JPH022592B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides novel aminocyclitols, i.e. The present invention relates to a compound represented by (hereinafter referred to as "epivariolamine").

The present inventors have discovered that Streptomyces hygroscopicus var.limoneus,
IFO No. 2703, Ferm No. 468, ATCC No. 21143 (1) Among the various compounds obtained by culturing under aerobic conditions, (1 (CH ₂ OH), 2, 4, 5/1
(OH), 3)-5-amino-1-hydroxymethyl-1,2,3,4-cyclohexanetetrol, that is, epivaliolamine, exists and has an α-glucosidase inhibitory effect. As a result of this discovery and further intensive research, the present invention was completed.

That is, the present invention provides (1) epivariolamine or a salt thereof; (2) a microorganism belonging to the genus Streptomyces and having the ability to produce epivariolamine is cultured in a medium, and the above-mentioned epivariolamine is added to the culture. producing variolamine and forming salts if necessary;
The present invention relates to a method for producing epivariolamine or a salt thereof, which comprises collecting epivariolamine or a salt thereof, and (3) an α-glucosidase inhibitor containing epivariolamine or a salt thereof.

The physicochemical substances of aminocyclitol or epivariolamine of the present invention are as follows.

(1) Appearance Hygroscopic white powder (2) Elemental analysis value (molecular formula) C ₇ H ₁₅ NO ₅・H ₂ O Calculated value (%): C39.80; H8.11: N6.63 Experimental value (%) : C39.55; H8.32; N6.50 (3) Specific rotation [α] ²⁵ _D +18.2° (c=1, H ₂ O) (4) Ultraviolet absorption spectrum Aqueous solution has a wavelength between 200 and 360 nm. It does not show a characteristic absorption maximum in the region, but only shows terminal absorption.

(5) Infrared absorption spectrum The spectrum measured by the KBr method is shown in Figure 1. The wave numbers of the main absorption maxima are shown below.

3340, 2920, 1570, 1500-1300, ^{1045cm -1} (6) ¹ H nuclear magnetic resonance spectrum Second spectrum measured at 100MHz in heavy water
As shown in the figure. In addition, chemical shift δ value (ppm, reference material: DSS (sodium 2,2-dimethyl-
2-silapentane-5-sulfonate;
(CH ₃ ) ₃ Si (PCH ₂ ) ₃ SO ₃ Na) and coupling constant J
(Hz) is shown below.

δ: 1.72 (1H, double doublet, J=4.6,
14.3), 1.96 (1H, double doublet, J=
6.6, 14.3) 3.42 (1H, multiplet), 3.58
(1H, double doublet, J=6.6), 3.60
(2H, double doublet, J=3.1), 3.76 (1H, double doublet, J=4.0, 6.6), 4.00 (1H,
triplet, J = 6.6).

(7) ^13H nuclear magnetic resonance spectrum Chemical shift δ value (ppm, reference material: under decouple conditions measured at 100MHz in heavy water)
DSS (sodium 2.2-dimethyl 2-silapentane-5-sulfonate; (CH ₃ ) ₃ Si
(CH ₂ ) ₃ SO ₃ Na) and the splitting pattern measured under off-resonance conditions are shown below.

δ: 37.16 (triplet) 48.79 (doublet),
67.93 (triplet) 74.27 (doublet),
75.44 (double), 77.03 (double),
77.85 (singlet).

(8) Solubility in solvents Soluble in water, dimethyl sulfoxide, and methanol. ethanol, ethyl acetate, chloroform,
Slightly soluble or insoluble in acetone.

(9) Color reaction Ninhydrin reaction: Positive Greig-Leaback
Reaction with reagent: Positive Naftresorcin/sulfuric acid reaction: Negative (10) Thin layer chromatography TLC plate, silica gel, 60F ₂₅₄ (manufactured by Merck & Co., West Germany) with n-propyl alcohol, acetic acid, water (4:1: The Rf values measured using Validamine and Variolamine as comparative samples using 1) as a developing solvent are shown below.

Epivariolamine 0.37 Valiolamine* 0.25 Valiolamine 0.31 (*valiolamine=(1(OH), 2, 4, 5/1
(CH ₂ OH), 3)-5-amino-1-hydroxymethyl-1,2,3,4-cyclohexanetetral) Epivariolamine has almost no toxicity (rat LD ₅₀ 500 mg/Kg or more) and In addition to exhibiting α-glucosidase inhibitory activity, it is also a useful compound as a raw material for producing epivariolamine derivatives, such as N-substituted derivatives.

That is, epivariolamine or a salt thereof of the present invention has an α-glucosidase inhibitory effect and suppresses carbohydrate metabolism in humans and non-human animals. Various diseases caused by blood sugar symptoms and hyperglycemia, such as obesity, obesity, hyperlipidemia (arteriosclerosis), diabetes, prediabetes, and diseases caused by sugar metabolism by oral microorganisms, such as tooth decay, etc. It is a useful compound for prevention. Furthermore, foods produced by adding epivariolamine or its salts are suitable for healthy people as food for patients with metabolic disorders and as preventative food for metabolic disorders. It is also useful as a feed additive for livestock to obtain low-fat, high-quality edible meat. Therefore, epivariolamine or a salt thereof can be used alone or together with a suitable carrier as an α-glucosidase inhibitor useful as a pharmaceutical, food additive, or animal feed additive. Such α-glucosidase inhibitors are administered orally or parenterally, preferably orally.

Epivariolamine can be obtained as a free base or a hydrate, or as any acid addition salt with a pharmaceutically acceptable acid by conventional methods. Examples of such acids include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and nitric acid, acetic acid, propionic acid, malic acid, citric acid,
Organic acids such as fumaric acid, maleic acid, ascorbic acid, mandelic acid, and methanesulfonic acid are used.

2,4-epivariolamine or a salt thereof is used alone or in combination with a non-toxic carrier. For example, it may be used with liquid or solid foods such as coffee, soft drinks, fruit juices, beer, milk, jam, and raw bean paste, seasonings, or various staple foods and side foods, and can be used directly or in the form of food coloring additives. or can be taken before or after meals. Furthermore, it can also be used as a feed additive for livestock to obtain low-fat, high-quality edible meat.

The α-glucosidase inhibitor of the present invention can be diluted with a non-toxic carrier such as a liquid carrier such as water, ethanol, ethylene glycol, or polyethylene glycol, or a solid carrier such as starch, cellulose, or polyamide powder, and then prepared into ampoules or granules. tablets, pills,
Capsules, syrups, etc. can be prepared according to conventional methods and used for the various uses mentioned above. In addition, sweeteners, preservatives, dispersants, and coloring agents can also be used.

Specifically, for example, by taking a preparation containing about 20 to 500 mg of epivariolamine or its salt after every meal, it is possible to suppress the increase in blood glucose concentration caused by eating. Also, for example, 0.01 to 1% of the carbohydrate content in food
A certain amount of epivariolamine or its salts may be added to various foods.

When mixed with feed, check the carbohydrate content of the feed.
0.001 to 1% is desirable.

Epivariolamine or a salt thereof of the present invention is a novel compound that has not been described in any literature, and is produced by the following method. That is, Streptomyces hygroscopicus limoneus, which belongs to the genus Streptomyces.
hygroscopicus var.

Streptomyces hygroscopicus var. limoneus is IFO to the Fermentation Research Institute.
12703, Ferm at the Institute of Microbial Technology, Agency of Industrial Science and Technology
No.468, The American Type Culture
Collection (The American Type Culture
Collection) as ATCC21431.

Note that, as a general property of microorganisms, the above-mentioned bacterial strains can also mutate naturally or by using mutagens. For example, mutant strains obtained by irradiation with radiation such as X-rays, gamma rays, and ultraviolet rays, monospore isolation, treatment with various drugs, cultivation on a medium containing drugs, or other means; Even naturally occurring mutant strains that have the property of producing epivariolamine can be used in the method of the present invention.

The medium used for culturing the above-mentioned microorganisms in the method of the present invention may be either liquid or solid as long as it contains a nutrient source that can be used by the above-mentioned strains, but it is more appropriate to use a liquid medium when processing a large amount. It is. The medium contains a carbon source that can be assimilated by the above-mentioned microorganisms, a nitrogen source that can be digested, inorganic substances, micronutrients, and the like as appropriate. Examples of carbon sources include glucose lactose, sucrose, maltose, dextrin, starch, glycerin, mannitol, sorbitol,
fats and oils (e.g. soybean oil, lard oil, chicken oil, etc.); nitrogen sources such as meat extract, yeast extract, dried yeast, soybean flour, corn steep liquor, peptone, cottonseed flour, blackstrap molasses, urea; , ammonium salts (for example, ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium acetate, etc.), and others are used. In addition, sodium, potassium, calcium, magnesium, iron,
Metal salts such as manganese, zinc, cobalt, and nickel; salts of inorganic acids such as phosphoric acid and boric acid; and organic acids such as acetic acid and propionic acid; and salts of organic acids such as acetic acid and propionic acid. In addition, amino acids (e.g., glutamic acid, aspartic acid, alanine, valine, lysine, methionine, proline, etc.), peptides (e.g., dipeptides, tripeptides, etc.), vitamins (e.g., B ₁ , B ₂ , nicotinic acid, B ₁₂ , C, etc.), and nucleic acids (eg, purines, pyrimidines, and derivatives thereof). Of course, inorganic or organic acids, alkalis, buffers, etc. may be added for the purpose of adjusting the pH of the medium, or appropriate amounts of oils and fats, surfactants, etc. may be added for the purpose of defoaming.

As a means for culturing, methods such as static culture, shaking culture, and aerated agitation culture are used. It goes without saying that for large-scale treatment, it is desirable to use so-called deep aeration agitation culture. It goes without saying that culture conditions vary depending on the state and composition of the medium, the type of strain, the culture method, etc., but they are usually selected at a temperature of 20° to 445°C and an initial pH of around 6 to 8. It is better. In particular, it is desirable that the temperature during the middle stage of the culture is 24° to 37°C, and the initial pH is 6.5 to 8.5. The culture time is also variable depending on the above conditions, but it is preferable to culture until the concentration of epivariolamine reaches its maximum. The time required for this is usually about 24 to 192 hours in the case of shaking culture or aerated agitation culture using a liquid medium, preferably 72 to 168 hours.
It takes about an hour. Note that the "culture" used in the present invention is obtained by the above-mentioned culture. Aminocyclitols are contained in the culture, and can be used as an α-glucosidase inhibitor as is or after being appropriately purified.

To collect the target product from the reaction solution, the means normally used for collecting microbial metabolites are used alone, in combination in any order, or repeatedly. That is, for example, filtration, centrifugation, concentration, drying, freeze-drying, adsorption, desorption, methods that utilize differences in solubility in various solvents (for example,
(precipitation, crystallization, etc.), chromatography, etc. In addition, by utilizing the fact that epivariolamine is a water-soluble basic substance, methods used for the isolation and purification of so-called water-soluble basic substances, such as ion exchange resins, activated carbon, high bolus polymers, Cephadex, and Cephadex, can be applied. Chromatography and adsorption/desorption methods using ion exchangers, cellulose, ion exchange cellulose, silica gel, alumina, etc. are advantageously used.

More specifically, for example, after removing the bacterial cells from the culture solution by centrifugation or filtration, preferably adjusting the pH of the supernatant or solution to basicity,
The target substance can be adsorbed onto a chroma column of activated carbon and eluted with an aqueous solution containing lower alcohols such as methanol, ethanol, n-propyl alcohol, iso-propyl alcohol, and n-butyl alcohol, or acetone.
In addition, the target substance is adsorbed onto a cation exchange resin (for example, a carunic acid type ion exchange resin such as Amberlite IRC-50, a sulfonic acid type ion exchange resin such as Dowex 50W x 8), and an acid (for example, hydrochloric acid) or alkali (For example, aqueous ammonia or sodium hydroxide solution) can also be used for elution. After concentrating the eluate thus obtained, if necessary, it can be further purified by appropriately repeating or combining activated carbon chromatography or chromatography using an ion exchange resin. Also, Dowex 1×2
The target product can be purified and isolated by subjecting it to chromatography using an anion exchange resin such as (OH ⁺ type) and eluting with water, for example.

The content of the present invention will be explained in detail by referring to Reference Examples and Examples below.

Reference example Method for measuring glucosidase inhibitors Maltase and satucalase [Borgström (B.
Borgstrom) and Dahlquist (A.
Acta Chemica Scandinavica (112 volumes) by Dahlqvist,
1997-2006, prepared according to the method described in 1958], the inhibitory activity to be tested is determined by adding the inhibitor to be tested to 0.25 ml of an enzyme solution appropriately diluted with 0.02 M phosphate buffer solution (PH 6.8) in the same buffer. of solution 0.5ml and substrate
Add 0.25 ml of the same buffer solution of 0.05 M maltose or 0.05 M sucrose, react this mixture at 37°C for 10 minutes, and add glucose B-test reagent (glucose oxidase reagent for glucose measurement, manufactured by Wako Pure Chemical Industries, Ltd., Japan). Add 3 ml, further heat at 37°C for 20 minutes, and measure the absorbance of the reaction solution at 505 nm to calculate.
The 50% inhibitory concentration is calculated by determining the inhibition rate (%) for samples of 3 to 5 different concentrations of the inhibitor.

α of epivariolamine measured by this method
- 50% inhibitory concentration for glucosidase [IC ₅₀
(M)] showed 1.4×10 ⁻⁴ M for maltase; 2.3×10 ⁻⁵ M for satucarase.

Example 1 (a) Cultured on glucose-asparagine agar medium (3% glucose, 2.2% raw soybean flour, 0.3% peptone, 0.4% calcium carbonate, 500 ml water, pH 7) in a sterilized two-sakaguchi flask. Streptomyces hygroscopicus var limoneus (IFO, 12703, Ferm.468,
One loopful of ATCC21431) was inoculated and cultured at 28°C on a reciprocating shaker for 48 hours. This culture solution 500
ml of culture medium (glucose 5%, raw soybean flour 3.6%,
Peptone 0.5%, calcium carbonate 0.4%, water 30%
, PH7) and cultured at 28°C for 114 hours with aeration and agitation.

(b) After adjusting the pH of the culture solution obtained in Example 1.(a) to 9, a filtering agent was added and filtered, the solution (25) was adsorbed on an activated carbon column (10), and after washing with water,
Elution was performed with % n-butyl alcohol water (25). The eluate was concentrated under reduced pressure, and the concentrated solution was adsorbed on a column (5) of Amberlite CG-50 (NH ⁺ ₄ type, manufactured by Rohm and Haas, USA).
After washing with water, it was eluted with 0.1N aqueous ammonia (15). The eluate was concentrated under reduced pressure, and the concentrated solution was subjected to column chromatography using Dowex 1×2 (OH ^- type, Dow Chemical Co., USA) (1), eluted with water, and the eluted fraction positive for ninhydrin reaction was concentrated under reduced pressure. The mixture was concentrated to dryness to obtain 29 g of a colored coarse powder. Dissolve 10 g of this coarse powder in water (500 ml) and add it to a column (550 ml) of Amberlite CG-50 (NH ⁴⁺ type).
After washing with water, it was eluted with 0.05N ammonia water and fractionated into fractions of 20 ml. Validamine [1L-(1,3,4/2,6)-4- Amino-6
-Hydroxymethyl-1,2,3-cyclohexanetriol] is added to fraction No. 55 to 60 by valienamine [1L-(1,3,4/2)-4-
amino-6-hydroxymethyl-5-cyclohexene-1,2,3-triol] and epivariolamino were eluted in fractions No. 64 to 83. After concentrating the eluted fraction of epivariolamine under reduced pressure, Amberlite was added again.
Adsorb onto a CG-50 (NH ⁴⁺ type) column (550ml), wash with water, and elute with 0.05N ammonia water.
The eluted fraction of epivariolamine was concentrated to dryness under reduced pressure to obtain epivariolamine.

Example 2 Epivariolamine obtained in Example 1 (50
mg) in water (5 ml), adjusted to pH 3 with 0.1N sulfuric acid, and then subjected to activated carbon column chromatography (25
ml) and elute with water. The eluted fractions are collected, concentrated under reduced pressure, and then lyophilized to obtain epivariolamine sulfate.

Example 3 50 mg of epivariolamine sulfate was added to 200 ml of fruit juice-containing beverage and dissolved with stirring to obtain a fruit juice-containing beverage.

Example 4 After completing the strawberry jam manufacturing process (boiling heat treatment) using a conventional method, when the product temperature decreased to approximately 55°C, epivariolamine was added to the weight of the finished product.
After uniformly mixing at a ratio of 0.1%, the mixture is cooled to obtain a strawberry jam product.

Example 5 1 part by weight of epivariolamine sulfate and 100 parts by weight of lactose are mixed uniformly and made into a powder or fine granules.

[Brief explanation of drawings]

FIG. 1 shows an infrared absorption spectrum of epivariolamine, and FIG. 2 shows a nuclear magnetic resonance spectrum of epivariolamine.

Claims (1)

[Claims] 1 formula A compound represented by or a salt thereof. 2 Belongs to the genus Streptomycells, formula A method for producing the above-mentioned compound or its salt, characterized by culturing a microorganism capable of producing the compound represented by in a medium, producing the above-mentioned compound in the culture, forming a salt if necessary, and collecting the salt. Manufacturing method. 3 formulas α- containing the compound represented by or its salt
Glucosidase inhibitor.