JPS6234396B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing glycoside-hydrolase inhibitors from microorganisms, particularly microorganisms of the order Actinomycetales. It is known that α-amylase can be inhibited by various low molecular weight substances such as salicylic acid and abiscisin [T. Hemberg, J. Larsson, Physiol. Plant. Volume 14, page 861 (1961),
T. Hemberg, Acta Chem. year) and
Bioch. J. 28, p. 296 (1934)] or by enzyme denaturation and precipitation [BS Miller,
E. Kneen, Arch.BIochem. vol. 15, p. 251
1947, DH Struhmeyer,
MH Malin, Blochem.Biophys.
Acta Vol. 184, p. 643 (1969)] It is also known that there are polymeric substances that can non-specifically inhibit the action of certain amylases. It has also been observed that a substance can be eluted from wheat with distilled water that reduces the dextrinizing action of salivary amylase but has little effect on the action of pancreatic amylase [E .Neen, RM Sandstadt,
Arch.Bioch.9, p.235 (1946)]. The inhibitory effect on amylase is nonspecific, or the inhibitory effect on pancreatic amylase is extremely small, as shown by the research of the present inventors, that is, the inhibitor:enzyme ratio is extremely small. It is a disadvantage of these known inhibitors that almost complete inhibition of amylase (up to 90% and more) is achieved only at high concentrations. One of the previous proposals (UK patent application 20581/71)
relates to amylase inhibitors. In fact, previous proposals have been proposed to prepare water-alcohol (C ₁ -C ₃ -alcohol) by using an aqueous electrolyte solution, preferably dilute acids, or particularly preferably at acidic PH values.
A highly active inhibitor of pancreatic amylase which does not exhibit the above-mentioned disadvantages by using a mixture of wheat (coarse wheat, wheat flour or wheat gluten)
This shows that it can be extracted with high yield from. The substances thus obtained inhibit pancreatic amylase to the extent of more than 90% even at very low inhibitor:enzyme ratios. The present invention relates to inhibitors of glycoside-hydrolase from actinomycetes, and in particular to glycoside-hydrolase, preferably gastrointestinal carbohydrate-degrading enzymes, obtained from actinomycetes. These inhibitors are produced by strains of the genus Actinobifida (according to Bergey's 7th edition classification, Micromonospora or Thermoactinomyces). Accordingly, the present invention provides glycoside-hydrolase inhibitors of microbial origin as novel products. The present invention provides a method for producing a glycoside-hydrolase inhibitor, which comprises culturing a glycoside-hydrolase inhibitor-producing bacterium belonging to the genus Actinobifida and extracting the hydrolase inhibitor from the resulting culture. I will provide a. Effective bacterial strains can be searched for using the method below. Strains of the order Actinomycetes, in particular of the families Streptomycetes and Actinoplanaceae, are isolated by known methods from strains of these orders obtained from microorganism depositories or from soil samples. This type of bacterial strain is inoculated into a culture flask containing a culture solution capable of growing this type of bacterial strain, and then cultured. For example, 2% glycerin, 0.25% glycine, 0.1% NaCl, 0.1% according to von plotho.
_K2HPO4 , 0.01% _FeSO4・_7H2O , _0.01 % _MgSO4・
Glycerin with the composition of 7H ₂ O and 0.1% CaCO ₃ -
A glycine culture solution can be used. For even more rapid growth, this type of culture may be supplemented with complex carbon sources, such as corn soaking liquid or soybean flour or yeast extract or protein hydrolysates, such as NZ-amines, or mixtures of these substances. It is also recommended to add In these cases, the pH of the culture medium must be adjusted. The initial pH of the culture solution is preferably between 6.0 and 8.0, particularly between 6.5 and 7.5. Instead of cultured glycerin it is also possible to use other carbon sources, such as glucose or sucrose or starch or mixtures of these substances. Instead of glycine, it is also possible to use other nitrogen sources, such as yeast extract, soybean flour, NZ-amines, pharmaceutical media and others. The concentrations of the carbon and nitrogen sources and also of the salts can be varied within wide limits. FeSO ₄ , CaCO ₃ and MgSO ₄ can also be omitted completely. For example, 100-200ml of culture solution
was introduced into a 1 liter Erlenmeyer flask, sterilized by known methods and inoculated with the strain to be searched, and the flask was heated at 15-60°C.
Culture is preferably carried out on a shaker at 24-50°C. If a culture shows growth, it is generally 1-
After 10 days, which occurs in most cases after 3 to 5 days, for example, a 5 ml sample is taken and the mycelium of this sample is separated by filtration or centrifugation. In the following tests, 0-100 μ of culture fluid was used and 1 ml
Calculate the inhibition potential per hit. Add 5 volumes of acetone to mycelium (1 volume of mycelium)
(per volume) twice and then with 1 x 5 volumes of diethyl ether, the extracted mycelial residue was dried in vacuo at 20°C and the resulting dry mycelium powder was extracted with dimethyl sulfoxide. (DMSO) 4～
Extract using 8 parts by weight. The two acetone and ether extracts are combined and concentrated in vacuo to near dryness. The residue from these extracts is extracted from the dry powder.
Combine with DMSO extract and use 0-100μ in the following tests. Amylase Test One amylase inhibitor unit (1 AIU) is defined as the amount of inhibitor that inhibits two amylase units to an extent of 50%. One amylase unit (AU) is the amount of enzyme that breaks down 1 μ equivalent of glycosidic bonds in starch in 1 minute under the following test conditions. The μVal of the cleaved bond is determined by a colorimetric method using dinitrosalicylic acid as the μVal of the reducing sugar produced, and is expressed as the μVal of maltose equivalent using a maltose calibration curve. To perform the test, add 0.1 ml of amylase solution (20-22 AU/ml).
0.02M sodium glycerophosphate buffer/
0-400 μg of inhibitor to be tested in 0.4 ml of 0.001 MCaCl ₂ PH6.9 or 0-100 μg of culture fluid
Alternatively, the mycelium extract is used to equilibrate the mixture at 35° C. for about 10-20 minutes in a water bath. Then preheat it to 35℃
A 1% starch solution (Messrs.Merck.Darmstadt) No.
1252 soluble starch] for 5 minutes at 35°C and then treated with 1 ml of dinitrosalicylic acid reagent [P. Bernfeld, Colowick.
- Kaplan), Meth. Enzymol., Vol. 1, p. 149]. To develop the color, the batch was heated on a boiling water bath for 5 minutes, then cooled and soaked in distilled water.
Process using 10ml. The absorbance at 540 mμ is measured for a suitably mixed blank sample containing no amylase. For evaluation, the amylase activity still active after addition of the inhibitor is read from a previously recorded standard curve of amylase, and the percent inhibition of the amylase used is calculated therefrom. The percent inhibition is plotted as a function of the quotient μg of inhibitor ⁺ /AU ⁺⁺ (+ AU in the uninhibited mixture of the same system relative to the solid content), and the 50% inhibition point is read from the curve and Convert to AIU/mg of inhibitor. Satucalase Test One Satucalase Inhibitor Unit (SIU) is defined as the amount of inhibitor that inhibits two Satucalase Units by approximately 50%. 1 sucarase unit (SU) means 1 micron of sucrose per minute under the following test conditions.
It is the amount of enzyme that breaks down moles into glucose and fructose. μ of decomposed sucrose
mol of glucose and fructose produced by a colorimetric method using dinitrosalicylic acid and calculated using a glucose/fructose calibration curve. To carry out the test, satucalase solution 1)
(0.8-0.4SU/ml) in 0.1ml of 0.1M Sodium Maleate PH6.0 with 0-400 μg of inhibitor.
Alternatively, mix with 0-50μ of the culture solution of the mycelial extract to be studied and place the mixture in a water bath.
Keep the temperature at 35℃ for about 10-20 minutes. Next, it was mixed with a 0.056M Shurose solution (Shucrose: Melta, Darmstadt,
No.7652) 0.2ml and cultured at 35℃ for 60 minutes.
It is then treated with 0.5 ml of dinitrosalicylic acid reagent (according to P. Bernfelt, Kolovik-Kaplan, Meth. Enzymol., Vol. 1, p. 149). To develop the color, heat the mixture on a boiling water bath for 5 minutes, then cool and add 5 ml of distilled water.
Process using. Measure at 540 nm against a suitable blank without satucalase. 1 B. Borgstrom, A. Dahlguist, Acta Chem.Scand.12
Solubilized satucarase from porcine intestinal mucosa according to Vol. 1997 (1958). For evaluation, the units of sutucalase that are still active after addition of the inhibitor are determined from the standard curve and the percent inhibition of the sutucalase used is calculated therefrom. Percent inhibition is the quotient μg/SU of inhibitor ^{+ +} relative to solids + + in an uninhibited mixture of the same strain.
It is plotted as a function of SU and the 50% inhibition point is read from the curve and converted to SIU/mg of inhibitor. Maltase Reagent One maltase inhibitor unit (1 MIU) is defined as the amount of inhibitor that inhibits two maltase units to an extent of 50%. One maltase unit (MU) is the amount of enzyme that decomposes 1 μ equivalent of glucoside bond in p-nitrophenyl-α-D-glucopyranoside in 1 minute under the following test conditions. The μVal of the cleaved bond is determined spectrophotometrically as the μVal of p-nitrophenolate. To conduct the test, maltase solution 1)
(0.09-0.12MU/ml) 0.05ml inhibitor 0-400μ
0-20 μg or the culture fluid or mycelium extract to be studied in 0.05 ml of 0.1 M sodium maleate buffer at PH 6.0 for about 10-20 minutes and kept constant in a 35° C. water bath. Then add the mixture in advance to 35
p-Nitrophenyl-α-D-glucopyranoside [Celva, Heidelberg (Messrs.
Heidelberg) No. 30716] was incubated at 35°C for 30 minutes with 0.1ml of a solution of 0.4%
And then 2 ml of 0.565M Tris buffer with pH 7.6
It is stopped by adding . In contrast to a suitably mixed blank sample containing no maltase.
Immediately measure absorbance at 403 nm. 1 B. Borgström, A. Dahlquist,
Solubilized maltase from pig intestinal mucosa or freeze-dried product of human pancreatic juice according to Acta Chem. Scand. Vol. 12, p. 1997 (1958). For evaluation, maltase units that are still active after addition of the inhibitor are calculated based on a molar extinction coefficient of E ₄₀₃ ]13.2×10 ³ for p-nitrophenolate anion at pH 7.6, and From the active maltase units, calculate the percent inhibition of maltase used. Percent inhibition is plotted as a function of the quotient μg of inhibitor ⁺ /MU ⁺⁺ + solids vs. MU in the uninhibited mixture and the point of 50% inhibition is read from the curve and MIU/mg of inhibitor. Convert. Since in this simple routine test an artificial substrate (maltose) is used rather than a real substrate for maltase, the formulation is further modified by Dahlquist (Enzyme.biol.clin. vol. 11, p. 52 (1970).
It is tested for maltase inhibitory activity by a more complex maltase test described by (2011). In the present invention, glucose produced during the action of maltase on maltose is measured by a colorimetric method using enzymatic action using glucose oxidase, peroxidase and dianisidine. All of the maltase inhibitors described here also exhibit an inhibitory effect in this test. A whole lineage of bacterial strains from various families and genera of the order Actinomycetes was tested according to the method described above. In that study, distinct, sometimes weak, glucoside-hydrolase inhibitory effects were found for various families and genera. Strains of the genera Micromonospora and Thermoactinomycetes of the Streptomycetes family have been shown to be beneficial. Of the strains tested, the strains listed below were shown to be particularly active in one or more of the specified tests.

【table】

[Table] The strains listed in Table 1 were collected at The Central Bieureau in Baarn, the Netherlands.
It was deposited with Ball Schimmel Cultures under the above CBS number. Further, the deposit number to the Microbial Technology Research Institute is as listed in the deposit number column of Table 1. In addition, these strains are recognized as strains belonging to the genus Micromonospora or the genus Thermoactinomyces according to the classification of Bergey, 7th edition. To obtain glucoside-hydrolase inhibitors,
The above bacterial strain is cultured in the above culture solution. When doing so, it should be kept in mind that virtually every strain requires a different culture medium with a different composition of nature and quantity to obtain optimal production. 1-10 at 15-60°C, preferably 24-50°C in fermenters or shake flasks of various sizes.
After culturing for a day, the mycelia are separated from the culture fluid and, depending on the occurrence of the inhibitor, the active element components are concentrated using the culture fluid and/or the mycelia. The inhibitors are obtained from the culture fluid by lyophilization or by precipitation with salts or water-soluble organic solvents (such as lower alcohols and ketones) or by adsorption of the active substance onto ion exchangers. Inhibitors are obtained from mycelium by extraction with organic solvents such as alcohols, ketones, ethers, esters and sulfoxides. For this purpose, the fermentation liquid is heated at 3000-20000 per minute.
Rotation, preferably 10 to 60 at 6 to 10,000 revolutions per minute
The mixture is centrifuged for a minute, preferably 30 minutes, or preferably under pressure and using a supercharging agent, such as Claricel, and thereby the culture liquid and mycelium residue are separated. Inhibitors can be isolated from specific culture fluids by a variety of methods, including the following. a About the initial volume at a bath temperature of 20-100°C, preferably 40-80°C under reduced pressure (10-50 mmHg)
Concentrate the culture solution to 1/5 to 1/50. The concentrated extract is filtered or centrifuged and the clear liquid (or clear supernatant), if necessary after desalting beforehand, is lyophilized. b. Inhibition from the culture broth (or culture broth concentrated according to a) by adding water-soluble organic solvents such as alcohols and ketones, preferably methanol, ethanol or acetone, to a content of 60-90%. precipitate the agent. Since inert turbid substances precipitate at low concentrations of solvent, this precipitation operation is particularly suitable for fractional precipitation to remove undesired turbid substances. c Salting out the inhibitor from the extract (or extract concentrated according to a)) using, for example, ammonium sulphate, sodium chloride and the like. The precipitate formed is collected by centrifugation or filtration and washed directly with acetone and ether and dried under vacuum or redissolved in water, dialyzed and lyophilized. d Adsorb the inhibitor onto the ion exchanger. This method is suitable for separating charged inhibitors due to their chemical nature. Inhibitors can be desorbed by changing the ionic strength or PH value of the elution medium. In addition to inhibitors, undesirable turbid substances are often present in the culture medium. These turbid substances can be removed by various methods, for example by denaturing the turbid substances with heat in the case of heat-safe inhibitors, or by dialysis through suitable membranes in the case of low molecular weight inhibitors. by retaining undesirable turbid substances in the membrane or by fractional precipitationb.
) or by adsorbing the turbid substance on an ion exchanger. Inhibitors can be obtained by repeated extraction of mycelia with organic solvents,
It is preferably obtained from the mycelium by two extractions for 10 to 20 minutes with 3 to 5 volumes of acetone (based on the wet mycelium volume), followed by one extraction with ether for 5 to 10 minutes. The mycelium extracted by this method is dried in vacuo and then extracted with 3 to 10 parts by weight of dimethyl sulfoxide for 2 to 8 hours with stirring, after which the
Centrifuge at 20,000 rpm. The acetone and ether extracts are concentrated to dryness in vacuo and combined with the DMSO extract. Instead of extracting the dried mycelium powder using DMSO, it can also be extracted using water or a dilute electrolyte solution for a longer time, preferably 12 to 24 hours. The new substance is highly soluble in water. A group of inhibitors are heat stable at neutral PH values, acid (PH2) stable, alkali (PH12) stable and can be slowly dialyzed. Inhibitors of this type are not inactivated by trypsin and pepsin and furthermore do not inhibit the enzymes mentioned above. They are not stained by typical protein dyes and show no characteristic absorption in UV light up to 250 nm. These inhibitors cannot be inactivated by urea and β-mercaptoethanol. According to estimates from gel filtration, the molecular weights of these inhibitors are over 500 and
6000 or less. Hydrolytic decomposition yields monosaccharides such as glucose. For example, these inhibitors are oligosaccharides or polysaccharides or derivatives thereof. The best inhibitors of this group are against amylase
Shows an inhibitory ability of 8000 AIU/mg. Additionally, one class of inhibitors is heat labile and cannot be dialysed, or only very poorly dialysed. These inhibitors are more or less rapidly inactivated by trypsin. urea and alpha
-Mercaptoethanol also inactivates most of these inhibitors. This type of inhibitor is probably a peptidic substance. The best inhibitors of this group are against amylase
Shows an inhibitory ability of 80 AIU/mg. For animals and humans, carbohydrate-containing foods and vegetables (e.g. corn starch, potato starch,
Hyperglycaemias are known to occur after ingestion of fruits, fruit juices or thiocholates, which are caused by the ingestion of carbohydrates by glycoside-hydrolytic enzymes (e.g. salivary and pancreatic amylases, maltase and succalases). by

[Table] This is due to the rapid decomposition according to the formula. This hyperglycemic symptom is particularly strong and lasts for a long time in cases of diabetes. In the case of a fatty diet, dietary hyperglycemic episodes often result in a particularly strong secretion of insulin, which further results in increased fat synthesis and decreased fat breakdown. In conjunction with this type of hyperglycemic condition, hypoglycemia as a result of insulin secretion often occurs in metabolically healthy and obese individuals. It is known that not only hypoglycemia but also residual juices in the stomach stimulate the production of gastric juices, which promotes or stimulates the development of gastritis or gastric or duodenal ulcers. In the present invention, the glycoside-hydrolase inhibitor according to the present invention, obtained and isolated according to the above-described method, causes dietary hyperglycemia after administration of wheat starch or sucrose or maltose to mice and/or humans; It has been found to significantly reduce hyperinsulinism and hypoglycemia and to speed up the passage of carbohydrates through the stomach. Furthermore, it is known that carbohydrates, particularly sucrose, are decomposed by microorganisms in the oral cavity and that this promotes the development of cavities. Therefore, glycoside-hydrolase inhibitors can be used to treat diseases such as obesity, steatosis, hyperlipidemia (atherlos-clerosis), diabetes, prediabetes, gastritis, gastric ulcers, duodenal ulcers, and caries. Suitable for use as a treatment for symptoms. Therefore, unless a surfactant is present,
Pharmaceutical compositions can be made that contain the inhibitors of the invention as active ingredients in admixture with liquid diluents other than solvents of molecular weight lower than 200 (preferably 350). Furthermore, pharmaceutical compositions containing the inhibitors of the present invention as active ingredients can be prepared in the form of sterile or isotonic aqueous solutions. It is also possible to prepare a medicament in dosage unit form containing the inhibitor of the invention alone or in admixture with a diluent. Also, tablets (including lozenges and granules), dragees, capsules, pills, containing the inhibitor of the invention alone or mixed with a diluent;
The drug can be in the form of ampoules and suppositories. As used herein, the term "drug" refers to a physically discrete unit suitable for pharmaceutical administration. As used herein, "drug in dosage unit form" refers to a unit dose or several multiples (up to 4 times) or a fraction (up to 40 times) of a unit dose of an inhibitor of the invention.
(up to one-fold) in a physically separate form suitable for pharmaceutical administration. Whether the drug comprises a unit dose or, for example, 1/2, 1/3 or 1/4 of a unit dose, means that the drug is administered each once a day, and also, for example, twice, three or four times a day. It will depend on whether it is administered or not. A unit dose is the amount of inhibitor to be administered in one instance. The pharmaceutical compositions may be in the form of, for example, gels, pastes (e.g. toothpaste), creams, chewing gums, suspensions, solutions and emulsions of the active ingredient in aqueous or non-aqueous diluents, syrups, granules or powders. It can be done. Diluents used in pharmaceutical compositions (e.g. granules) to form tablets, dragees, capsules and pills include (a) fillers and bulking agents such as starch, sugar, mannitol and salicylic acid; (b) binder agents such as carboxymethyl cellulose and other cellulose derivatives,
alginates, gelatin and polyvinylpyrrolidone, (c) wetting agents such as glycerol, (d) disintegrants,
(e) dissolution inhibitors such as paraffin, (f) absorption enhancers such as quaternary ammonium compounds, (g) surfactants such as cetyl alcohol, glycerol monostearate, (h) adsorption carriers such as kaolin and bentonite; (i) lubricants such as talc, calcium and magnesium stearate and solid polyethylene glycol; (j) elastomeric binders such as chicle. Tablets, dragees, capsules and pills made from the present pharmaceutical compositions may have conventional coatings, casings and protective bases, and they may contain opacifying agents. They can be made so that they release the active ingredient only or preferably in a certain part of the intestinal tract, perhaps over a period of time.
Coatings, envelopes and protective bases can be made of polymeric materials or waxes, for example. The components can also be combined in microencapsulated form with one or more of the diluents mentioned above. Diluents used in the pharmaceutical compositions employed to make suppositories include conventional water-soluble diluents such as polyethylene glycols and fats such as cocoa oil and higher esters such as C ₁₄ alcohols and C ₁₆ fatty acids. Alternatively, it can be a water-insoluble diluent or a mixture of these diluents. The pharmaceutical compositions, which are pastes, creams and gels, may, for example, contain the usual diluents such as animal and vegetable fats, waxes, paraffin, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide. or a mixture of these substances. The pharmaceutical compositions, which are powders, can contain, for example, customary diluents such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder or mixtures of these substances. The pharmaceutical compositions, which are solutions and emulsions, may contain, for example, the usual diluents (excluding, of course, solvents having a molecular weight below 200, except in the presence of surfactants as mentioned above), such as solubilizers and emulsifiers. Specific examples of such diluents include water, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, Fatty acid esters of oils (eg ground walnut oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and sorbitol, or mixtures thereof. For parenteral administration, solutions and emulsions must be sterile and, where appropriate, isotonic with blood. The pharmaceutical composition, which is a suspension, contains liquid diluents such as water, ethyl alcohol, propylene glycol, surfactants such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, meth-water Conventional diluents such as aluminum oxide, bentonite, agar and tragacanth or mixtures thereof may be included. The pharmaceutical compositions can also contain coloring agents and preservatives, as well as flavoring and aroma-adding agents such as linseed oil and eucalyptus oil and sweetening agents such as saccharin. In particular, chewing gum and toothpaste contain fragrances. The pharmaceutical composition preferably comprises about 0.1 to 99.5%, more preferably about 0.5%, based on the weight of the total composition.
Contains up to 95% inhibitor. Besides the inhibitors of the invention, the pharmaceutical compositions can also contain other pharmaceutically active compounds. They can also contain two or more different inhibitors of the invention. Particular examples of other pharmaceutically active compounds of this type are oral antidiabetic agents such as β-citotropic sulfonyl-urea derivatives and biguanides, which influence blood sugar levels. The diluent in the medicament can be any of those described above with respect to the pharmaceutical composition. This type of drug can contain a solvent with a molecular weight of less than 200 as the sole diluent. Separate and unitary parts of the drug (whether in unit dosage form or neat) may include, for example, tablets (including lozenges and granules), pills, dragees, capsules. , suppositories and ampoules. Certain forms of this type can be made to provide sustained release of the inhibitor. Some, such as capsules, include a protective envelope that provides physical separation and uniformity to each portion of the drug. The preferred unit dosage for the drug of the invention is
5000~500000AIU, 2.5~250MIU, or 100
~10000SIU of inhibitor. The unit dosage will be administered orally, usually once or several times a day just before, during or after meals. It is intended that the inhibitor be administered orally. Preferred drugs are therefore those adapted for oral administration, such as tablets, dragees and chewing gum parts. The toxicity of some glucoside-hydrolase inhibitors of this type is extremely low. The active substances obtained from Examples 14 and 37 were tolerated up to a dose of 5×10 ⁶ AIU/Kg in rats or mice for 20 days when administered orally without showing symptoms. For intravenous injections, 20-day mice and mice
Tolerable up to 10 ⁶ AIU/Kg. The following examples illustrate methods of making inhibitors according to the invention. Example 1 Erlenmeyer containing the medium listed in the table
Flasks were inoculated with the corresponding strains and shaken on a rotary shaker at the indicated temperature. As a result, after a few days, a culture solution was obtained and the mycelium was separated by centrifugation to obtain a mycelium extract having the activity listed in the table. In addition, in the table, the culture medium of Example 44 is as follows. 0.5% bactopeptone, 0.5% meat extract, 0.2% yeast extract, 0.03% casein hydrolyzate, 11% i-
Isositol, 1% sorbitol, 1% D-mannitol, 1% glycose, 0.1% K ₂ HPO ₄ 0.05%
The composition was MgSO ₄ , 0.05% KCl and 0.01% FeSO ₄ and the pH was adjusted to 7.2. Furthermore, in the table, mycelium extracts were prepared and tested as follows. The mycelium was treated with 50 ml of acetone and treated with an Ultraturrax homogeniser (Jahnke & Co., Ltd.).
Messrs. Janke and Kunkel, Staufen,
Breisgau] for 1 minute and the mixture was then centrifuged at 3000 rpm for 10 minutes. The residue was extracted again in the same way with 50 ml of acetone, then once with 50 ml of ether, and the three extracts were combined and extracted with approx.
It was concentrated to almost dryness in a rotary evaporator at 20 mm Hg and a water bath temperature of 37°C. The mycelial residue was dried in vacuo and then dimethyl sulfoxide 15
ml, homogenized for 2 minutes using an Ultraturax and extracted for 2 hours with stirring (magnetic stirrer). The mixture was then centrifuged at 20000 rpm for 30 minutes. next
The DMSO extract was separated from the extracted mycelium by decanting,
The acetone/ether extraction residue was added and the mixture was stirred (magnetic stirrer) for about 30 minutes and centrifuged again at 20000 rpm for 1 minute, and the supernatant was tested as mycelial extract.

[Table] Example 2 Experimental method to demonstrate the effects of glycoside-hydrolase inhibitors in mice and humans Mice (n=
6), 2.5 g/kg of starch, maltose, or sucrose was orally administered as a solution or suspension (comparison animals). Six other groups of mice were administered orally one of the carbohydrates listed above as well as the indicated dose of a glycoside-hydrolase inhibitor. retro-orbital venous plexus
Auto-Analyzer (Technicon) measures blood glucose levels from the venous plexus at short time intervals after carbohydrate administration.
, following Hoffman: J.biol.
Chem. Vol. 120, p. 51 (1937)]. 50 to cause dietary hyperglycemia in humans.
g starch/vp was administered orally as an aqueous suspension. Blood glucose levels in capillary blood from the fingertips were measured by the method described above immediately before and at short time intervals after the start of the experiment. In yet another experiment,
The active substance was added to the starch suspension. Insulin in the serum of a group of six mice was measured at short time intervals after carbohydrate administration, with one group of mice receiving 2.5 g starch/Kg orally (comparison) and the other group receiving additional doses. Glucoside-hydrolase inhibitors were given. Hales and Randle
Insulin in serum was measured by a radioimmunoassay based on the double antibody method of J. Randle [Biochem. J. Vol. 88, p. 137 (1963)]. Measurement of starch in the gastrointestinal tract of mice using 300mg starch/
The tests were carried out at short time intervals after oral administration to mice. For this purpose, individual parts of the gastrointestinal tract were prepared and washed after killing the animals, and the undigested starch contained therein was determined as glucose after acid hydrolysis. Figures 1 to 3 (Example 2) Figures 1 to 3 are obtained using a throat probe.
Average starch content mg in the stomach (Fig. 1), small intestine (Fig. 2) and large intestine (Fig. 3) at various times (horizontal axis) after oral administration of 300 mg raw wheat starch/mouse.
The number (vertical axis)/mouse (n=6) is shown. Figures 1-3
In , curve 1 shows the results for comparison animals (group 1) fed starch without amylase inhibitors, while curve 2 shows the results for comparison animals (group 1) fed starch without amylase inhibitors, while curve 2 shows the results for comparison animals (group 1) fed starch without amylase inhibitors, while curve 2
Results are shown for animals fed the same dose of starch supplemented with amylase inhibitor (group 2). Results: The content of wheat starch in the stomach 15+60 minutes after administration (P<0.001 to P<0.02) with amylase inhibitors in group 2 (Fig. 1) is significantly lower than in group 1. It can also be seen from FIG. 2 that starch passes through the stomach more quickly after the addition of the amylase inhibitor. In this case, the starch content in the small intestine of group 2 is 150~
It becomes significantly higher after 180 minutes (P<0.001). Figure 3
shows that when amylase inhibitors are added to starch at the above doses, the starch reaches the mouse colon undigested. Figures 4 to 6 (Example 2) In Figures 4 to 6, for human experiment subjects,
After oral administration of 60 g of cooked starch,
Blood glucose mg/initial value (0)
100 ml (vertical axis) (Fig. 4), the serum concentration of immunoreactive insulin (μU/ml) (vertical axis) (Fig. 5), and the concentration of unesterified fatty acids (microequivalents/plasma 1 Little) (vertical axis) (Figure 6)
It is shown. Curve 1 shows the effects of the above factors (blood sugar, serum insulin, plasma
This shows the evolution of UFA). Curve 2 shows the changes in the above factors after administering the same dose of starch supplemented with 0.25 mg AIU amylase inhibitor. Curve 3 shows the changes in the above factors after adding 0.5 mega AIU amylase inhibitor to 60 g of cooked starch per person. Results: After a temporary hyperglycemic episode between 0+30 minutes in Figure 4, blood sugar drops to a value significantly lower than the initial value after 45-180 minutes. As a result of early hyperglycemic symptoms, the concentration of insulin in the serum in the comparative experiment (curve 1) rises rapidly. 0.25 to starch
Addition of mega AIU (curve 2) or 0.5 mega AIU (curve 3) has the effect of quickly attenuating hyperglycemic symptoms (Figure 4) and hyperinsulinic symptoms (Figure 5). The ratio of unesterified fatty acids in plasma was essentially equal in all three experiments.

[Brief explanation of the drawing]

Figures 1 to 3 show various times (horizontal axis) after oral administration of 300 mg raw wheat starch/mouse using a throat probe.
Figure 1 shows the average starch content in mg (vertical axis)/mouse (n=6) in the stomach (Figure 1), small intestine (Figure 2), and large intestine (Figure 3). Figures 4 to 6 show blood glucose (mg) relative to the initial value (0) after oral administration of 60 g of cooked starch to human experimental subjects.
Change in number/100ml (vertical axis) (Figure 4), concentration of immunologically reactive insulin in serum (μU/ml)
(vertical axis) (FIG. 5) and the concentration of unesterified fatty acids (microequivalents/liter of plasma) (vertical axis) (FIG. 6).

Claims (1)

[Claims] 1. A glycoside-hydrolase inhibitor characterized by culturing a glycoside-hydrolase inhibitor-producing bacterium belonging to the genus Actinobifida and extracting a glycoside-hydrolase inhibitor from the resulting culture. manufacturing method.