 20, 21st, page 125].

Further, when the above-mentioned acarbose is taken as a medicine, it has a drawback that it has side effects such as belly noise and flatus ("Medical Ayumi" Vol. 156, No. 1).
No. 3, pp. 1039-1042).

[0012] On the other hand, 6 3 ^- With respect to the deoxy -D- maltotriose, which has been reported only may not be representative of substrate inhibition effect on Taka-amylase A [ "Journal of Biochemistry (J. Bi
ochem. ) ”, Vol. 84, pp. 835-841], and is not known to have an α-amylase inhibitory action.

[0013]

The present invention overcomes the drawbacks of such conventional preventive or therapeutic agents, has an α-amylase inhibitor having a strong inhibitory effect on α-amylase, and no side effects, The purpose of the present invention is to provide a preventive or therapeutic agent for hyperglycemia such as obesity and diabetes.

[0014]

Means for Solving the Problems As a result of intensive studies for achieving the above-mentioned object, the present inventors have found that the 6-hydroxyl group of the non-reducing terminal glucose has a 6-deoxymalto-oligosaccharide substituted with a hydrogen atom. But α-amylase, especially pancreatic juice α
-Based on this finding, it was found to be useful as a potent inhibitor of amylase, and that it can be effectively used as a prophylactic or therapeutic agent for hyperglycemia without side effects and can achieve its purpose. As a result, the present invention has been completed.

That is, the present invention has the general formula

[Chemical 3] An α-amylase inhibitor containing 6-deoxymalto-oligosaccharide represented by the formula (n is an integer of 1 to 5) as an active ingredient, and a prophylactic or therapeutic agent for hyperglycemia using the same Is.

The 6-deoxymalto-oligosaccharide of the general formula (I) is a known substance described in, for example, JP-A-3-86701.

The 6-deoxymaltooligosaccharide of the general formula (I) may be either an α-anomer or a β-anomer.

Examples of such compounds include 6 ³ -deoxy-D-maltotriose, 6 ⁴ -deoxy-D-maltotetraose, 6 ⁵ -deoxy-D-maltopentaose and 6 ⁶ -deoxy-D- Marto Hexaose, 6 ⁷
-Deoxy-D-maltoheptaose. And these compounds are used 1 type or in combination of 2 or more types.

In the above, the symbols 6 ^3- , 6
⁴ - 6 5 ^-, etc., from the reducing end glucose units constituting the maltooligosaccharides, third, fourth, fifth glucose residues (i.e., glucose residue of the non-reducing end) the 6-hydroxyl group of Indicates that it has been replaced.

Next, the 6-deoxymalto-oligosaccharide of the general formula (I) used in the present invention can be produced, for example, by the method described in JP-A-3-86701, but other methods. It may be manufactured by

According to the above method, mono-6-deoxycyclodextrin is cleaved with a known cyclodextrinase [ie (a) cyclodextrin to form maltooligosaccharide derived from the glucose polymerization degree of cyclodextrin. Cyclodextrinase having an action and a higher substrate specificity than (b) a hydrolysis rate or affinity for cyclodextrin or a linear oligosaccharide having the same degree of polymerization as cyclodextrin] (JP-A-3-15384) And the above-mentioned JP-A-3-86701
(See Japanese Patent Laid-Open Publication No. 1993), at the same time as or after the action,
It can be produced by reacting with α-glucosidase and the like, and if necessary, purification using, for example, activated carbon column chromatography, octadecylated silica gel (ODS) column chromatography and the like.

The compound of the general formula (I) used in the present invention has a strong inhibitory effect on α-amylase, especially pancreatic juice α-amylase, and therefore can be used as an α-amylase inhibitor. Above all, 6 ³ -deoxy-D
-Maltotriose, 6 ⁴ -deoxy-D-maltotetraose and 6 ⁵ -deoxy-D-maltopentaose have a particularly strong inhibitory effect.

The dose of the α-amylase inhibitor of the present invention varies depending on the administration method, the degree of symptoms, the age and weight of the patient, etc.
It is selected in the range of 01 to 20 g, preferably 0.5 to 10 g.

It can be administered either orally or parenterally, but it is preferably administered orally.

Although the α-amylase inhibitor of the present invention can be administered alone, it is usually used as an excipient, a lubricant, a diluent, a stabilizer, a pH adjuster, a preservative, a sweetener, and an aroma. Agent,
Tablets, powders, granules, capsules, syrups, etc. using flavoring agents, coloring agents, preservatives, emulsifiers, thickeners, bases, etc.
It is used in the form of suppositories, injections, drops, and other preparations.
These preparations are carried out by a conventional method.

Excipients include, for example, potato starch, lactose, crystalline cellulose, mannitol, and lubricants such as magnesium stearate, talc,
Examples of sweeteners, aromatics and corrigents such as hydrogenated oil include salt, saccharin, orange oil, citric acid, menthol and malic acid.

[0027]

INDUSTRIAL APPLICABILITY The 6-deoxymalto-oligosaccharide of the general formula (I) of the present invention has a strong inhibitory action on α-amylase, particularly pancreatic juice α-amylase, and therefore, hyperglycemia such as obesity , Has a medicinal effect as a preventive or therapeutic agent for diabetes and has no side effects.

Therefore, it is also effective as a preventive or therapeutic agent for hyperlipidemia, fatty liver, arteriosclerosis, etc. due to this hyperglycemia, and the inhibitor is expected to have an immunostimulatory action, and is expected in the future. It is an important physiologically active substance.

[0029]

EXAMPLES The present invention will be described in more detail with reference to examples.

Reference Example 1 Production of 6 ³ -deoxy-D-maltotriose (1) Production of 6-deoxy-β-cyclodextrin Commercially available β-cyclodextrin [Wako Pure Chemical Industries, Ltd.]
Manufactured] 5.00 g (4.41 mmol) of pyridine 50 m
It dissolves in 1 and 10.0 g of tosyl chloride (52.4 mm
ol) was added, and the mixture was reacted at room temperature for 5 hours while stirring. Next, the pyridine in this reaction solution was distilled off under reduced pressure, and then 100 ml of water and 150 m of benzene were added to the residue.
1 was added with stirring to precipitate a solid. Next, the solid matter was filtered off with a glass filter and washed twice with 50 ml of acetone, and then the filtered matter was purified by ODS column chromatography to obtain an ethanol-water mixture (volume ratio 1:
The target fraction eluted in 9) was concentrated and recrystallized from water to give 6-O-tosyl-β-cyclodextrin with 1.6
3 g (1.26 mmol, yield 28.6%) was obtained.

The physical properties of this product are as follows. Melting point (° C): 172.0 to 174.0 (decomposition) Infrared absorption spectrum (cm ^-1 ): 3400, 293
0,1642,1632,1600,1424,136
0,1300,1178,1156,1078,102
8

Nuclear magnetic resonance spectrum (200 MHz) p
pm (DMSO-d ₆ ): 2.44, (3H, s),
3.15 to 4.45 (m), 4.76 (2H, br.
s), 4.85 (5H, br.s), 7.44 (1H,
d, J = 8.8 Hz), 7.75 (1H, d, J = 8.
8Hz)

Next, the 6-O thus obtained is obtained.
-Tosyl-β-cyclodextrin 1.27 g (0.9
85 mmol) to dimethyl sulfoxide (DMSO) 2
After dissolving in 0 ml, 384 mg (10.2 mmol) of sodium borohydride (NaBH ₄ ) was added, and the mixture was reacted at 50 ° C. for 12 hours. Then, 1000 ml of water was added to this reaction solution, DMSO was removed by ODS column chromatography, and the target fraction eluted with an ethanol-water mixture (volume ratio 1: 9) was concentrated and recrystallized with methanol. As a result, 6-deoxy-β-cyclodextrin 839.6 mg (0.750 mmo
1, yield 76.1%) was obtained.

The physical properties of this product are as follows. Melting point (° C): 280.0 to 281.0 (decomposition) Infrared absorption spectrum (cm ^-1 ): 3370, 292
0,1152,1080,1020

Nuclear magnetic resonance spectrum (200 MHz) p
pm (DMSO-d ₆ ): 1.20 (3H, d, J =
6.1 Hz), 2.80 to 4.05 (m), 4.84
(7H, br.s)

(2) Production of 6 ³ -deoxy-D-maltotriose 15 g of 6-deoxy-β-cyclodextrin obtained in (1) above was added to 100 mM phosphate buffer (pH = 7.0).
It was poured into 1.0 l while stirring and completely dissolved.
To this, add 24 units of cyclodextrinase
The reaction was performed while stirring at 48 ° C for 48 hours. After the reaction was completed, hydrochloric acid was added to the reaction solution to stop the reaction by adjusting the pH to about 2.0, and then a sodium hydroxide solution was added to neutralize the solution. Then, this was passed through an ODS column, Unreacted 6-deoxy-β-cyclodextrin was adsorbed to obtain a flow-through fraction. Repeat this operation for a total of 63
g of 6-deoxy-β-cyclodextrin was treated. This flow-through fraction was mixed with 1/10 volume of 100 mM acetate buffer (pH 4.5) and then 100 mM.
The pH was adjusted to 4.5 with acetic acid. Next, 2500 units of glucoamylase was added to this and the enzyme reaction was carried out at 40 ° C. for 8 hours, and then the reaction was stopped by adding hydrochloric acid to bring the pH to about 2.0. A sodium hydroxide solution was added to neutralize. Next, after passing this solution through an activated carbon column, 6-deoxymalto-oligosaccharide was eluted with an ethanol gradient of 0 to 35%, and about 21% of this ethanol-eluted fraction was lyophilized to a purity of about 98%. % ^{6 3} - was obtained deoxy -D- maltotriose about 1.1 g.

The physical properties of this product are as follows. Infrared absorption spectrum (cm ^-1 ): 3400,295
0,1690,1146,1042

High performance liquid chromatography [Tosoh Corporation]
TSKgel Amide-80 column (4.6 mm)
ID × 250 mm), RI detection, eluent: acetonitrile / water = 3/2 (v / v), flow rate: 1.0 ml / mi
n]: t _R = 5.9 min

Nuclear magnetic resonance spectrum (200 MHz) p
pm (D ₂ O): 1.28 (3H, d, J = 6.1H
z), 3.17 (1H, t, J = 8.9Hz), 3.2.
9 (1H, t, J = 8.9Hz), 3.50 to 4.05
(M), 4.65 (ca.0.5H, d, J = 7.8H
z), 5.35 (ca.0.5H, d, J = 3.7H)
z), 5.27 (1H, d, J = 3.0 Hz), 5.3
6 (1H, d, J = 3.9Hz)

[0040] Reference Example 2 6 4 ^- In deoxy -D- maltotetraose the preparation of Reference Example 1 (2), except that was collected about 23% ethanol eluted fraction with ethanol gradient, likewise manner as in Reference Example 1. Was performed to obtain about 2.4 g of 6 ⁴ -deoxy-D-maltotetraose having a purity of 99.5%.

The physical properties of this product are as follows. Infrared absorption spectrum (cm ⁻¹ ): 3420, 293
0, 1636, 1410, 1366, 1148, 107
8,1038

High performance liquid chromatography [Tosoh Corporation]
TSKgel Amide-80 column (4.6 mm)
ID × 250 mm), RI detection, eluent: acetonitrile / water = 3/2 (v / v), flow rate: 1.0 ml / mi
n]: t _R = 7.2 min

Nuclear magnetic resonance spectrum (200 MHz) p
pm (D ₂ O): 1.27 (3H, d, J = 6.3H
z), 3.14 (1H, t, J = 9.0 Hz), 3.2
6 (1H, t, J = 9.0 Hz), 3.50 to 4.10
(M), 4.63 (0.5H, d, J = 8.1Hz),
5.22 (0.5H, d, J = 3.4Hz), 5.25
(1H, d, J = 2.9 Hz), 5.33 (2H, d,
J = 1.5Hz)

Reference Example 3 Production of 6 ⁵ -deoxy-D-maltopentaose The same procedure as in Reference Example 1 except that in (2) of Reference Example 1 an about 25% ethanol elution fraction was collected by an ethanol gradient. It was carried out, 98% pure ^{6 5} - to give a deoxy -D- maltopentaose about 2.5 g.

The physical properties of this product are as follows. Infrared absorption spectrum (cm ⁻¹ ): 3420, 292
5,1628,1412,1368,1150,108
0,1040

High performance liquid chromatography [Tosoh Corporation]
TSKgel Amide-80 column (4.6 mm)
ID × 250 mm), RI detection, eluent: acetonitrile / water = 3/2 (v / v), flow rate: 1.0 ml / mi
n]: t _R = 8.7 min

Nuclear magnetic resonance spectrum (200 MHz) p
pm (D ₂ O): 1.27 (3H, d, J = 6.3H
z), 3.16 (1H, t, J = 9.0 Hz), 3.2
9 (1H, t, J = 9.0 Hz), 3.50 to 4.10
(M), 4.65 (0.5H, d, J = 8.1Hz, α
-H), 5.23 (0.5H, d, J = 3.4Hz, β
-H), 5.27 (1H, d, J = 3.2 Hz), 5.
35 (3H, d, J = 3.9Hz)

Reference Example 4 Production of 6 ⁶ -deoxy-D-maltohexaose The same operation as in Reference Example 1 except that in (2) of Reference Example 1 an approximately 27% ethanol elution fraction was collected by an ethanol gradient. Was performed to obtain about 2.2 g of 6 ⁶ -deoxy-D-maltohexaose having a purity of 97%.

The physical properties of this product are as follows. Infrared absorption spectrum (cm ⁻¹ ): 3400, 293
0, 1640, 1412, 1360, 1150, 107
8,1036

High performance liquid chromatography [Tosoh Corporation]
TSKgel Amide-80 column (4.6 mm)
ID × 250 mm), RI detection, eluent: acetonitrile / water = 3/2 (v / v), flow rate: 1.0 ml / mi
n]: t _R = 10.1 min

Nuclear magnetic resonance spectrum (200 MHz) p
pm (D ₂ O): 1.27 (3H, d, J = 6.1H
z), 3.16 (1H, t, J = 8.7 Hz), 3.2
8 (1H, t, J = 8.7 Hz), 3.45 to 4.15
(M), 4.65 (0.5H, d, J = 8.3Hz, α
-H), 5.22 (0.5H, d, J = 3.9Hz, β
-H), 5.27 (1H, d, J = 2.9 Hz), 5.
35 (4H, d, J = 2.9Hz)

Reference Example 5 Production of 6 ⁷ -deoxy-D-maltoheptaose The same operation as in Reference Example 1 except that in (2) of Reference Example 1 an about 29% ethanol elution fraction was collected by an ethanol gradient. It was carried out, ^{6 7} purity 99.5% - was obtained deoxy -D- maltoheptaose about 2.7 g.

The physical properties of this product are as follows. Infrared absorption spectrum (cm ⁻¹ ): 3420, 293
0, 1628, 1412, 1364, 1150, 107
8,1022

High performance liquid chromatography [Tosoh Corporation]
TSKgel Amide-80 column (4.6 mm)
ID × 250 mm), RI detection, eluent: acetonitrile / water = 3/2 (v / v), flow rate: 1.0 ml / mi
n]: t _R = 12.1 min

Nuclear magnetic resonance spectrum (200 MHz) p
pm (D ₂ O): 1.25 (3H, d, J = 6.4H
z), 3.16 (1H, t, J = 8.7 Hz), 3.2
7 (1H, t, J = 8.7 Hz), 3.45 to 4.10
(M), 4.64 (0.5H, d, J = 8.0Hz, α
-H), 5.20 (0.5H, d, J = 3.5Hz, β
-H), 5.26 (1H, d, J = 3.1 Hz), 5.
35 (5H, d, J = 3.9Hz)

[0056] Reference Example 6 6 2 ^- In deoxy -D- maltose preparation of Reference Example 1 (2), except that was collected about 19% ethanol eluted fraction with ethanol gradient, perform the same procedure as Reference Example 1 , ^{6 2} purity 98.5% - was obtained deoxy -D- maltose about 1.5 g.

The physical properties of this product are as follows. Infrared absorption spectrum (cm ^-1 ): 3422,293
0,1630,1150,1050

High performance liquid chromatography [Tosoh Corporation]
TSKgel Amide-80 column (4.6 mm)
ID × 250 mm), RI detection, eluent: acetonitrile / water = 3/2 (v / v), flow rate: 1.0 ml / mi
n]: t _R = 4.7 min

Nuclear magnetic resonance spectrum (200 MHz) p
pm (D ₂ O): 1.28 (3H, d, J = 6.4H
z), 3.16 (1H, t, J = 8.5 Hz), 3.2
8 (1H, t, J = 8.4 Hz), 3.50 to 4.00
(M), 4.65 (ca.0.5H, d, J = 7.8H
z), 5.22 (ca.0.5H, d, J = 3.4H)
z), 5.27 (1H, d, J = 3.1Hz)

Reference Example 7 Preparation of cyclodextrinase 1% (w / v) β-cyclodextrin, 1% (w / v)
v) peptone, 0.5% (w / v) NaCl and 0.1
100 ml of a liquid medium (using tap water, pH 7.0) consisting of% (w / v) yeast extract was placed in a 500 ml Sakaguchi flask and sterilized at 120 ° C. for 20 minutes.
In addition, Bacillus sphaericus E-244 (FERM
One platinum loop was inoculated from the stored slant of BP-2458), and cultured at 30 ° C. for 1 day with shaking. 50m of this culture
1 ml was inoculated into a 3000 ml mini jar containing 2000 ml of medium prepared by the same medium composition and sterilization conditions as above, and aerated and agitated at 30 ° C., 1 vvm and 350 rpm for 2 days. The cells were separated from the culture solution by centrifugation at 8000 rpm for 20 minutes, and 500 ml of 10 mM phosphate buffer (pH 7.0) containing 2% (w / v) Triton X-100.
The microbial cells were suspended and stirred at 25 ° C for 1 day. After the bacterial cells were removed from the suspension by centrifugation at 12000 rpm for 20 minutes, the supernatant was dialyzed against 10 mM phosphate buffer (pH 7.0) for 16 hours. The dialyzed product obtained was centrifuged at 12000 rpm for 20 minutes to remove insoluble materials, and the supernatant was used as a crude enzyme solution (1).

Then, about 500 ml of this crude enzyme solution (1)
(Total activity 200 units, protein amount 2083 mg, specific activity 0.1, pH 7.0) in 10 mM phosphate buffer (pH
It was applied to a DEAE sepharose packed column (φ34 × 170 mm) equilibrated with 7.0) to adsorb the enzyme, and then elution was performed with a gradient gradient of 0 to 1.5 M NaCl. The active fractions thus obtained were collected to obtain 105 ml of crude enzyme solution (2) (total activity 145 units, specific activity 0.58, yield 72.5%).

Subsequently, 20 ml of this crude enzyme solution (2) (total activity 31 units, protein amount 29 mg) was equilibrated with 100 mM phosphate buffer solution (pH 7.0) containing 1 M sodium sulfate, and packed with an ether 5PW column (φ21. 5 x 150
mm) to adsorb the enzyme, and then elution was performed with a gradient of 1M to 0M sodium sulfate. The active fractions thus obtained were collected and 50 ml of the crude enzyme solution (3) (total activity 72 units, specific activity 2.93,
Yield 36%) was obtained.

[0063] Test Example 1 In Vitro α- amylase inhibitory activity (1) of the substrate solution prepared commercially available 2-chloro-4-nitrophenyl = beta-D-maltopentaoside _(M W 983) of the enzyme reaction solution 40 mM NaCl and 2 mM so that the concentration becomes 2.0 mM
-50 mM phosphate buffer containing MgCl ₂ (pH
It was dissolved in 7.0). This concentration is used for the human pancreatic juice α-amylase (hereinafter, human P
Type α-amylase) and human salivary α-amylase (hereinafter referred to as human S-type α-amylase), each of which is equivalent to 5 times the Km value. is there.

(2) Preparation of conjugate enzyme solution Commercially available α-glucosidase derived from yeast and β-glucosidase derived from almond were mixed at a concentration of 11 each.
40 mM to a concentration of 7 U / ml, 13 U / ml
-NaCl and 50mM containing 2mM-MgCl ₂
It was mixed with a phosphate buffer (pH 7.0) and dissolved. In addition, Toyobo Co., Ltd. product was used for these commercially available α- and β-glucosidases.

(3) Preparation of Human α-Amylase Solution Commercially available human P-type α-amylase and human S-type α-amylase (in the table, indicated as HPA and HSA, respectively) 50
Add 150 mM / l phosphate buffer (pH 7.0)
A human α-amylase solution was prepared by dissolving the solution at a concentration of.

The commercially available human α-amylase used was Calibzyme AMY manufactured by International Reagents Co., Ltd. Moreover, the activity of α-amylase is 1 μm per minute at 37 ° C
The amount of enzyme that decomposes 2-chloro-4-nitrophenyl = β-D-maltopentaoside (commercially available product) of ol was defined as 1 unit (U).

(4) Preparation of mouse pancreatic juice Commercially available ICR-CRJ: C weighing 13.2-15.8 g
D-1 mice [male, 3 weeks old, sold by Japan Charles River Co., Ltd.] were taken out of pancreas for 7 mice, homogenized by adding 50 mM phosphate buffer (pH 7.0), and then 100,
Centrifugation was performed at 000 × g for 1 hour to obtain a supernatant.
The activity of the supernatant was determined to be 2-chloro-4-nitrophenyl = β
-D-maltopentaoside (commercially available) was measured as a substrate, and the activity was adjusted to 50 U / l at a concentration of 150 U / l.
M phosphate buffer (pH 7.0) was added and diluted to obtain mouse pancreatic juice.

The enzyme activity of this mouse pancreatic juice is α-
Amylase (indicated as MPA in the table) activity.

(5) Preparation of Inhibitor Solution Various 6-deoxymalto-oligosaccharides obtained by the above-mentioned Reference Example [in the table, compounds of the general formula (I) with n = 1 to 5 are shown as DOG3 to DOG7] and 6 ² - (in Table indicated as DOG2) deoxy -D- maltose inhibiting solution was prepared using 50mM phosphate buffer (pH7.0) so that each a concentration of 7,14MM. Note that the concentrations (final concentrations) of the various inhibitors at the time of measuring the inhibitory action are shown in Table 1.

(6) Measurement of human α-amylase inhibitory action and mouse pancreatic juice inhibitory action in inhibitor solution To 250 μl of human P-type α-amylase, human S-type α-amylase or mouse pancreatic juice, 1.0 ml of the conjugate enzyme solution was added. After stirring and heating at 37 ° C. for 1 minute, the substrate solution 2.
Add 0 ml and 250 μl of inhibitor and stir, 37 ℃
After heating for 2 minutes, the amount of change in absorbance at 400 nm for 2 minutes was measured.

As a blank, instead of the inhibitor, 50 m
M phosphate buffer (pH 7.0) was used.

The α-amylase inhibition rate (%) is
[1- (Ai / Ao)] × 100, where Ao is the amount of change in absorbance of the blank and Ai is the amount of change in absorbance when the inhibitor was added
Was calculated. The results are shown in Tables 1 and 2.

[0073]

[Table 1]

[0074]

[Table 2]

From Tables 1 and 2, the 6-deoxymaltooligosaccharides of the present invention were identified as human α-amylase, especially human P-type α.
-Amylase and mouse pancreatic juice enzyme activity are strongly inhibited, and for human P-type α-amylase, 6 ³ -deoxy-D-maltotriose (DO
G3) ~6 ⁵ - deoxy -D- maltopentaose (D
OG5) Whereas showing a particular strong inhibitory effect, ^{6 2} - deoxy -D- maltose (DOG2) it can be seen that show no inhibitory effect.

Test Example 2 Inhibitory Action on Weight Gain of α-Amylase Inhibitor (1) Animal Used Commercially available ICR-CRJ: C weighing 11.6 to 16.6 g
D-1 mouse male and female [3 weeks old, sold by Japan Charles River Co., Ltd.]

(2) Experimental method The administration group consisted of 10 male and 3 week-old mice, each containing 10 male and female mice, and a standard solid feed (MF) (carbohydrate content 42-45%) [Oriental Yeast Industry Co., Ltd.] It is bred for a day, and the α-amylase inhibitor is dissolved in distilled water, and 2 mg (α
-Amylase inhibitor amount) / mouse was administered. The administration method was free oral intake. The control group was bred in the same manner as above except that the α-amylase inhibitor was not administered. Body weight administered group and the control group, feed intake, measured fecal weight and dry feces amount, as α- amylase inhibitor 6 ³ - deoxy -D-
The results of administration of maltotriose (DOG3) are shown in Table 3, and 6 ⁵ -deoxy-D-maltopentaose (D
The results when OG5) was administered are shown in Table 4. The effect of suppressing weight gain was determined by t-test of "weight gain (g)" on 5 days after administration.

[0078]

[Table 3]

[0079]

[Table 4]

From Table 3 and Table 4, 6 ³ -deoxy-D-maltotriose and 6 used in the present invention (administration group)
⁵ -Deoxy-D-maltopentaose markedly increased body weight compared to the control group, although no decrease in feed intake and no diarrhea (fecal volume, dry fecal volume and observation) It can be seen that it is suppressed, that is, it is effective as a prophylactic or therapeutic agent for obesity.

Since the amount of dry feces in the administration group was higher than that in the control group, it can be seen that the digestion is suppressed by the α-amylase inhibitory action.

Test Example 3 Antidiabetic Effect of α-Amylase Inhibitor (1) Animal Used Hereditary obese diabetic mouse having a body weight of 32.0 to 35.2 g [Yellow KK mouse, male, 10 weeks old, CLEA Japan (strain) ) Sales]

(2) Experimental method In the administration group, 5 mice that had been fasted for 20 hours in advance were liquefied with α-amylase inhibitor and corn starch as a sugar in distilled water to obtain 0.1 g (α-amylase The amount of inhibitor) / body weight 1 kg and 1 g (corn starch amount) / body weight 1 kg were orally administered and forcibly ingested. The control group was the same as above, except that the α-amylase inhibitor was not administered.

Then, blood was collected from the orbital venous plexus of the mice before and 30 minutes after ingestion, the blood glucose level was measured, and the inhibitory effect on the increase in blood glucose level after ingestion of corn starch, that is, the antidiabetic effect was examined. As α- amylase inhibitor ^{6 3} - deoxy -D- maltotriose (DO
Results upon administration of G3) shown in Table 5, ^{6 5} - shows the result upon administration of deoxy -D- maltopentaose (DOG5) Table 6.

The inhibition rate (%) in each table is [1- (B
/ A)] × 100. The blood glucose level (mg / dl) was quantified by the glucose oxidase method and shown as the average value ± standard deviation.

[0086]

[Table 5]

[0087]

[Table 6]

From Tables 5 and 6, 6 ³ -deoxy-D-maltotriose and 6 used in the present invention (administration group)
^{It is found that 5} -deoxy-D-maltopentaose is significantly suppressed as compared with the control group, that is, it is effective as a preventive or therapeutic agent for diabetes.

Next, the test results for the acute toxicity of the compound of the general formula (I) used in the present invention are shown as Test Example 4.

Test Example 4 Acute toxicity test (1) Animal used Commercially available ICR-CRJ: CD-1 mouse having a body weight of 29 to 32 g [5 weeks old, Charles River Japan Co., Ltd.]
Sale]

(2) Experimental method “Revised Guidelines on Drug Toxicity Tests GLP Standards”
A single dose toxicity test was conducted by the Ministry of Health and Welfare (September 1989).

That is, 6 ³ -deoxy-D-maltotriose and 6 ⁵ -deoxy-D-maltopentaose were dissolved in 100 mg per 1 ml of physiological saline to dissolve 5 5-week-old ICR-CRJ: CD- 1 mouse (female 29
~ 32g), 0.5ml per 25g body weight (2.0g /
(kg) was orally administered and observed for 7 days.

No animals died and no abnormalities such as diarrhea and constipation were observed even with this extremely large dose. Again 7
No microscopic abnormalities of tissues or organs were observed even in the autopsy on the day, and the toxicity was judged to be extremely low.

[0094] The ^{6 3} - deoxy -D- maltotriose and ^{6 5} - deoxy -D- maltopentaose were dissolved by 100mg per saline 1 ml, this weight 25g per 0.25 ml (1.0 g / kg ) Was intraperitoneally injected and the progress was observed for 7 days, but no abnormality was observed.

From the above test results, the 6-deoxymalto-oligosaccharide of the general formula (I) has a strong α-amylase inhibitory action, is a potent α-amylase inhibitor, and is useful for hyperglycemia such as obesity and diabetes. It is recognized that it is useful as a preventive or therapeutic agent.

Example 1 Freeze-dried powder formulation 6 ³ -deoxy-D-maltotriose 250 g 10
After being dissolved in 00 ml of physiological saline, aseptically filtered using a membrane filter, 10 ml each of the obtained filtrate was filled in a sterilized glass container, which was sealed and freeze-dried by a conventional method, followed by freeze-drying. It was a powder formulation.

Example 2 Oral tablet (1) 6 ³ -Deoxy-D-maltotriose 250 g (2) Mannitol 200 g (3) Potato starch 47 g (4) Magnesium stearate 3 g (1) and (2) After mixing, (3) was added as 10% starch paste and granulated. Pass through a 60 mesh (BS) sieve, and further After screening with a 16-mesh (BS) sieve and mixing the particles with (4), a tablet having a diameter of 10 mm and a weight of 500 mg per tablet was made with a tableting machine.

Example 3 Oral tablet (1) 6 ⁵ -deoxy-D-maltopentaose 250 g (2) mannitol 200 g (3) potato starch 47 g (4) magnesium stearate 3 g (1) and (2) After mixing, (3) was added as 10% starch paste and granulated. Pass through a 60 mesh (BS) sieve, and further After screening with a 16-mesh (BS) sieve and mixing the particles with (4), a tablet having a diameter of 10 mm and a weight of 500 mg per tablet was made with a tableting machine.

Example 4 Capsule 6 ³ -deoxy-D-maltotriose 100 g or 6
100 g of ⁵ -deoxy-D-maltopentaose, 50 g of potato starch, 50 g of lactose and 10 g of crystalline cellulose were well mixed and filled into a capsule to give a capsule containing 200 mg of the active ingredient in each capsule.

[0100] Example 5 internal medicine 6 ³ - deoxy -D- maltotriose 20g or ^{6 5}
To 20 g of -deoxy-D-maltopentaose, 1 ml of 5% benzoic acid (45 v / v ethanol) and purified water were added to bring the total volume to 100 ml to give an internal liquid preparation.

Example 6 Injection Preparation 5 g of sterilized 6 ³ -deoxy-D-maltotriose or ⁵ g of 6 ⁵ -deoxy-D-maltopentaose was dissolved in distilled water for injection to a total volume of 300 ml to give 1 ampoule. It was aseptically sealed at a rate of 3.0 ml to prepare an injection.

Claims (3)

[Claims] 1. A general formula: An α-amylase inhibitor containing 6-deoxymaltooligosaccharide represented by the formula (n is an integer of 1 to 5) as an active ingredient. 2. A general formula: A prophylactic or therapeutic agent for hyperglycemia, which comprises 6-deoxymaltooligosaccharide represented by the formula (n is an integer of 1 to 5) as an active ingredient. 3. The preventive or therapeutic agent according to claim 2, wherein the hyperglycemia is obesity or diabetes.