JPS6216638B2 - - 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 Actinomyceta. It is known that α-amylase can be inhibited by various low molecular weight substances such as salicylic acid and abscisin [T. Hemberg, J. Larsson, Physiol. Plant. Volume 14, page 861 (1961),
T. Hemberg, ActaChem. Scand. vol. 21, p. 1665 (1967)]. Furthermore, by physical adsorption [T. Chrzaszcz, J. Janicki, Bioch. Z. 260 p. 354 (1933) 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, Biochem.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 Sandstedt,
Arch.Bioch.9, p.235 (1946)]. The inhibitory effect on amylase is non-specific, or the inhibitory effect on pancreatic amylase is minimal, as shown by the research of the present inventors, that is, the inhibitor:enzyme ratio is A disadvantage of these known inhibitors is that almost complete inhibition of amylase (up to 90% and more) is achieved only at very high concentrations. One of the previous proposals (UK patent application 20581/71)
relates to amylase inhibitors. In fact, previous proposals have been made for pancreatic amylases which do not exhibit the above disadvantages by using aqueous electrolyte solutions, preferably dilute acids, or especially preferably by using water-alcohol (C1-C3 alcohol) mixtures at acidic PH values. It has been shown that highly active inhibitors can be extracted from wheat (coarse wheat, wheat flour or wheat gluten) in high yields. 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 enzymes obtained from actinomycetes, and in particular to glycoside-hydrolase enzymes, preferably gastrointestinal hydrate-degrading enzymes, obtained from actinomycetes. These inhibitors are produced by microorganisms of the order Actinobacteria, in particular of the family Actinoplanacea. They are also highly produced by strains of the genera Actinoplanes, Ampullariella, Straptostosporangium, Pilimelia and Planomonospora. Accordingly, the present invention provides glycoside-hydrolase inhibitors of microbial origin as novel products. The present invention is directed to a glycoside-hydrolase inhibitor, which is characterized by culturing a glycoside-hydrolase inhibitor-producing bacterium belonging to the Actinoplanaceae family, and extracting the glycoside-hydrolase inhibitor from the resulting culture. A manufacturing method is provided. Effective bacterial strains can be searched for using the method below. Bacterial strains of the order Actinomycetes, particularly strains of the family 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, von, proto
plotho) 2% glycerin, _0.25 % glycine, 0.1% NaCl, 0.1% _K2HPO4 , 0.01% _FeSO4 .
_7H2O , 0.01% _{MgSO4 7H2O} and 0.1% _CaCO3
A glycerin-glycine culture solution having the following composition can be used. In order to grow even more quickly,
It is also recommended to add complex carbon sources to this type of culture medium, such as corn steeping liquid or soybean flour or yeast extract or protein hydrolysates, such as NZ-amines, or mixtures of these substances. In these cases, the pH of the culture medium must be adjusted. The initial pH of the culture solution is 6.0 and 8.0
preferably between 6.5 and 7.5. Instead of glycerin in the culture medium it is also possible to use other carbon sources, such as glycose or sucrose or starch or mixtures of these substances. Instead of glycine, it is also possible to use other nitrogen sources, such as yeast extracts, soybeans, 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, culture solution 100
~200 ml was introduced into a 1 liter Erlenmeyer flask, sterilized by known methods and inoculated with the strain to be searched, and the flask was inoculated with 15
Culture on a shaker at ~60°C, preferably 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, 0.1 ml of amylase solution (20-22 AU/ml)
0.02M sodium glycerophosphate buffer/
0-400 μg of the inhibitor to be tested in 0.4 ml of 0.001 MCaCl ₂ PH6.9 or 0-100 μg of culture fluid or mycelial extract and the mixture was incubated at 35° C. in a water bath. Equilibrate for approximately 10-20 minutes. Next, it was added to a 1% starch solution [Messrs.Merck.Darmstadt No. 1252] previously warmed to 35°C.
0.5 ml of soluble starch] was incubated at 35°C for 3 minutes, and then dinitrosalicylic acid reagent 1
ml [P. Bernfeld, Colowick-Kaplan]
- 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 nm is measured on 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 amylase calibration curve, 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 dinitrokyrithylic acid and calculated using a glucose/fructose calibration curve. To carry out the test, satucalase solution ¹⁾
(0.3-0.4 SU/ml) in 0.1 ml of 0.1M sodium maleate PH6.0 with 0-400μ of inhibitor.
g or the mycelial extract to be studied with 0 to 5 μ of the culture and the mixture is incubated at 35° C. in a water bath for about 10 to 20 minutes. It was then incubated for 60 minutes at 35°C with 0.2 ml of 0.056 M Shurose solution (Sucrose: Merck & Co., Darmstadt, No. 7652) prewarmed to 35°C, followed by 0.5 min of dinitrosalicylic acid reagent.
ml (according to P. Bernfelt, Kolovitzk-Kaplan, Meth. Enzymol., Vol. 1, p. 149). To develop the color, the mixture was heated on a boiling water bath for 5 minutes, then cooled and poured with distilled water 5 minutes.
Process using ml. Measure at 540 nm against a suitable blank without satucalase. As an evaluation, it still shows activity after addition of the inhibitor. 1 B. Borgstrom, A. Dahlguist, ActaChem.Scand.12 volumes
Solubilized satucarase from pig intestinal mucosa according to p. 1997 (1958). The sutucalase units 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 Test 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 carry out the test, maltase solution ¹⁾ (0.09
~0.12MU/ml) 0.05ml with 0-400μg of inhibitor or 0-20μ of the culture medium or mycelial extract to be studied and 0.1M sodium maleate buffer.
Maintain constant temperature in a 35°C water bath for about 10-20 minutes at PH6.0 in 0.05ml. The mixture was then preheated to 35°C with a pH of 0.1 M. 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). Add p-nitrophenyl-α-D-glucopyranoside [Messrs.Serva, Heidelberg] No. 1 to sodium maleate buffer.
30,716] dissolved at a concentration of 0.4% for 30 minutes at 35℃, and then the pH
Stop by adding 2 ml of 0.565M Tris buffer from 7.6. The absorbance at 403 nm is immediately measured against a suitably mixed blank sample containing no maltase. For evaluation, the maltase units that are still active after addition of the inhibitor are calculated based on the molar extinction coefficient of E ₄₀₃ = 13.2 × 10 ³ for p-nitrophenolate anion at pH 7.6; 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. In this simple routine test, an artificial substrate (maltose) rather than the actual substrate for maltase
The formulation is further described 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, glycose produced during the action of maltase on maltose is measured by a colorimetric method using enzymatic action using glycose oxidase, peroxidase and dianisidine. All of the maltase inhibitors described here also exhibit an inhibitory effect in this test. Strains of various families and genera of the order Actinomycetes were tested according to the method described above across the entire lineage. In that study, distinct, sometimes weak, glycoside-hydrolase inhibitory effects were found for various families and genera. In particular, strains of the Actinoplanaceae family were shown to be most beneficial in terms of yield. Among these families, particularly active strains were found in the genera Actinoplana, Ampoulariella and Streptosphorangiium. Of the strains tested, the strains listed below were shown to be particularly active in one or more of the specified tests. Regarding the mycological properties of the strains exemplified below, the known strains of ATCC19190 are described in more detail in, for example, C.Gupta.J.Antibiol., Ser.A., p. 18, 125 (1965). has been done. Also, regarding the ATCC12428 known strain,
Bergey's Manual, 8th edition, pages 712-713, furthermore, CBS367.66 (ATCC14539) and CBS191.64
(ATCC15349) for known strains, see J. Busha Mitunell, Sci. Soc., p. 79, 69 (1963) and J. Elisha Mitchell, Sci, Soc., respectively.
79, p. 61 (1953) in more detail. For the known strains of CBS190/64 (ATCC15348), see Bergey's manual, 8th edition, page 717.
Each is described in detail. Also, KCC-A0027 (CBS43261), KCC-A0025
(CBS42961) and KCC-A0028 (CBS43361) known strains are described by Nomura and Ohara, J. Ferm.
Techn., 38, p. 405 (1960), and the CBS193.64 known strain is further described in JNCouch and Elisha Mitchell, Sci.Soc.
, 79, p. 54 (1963).

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[Table] SB18/4 strain (Actinoplanes species)
It has almost the same mycological properties as the SB18 strain (Actinoplanetaceae species), but
It produces more aerial mycelia on CPC agar medium than the SB18 strain. In addition, the SB18/5 strain (Actinoplanes species) has almost the same mycological properties as the SB18 strain, but its growth on CPC agar medium is slower than that of the SB18 strain. It's late.

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[Table] The strains listed in Table 1 are from the American Type Culture Collection.
type Chlture Collection) and the Centraalbureau voor Schimmelcultures
It was obtained from an established microbial depository such as , or deposited with the Central Bieureaud Bor Schimmel Cultures, Baarn, Netherlands, under the above CBS number. Furthermore, the deposit number to the Microbial Technology Research Institute is as stated in the deposit number column of the table. Furthermore, SB18/4Actino=Planes spec. (Actinoplana species) is also
SB18/5Actino-Planes spec. has been deposited as FRI Deposit No. 1259. Tables 2 and 3 list some characteristics of the above strains. To obtain glycoside-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 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 the 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 superjuvant, such as Claricel, and thereby the culture fluid and mycelial debris are separated. Inhibitors can be isolated from specific culture fluids by a variety of methods, including the following. a 20-100℃ under reduced pressure (10-50mmHg),
The culture liquid is concentrated to about 1/5 to 1/50 of the initial volume at a bath temperature of preferably 40 to 80°C. The concentrated extract is filtered or centrifuged and the clear liquid (or clear supernatant), if necessary after desalting beforehand, is lyophilized. b. From the culture broth (or the culture broth concentrated according to a)), 60 to 90% of a water-soluble organic solvent such as alcohol and ketone, preferably methanol, ethanol or acetone.
The inhibitor is precipitated by adding up to a content of . Since inert turbid substances precipitate at low concentrations of solvent, this precipitation operation is particularly suitable for fractional precipitation to remove undesirable 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-stable inhibitors, or by dialysis through suitable membranes in the case of low molecular weight inhibitors. The undesired turbid substances can be separated out by leaving them behind by means of a membrane, or by fractional precipitation (see b) or by adsorption of the turbid substances 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. According to the results of this experiment, 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,
Hyperglycemia is known to occur after ingestion of fruits, fruit juices or thiocholate, in which carbohydrates are absorbed by glycoside-hydrolytic enzymes (e.g. salivary and pancreatic amylases, maltase and sutucalase). 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 hypervascular 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 fluid in the stomach stimulates the production of gastric juice, 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 (atherios-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 form 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 fraction of a unit dose (40 times) 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. or 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. The separate, unitary parts that constitute the drug (whether in unit dosage form or neat) include, for example, tablets (including lozenges and granules), pills, dragees, capsules, It may be either a suppository or an ampoule. 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 of this class of glycoside-hydrolyzed oxygen inhibitors is extremely low. The active substances obtained from Examples 14 and 37 were tolerated up to a dose of 5×10 ⁵ 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 ⁵ AIU/Kg. The following examples illustrate methods of making inhibitors according to the invention. Example 1 Three one-volume Erlenmeyer flasks (sterilized at 121°C) containing 200 ml of culture medium with the following composition: 2% starch, 1% glucose, 0.5% NZ-amine, 1.0% yeast extract and _0.4 % CaCO3 (pH adjusted to 72 before sterilization) for 30 minutes before sterilization) and 1 ml of a preculture of strain SB2 (inoculated from a slant tube culture with oatmil agar and obtained in the same culture medium).
and the flasks were incubated at 28°C on a rotary shaker. After 5.5 days of culture time,
The contents of the three flasks were combined and the mycelium was separated and removed by centrifugation. 425 ml of supernatant containing 100 AIU/ml was obtained. The centrifuged supernatant was heated to 15-20 mmHg and approx.
It was concentrated to 60 ml using a rotary evaporator at a water bath temperature of 37°C. The viscous solution was poured into 8 volumes = 480 ml of ethanol with stirring. The precipitate formed was collected by centrifugation, redissolved in 60 ml of water, and dialyzed in distilled water for 6 hours. The dialysate was lyophilized. The yield was 1.6 g, containing 19×10 ³ AIU/g. Example 2 Using a batch according to Example 1, 440 ml of centrifugation supernatant containing 100 AIU/ml was obtained. 440 ml of this centrifuged supernatant was concentrated to 100 ml using a rotary evaporator. 8 volumes of the concentrate = 800ml
of ethanol with stirring and the precipitate was collected by centrifugation, washed twice with acetone and once with ether and dried in vacuo at room temperature. The yield was 2.2 g containing 15×10 ³ AIU/g. Example 3 Using a batch according to Example 1, 500 ml of centrifugation supernatant containing 120 AIU/mg was obtained after 5 days of cultivation. This 500 ml was directly freeze-dried. The yield is
It was 7.1g containing 8.3×10 ^-3 AIU/g. Example 4 3% glycerin, 3% soy flour and 0.2%
1 of 3 containing 200ml of culture solution with composition CaCO ₃
Erlenmeyer flasks (sterilization: 30 min at 121°C; pH 7.2 after sterilization) were each inoculated with a preculture of strain SB12 (from a slanted tube culture on Tzapetsk-peptone-casein agar and inoculated with identical 1 ml (obtained from a culture of 20%) was inoculated, and the flask was incubated for 3 days at 28° C. on a rotary shaker. After incubation, the contents of the flasks were combined and the mycelium was removed by centrifugation. 500 ml of supernatant containing 450 AIU/ml was obtained. The supernatant was treated with 250 g of ammonium sulphate in portions with stirring, and the mixture was then centrifuged for 10 minutes at 12,000 revolutions per minute.
Dissolve the precipitate in 100 ml of distilled water and add 4 volumes (=400
ml) of acetone was added with stirring. A well-settling precipitate was formed. The liquid was decanted and the precipitate was washed twice with acetone and once with ether and dried in vacuo. Yield: 13.4g containing 10×10 ³ AIU/g. Example 5 The precipitate obtained after precipitation with ammonium sulfate according to Example 4 was dissolved in 100 ml of water and dialyzed for 6 hours in distilled water. A dialysate was obtained, which was frozen and lyophilized. Yield: 80AIU/
1.5g containing mg. Example 6 When the mixture according to Example 4 was cultivated for 3 days at 32° C., a culture medium containing 350 AIU/mg was obtained after over-removal of the mycelia. Example 7 When the culture solution according to Example 1 was inoculated according to Example 4, a culture solution containing 270 AIU/ml was obtained after culturing at 28° C. for 3 days. Example 8 3% glucose, 3% soy flour and 0.2%
1 of 6 bottles each containing 100 ml of culture medium consisting of CaCO ₃
Erlenmeyer flask (sterilized: 121℃)
After sterilization, pH 7.2) was inoculated with 1 ml of a preculture of strain SB5 (obtained as in Example 1) for 30 min at 28°C on a rotary shaker. The cells were cultured for 4 days. The contents of the flasks were combined and the mycelium was separated off by centrifugation. The resulting 500 ml supernatant containing 150 AIU/ml was frozen and lyophilized. Yield: 6.9g containing 11×10 ³ AIU/g. Example 9 A preculture of strain SB11 (Example 4) was placed in 24 flasks each containing 120 ml of the mixture according to Example 4.
1.1 MIU/ml after centrifugation when inoculated and incubated for 5 days at 28°C
2.0 liters of supernatant containing . The 2 liters were concentrated to 200 ml using a rotary evaporator. Dispense 200 ml of the concentrate in 2 liters of distilled water at room temperature in Visking dialysis tubing [type 27/100FT,
The samples were dialyzed for 24 hours in Union Carbide Corporation. The outer medium containing the inhibitor was concentrated to 100 ml using a rotary evaporator and added dropwise to 900 ml of absolute ethanol with stirring. The almost inert precipitate that separated was centrifuged off and discarded, and the alcoholic supernatant was concentrated to 30 ml on a rotary evaporator. 1 ml of this concentrate contained 70 MIU/ml. For further purification, this solution was applied to an anion exchange column [Amberlite IRA410, acetate form, in 0.05M ammonium acetate, PH 7, 2.5
x 20 cm column] and the active fractions were combined and gel-filtered with water through a Sephadex ^R G75. The active fraction obtained by gel filtration was concentrated to 12 ml. 1 ml of this solution contained 150 MIU/ml. Mycelium (approx. 500ml) was mixed with 1 part of acetone and 2 parts.
Extracted twice with ether and once with ether and the extracts were combined and evaporated to dryness in vacuo using a rotary evaporator. The mycelium residue was vacuum dried at 20° C. and the resulting dry mycelium powder (approximately 51 g) was then extracted with 150 ml of DMSO for 2 hours at room temperature. After centrifugation (15000 rpm, 30 min), the evaporated acetone/ether extract was combined with the DMSO supernatant from the dried mycelium powder. Yield: 120ml containing 3MIU/ml. Example 10 When a 1 volume Erlenmeyer flask containing 120 ml of culture according to Example 1 was inoculated according to Example 8 and incubated for 6 days at 28° C. on a rotary shaker, 0.9 MIU/ml was obtained. A culture solution containing the following was obtained. Example 11 Five one-volume Erlenmeyer flasks (sterilized) each containing 120 ml of culture medium with the following composition: 2.5% starch, 0.5% glucose, 0.5% NZ-amine, 1.0% yeast extract and 0.4% _CaCO3 . 30 minutes at 121℃;
PH adjusted to 7.2 before sterilization) were each inoculated with 2 ml of a preculture of strain SB27 (obtained according to Example 1) and the flasks were incubated at 28°C on a rotary shaker for 4 hours. If cultured for 1 day, after combining the flasks and removing the mycelium by centrifugation,
500 ml of supernatant containing 70 AIU/ml was obtained. The centrifugation supernatant was frozen and lyophilized. Yield: 7.7g containing 3.5×10 ³ AIU/g. Example 12 When 2 ml of a preculture of mycelium SB18 (obtained according to example 1) was inoculated with a mixture according to example 1 and incubated for 3 days at 28° C., 1100 AIU/
ml, 0.17 MIU/ml and 1.0 SIU/ml were obtained. Example 13 Five experimental fermenters, each containing 8 liters of culture according to Example 1, were inoculated with 120 ml of the preculture (obtained according to Example 1) and incubated at 28° C. with stirring and aeration. When incubating for 65 hours, after combining the fermentation liquid and separating and removing the mycelium, the fermentation liquid
was gotten. Thirty liters of this centrifuged culture (0.57 MIU/ml, 6.2 SIU/ml and
8000 AIU/ml) was concentrated to 5 liters in vacuo at 20 mmHg at a bath temperature of 100°C, and 4 volumes of acetone (=20 liters) were added to the concentrate with stirring,
Then, remove the black sticky precipitate that is formed.
Collected by centrifugation at 6000 rpm for 30 minutes, the precipitate was dissolved in 2.5 liters of water and the black colored solution was moistened with Amberite IRA410.
(acetate form, pH 7) and stirred for 60 minutes with 500 g.
This mixture was centrifuged at 6000 rpm for 10 minutes to separate the supernatant and the amberite precipitate. In the same way, the supernatant was further stirred three times for 60 minutes each time with 500 g of amberite, and then further stirred with 500 g of amberite each time.
Stirred with 500 g overnight (about 15 hours). After this treatment, the supernatant exhibited a pale yellow color, while the black cloudy pigment was bound to the ion exchanger. The collected amberite residue was washed twice with 1.5 liters of water and the wash water was combined with the supernatant containing the inhibitor. The supernatant liquid combined with the washing water was heated to 15 mmHg and 80°C.
The mixture was concentrated to 1 liter in a rotary evaporator at a bath temperature of 100 liters and then added dropwise to 10 liters of acetone with vigorous stirring. This resulted in a white fluffy precipitate which was separated, washed with acetone and ether and dried in vacuo. Yield: 150 g of white powder containing 1×10 ⁵ AIU/g and 450 SIU/g. Example 14 In the culture medium according to Example 1, other sugars or sugar alcohols were substituted for glucose and in each shake flask containing 120 ml of culture medium strain SB18 (produced according to Example 1) was added. When inoculating 1 ml of a preculture of
obtained after culturing for 3 or 4 days.

[Table] Example 15 3% soybean flour, 2% starch, 1% glucose and
When inoculated into a medium consisting of 0.2% _CaCO3 and cultured according to example 14, after 4 days of fermentation
A culture fluid containing 5600 AIU/ml was obtained. Example 16 When a morphological variant of SB18, strain SB18/5, was used to inoculate and culture a mixture according to Example 12, a culture containing 27400 AIU/ml was obtained after 4 days of fermentation. Strain SB18/5 was distributed to Central Bureau Bor Schimmel Cultures in Baarn with CBS No.
It was deposited under the number 613.71. Example 17 When a morphological variant of SB18, SB18/4, was used to inoculate and culture a mixture according to Example 12:
After 4 days of fermentation, a culture solution containing 13900 AIU/ml was obtained. Strain SB18/4 was distributed to Central Bureau Bor Schimmel Cultures in Baarn with CBS No.
It was deposited under the number 612.71. Example 18 2% starch, 1% glucose, 0.3% glycine,
0.25% corn soaking liquid, 0.4% soy flour, 0.1%
NaCl, 0.1% _{K2HPO4 ,} 0.0.1% _FeSO4 and 0.01
When a culture solution with a composition of % _CaCO3 was inoculated and cultured according to Example 11, after 3 days of culture
A culture fluid containing 2900 AIU/ml was obtained. Example 19 Inject the strain into two flasks of the mixture according to Example 1.
When inoculating 1 ml of a preculture of SB46 and culturing for 4 days at 28°C on a rotary shaker,
250 ml of supernatant containing 250 AIU/ml was obtained after centrifugation. 250 ml of this supernatant was treated with 150 g of ammonium sulphate in portions with stirring, and the mixture was then centrifuged at 10,000 rpm for 15 minutes.
The residue was dissolved in 12 ml H ₂ O and dialyzed in distilled water for 3 hours. 20 ml of dialysate were precipitated with 6 volumes of acetone (=120 ml) in an ice bath and the precipitate was separated, washed with acetone and ether and then dried in vacuo. Yield: 0.28g containing 220×10 ³ AIU/g. Example 20 A 1 volume Erlenmeyer flask containing 120 ml of culture according to Example 1 was inoculated with 2 ml of a preculture of strain SE5 (produced according to Example 4) and placed on a rotary shaker at 28°C. When cultured above for 7 days, a culture solution containing 0.09 MIU/ml was obtained. The mycelium was treated with 50 ml of acetone and
rrax homogeniser) [Jahnke & Kunkel, Staufen, Breisgau (Messrs.
Janke and Kunkel, Staufen, Breisgan)] for 1 minute and the mixture was then
Centrifugation was performed at 3000 rpm for 10 minutes. The residue was extracted again in the same manner with 50 ml of acetone, then once with 50 ml of ether, and the three extracts were combined and heated to approximately 10-20 mm Hg and 37
It was concentrated to almost dryness in a rotary evaporator at a water bath temperature of .degree. The mycelial residue was dried in vacuo and then treated with 15 ml of dimethyl sulfoxide, homogenized for 2 minutes using an Ultraturax and stirred (magnetic stirrer) for 2 minutes.
Time extracted. The mixture was then centrifuged at 20000 rpm for 30 minutes. The DMSO extract was then separated from the extracted mycelia by decanting, added to the acetone/ether extraction residue, and the mixture was stirred (by magnetic stirrer) for about 30 minutes and again
Centrifugation was performed at 20000 rpm for 10 minutes and the supernatant was tested as mycelial extract. 0.14 MIU/
ml. Example 21 Two shake flasks according to Example 1 each containing 160 ml of input were injected with strain SE21 ((Example 1
200 ml of supernatant containing 360 AIU/ml after centrifugation if 1 ml of preculture is inoculated and incubated
was gotten. Add 100g of ammonium sulfate to the centrifuged supernatant.
was added in portions with stirring and centrifuged, the precipitate was dissolved in 20 ml of water and the solution was precipitated with 2 volumes (approximately 40 ml) of acetone. The precipitate was washed with acetone and ether and dried in vacuo. Yield: 2g containing 25×10 ³ AIU/g. Example 22 When a preculture of strain SE21 was used to inoculate and cultivate a mixture according to Example 13, a culture broth containing 140 AIU/ml was obtained after 65 hours of fermentation. Example 23 1 containing 120 ml of culture medium with the composition according to Example 4
When Erlenmeyer flasks were inoculated with 1 ml of a preculture of strain SE39 according to Example 1 and cultured according to Example 1, a culture containing 50 AIU/ml was obtained after only 3 days of fermentation. Obtained. Example 24 When using glycose instead of glycerin in the mixture according to example 23, after 3 days 60 AIU/ml
A culture fluid containing the following was obtained. Example 25 If the culture medium according to Example 1 was modified in the mixture according to Example 23 and the starch was replaced by glucose, a culture medium containing 140 AIU/ml was obtained after 3 days of cultivation. Example 26 120 ml of culture solution with the following composition: 3% glucose, 0.5% NZ-amine, 0.1% yeast extract and 0.4% _CaCO3
Add 1 ml of a preculture of strain SE39 (prepared according to Example 4) to five one-volume Erlenmeyer flasks (sterilization: 30 minutes at 121°C; pH 7.2 before sterilization) each containing When inoculated and cultured for 3 days on a rotary shaker at 28°C, the yield after centrifugation was 160 AIU/
500 ml of supernatant containing ml was obtained. 6 volumes (=3 liters) of acetone were added to 500 ml of the supernatant with stirring to cause precipitation, and the light brown precipitate was separated, washed with acetone and ether, and dried in vacuo. Yield: 3.5g containing 13.5×10 ³ AIU/g. Example 27 An Erlenmeyer flask containing 120 ml of culture according to Example 4 is inoculated with 1 ml of a preculture of strain SE50 (preculture prepared according to Example 4) and again according to Example 5. When cultured, 5
After a day, a culture solution containing 1.0 SIU/ml was obtained. Example 28 When using the culture medium according to Example 1 and operating according to the method of Example 27, after 5 days of culture,
26000AIU/ml and 0.18MIU/ml and
A culture fluid containing 1.8 SIU/ml was obtained. Example 29 When the method according to Example 28 is carried out using a 200 ml flask input, after 5 days of incubation
24500AIU/ml and 0.18MIU/ml (and
A culture solution containing 2.1 SIU/ml) was obtained. Example 30 2% starch, 1% glucose, 0.3% glycine,
0.25% corn soaking liquid, 0.4% soy flour, 0.1%
NaCl, _{0.1K2HPO4 ,} (0.01% _MgSO4 ) 0.01%
When the method of Example 27 was followed using a culture solution with a composition of CaCO ₃ and 0.001% FeSO ₄ , a culture solution containing 12900 AIU/ml was obtained after 5 days of fermentation. Example 31 When a sugar other than a sugar alcohol is used in place of glucose in the culture solution according to Example 1, and the solution is inoculated and cultured according to Example 27, a culture solution containing the following amylase inhibitor concentration: was gotten.

[Table] Example 32 When glucose was used instead of glycerin in the mixture according to Example 27, a culture fluid containing 330 AIU/ml was obtained. Example 33 When glucose was used instead of starch in the mixture according to Example 27, a culture fluid containing 660 AIU/ml was obtained. Example 34 If other complex nitrogen sources were used instead of NZ-amine in the mixture according to Example 28, a culture fluid containing the following amylase inhibitor units was obtained after 3 days of fermentation. Alternatives AIU/ml Soy flour 13500 Fish soluble components 16800 Tryptone 25200 Meat extract 32700 Pharmamedia 23100 Example 35 Morphological variant of strain SE50, i.e. strain SE50/12
When inoculating the mixture according to Example 28 using
After 3 days of fermentation, a culture fluid containing 51500 AIU/ml was obtained. Example 36 Culture medium according to Example 1 but containing 0.1% by volume of Bayer E100 antifoam 8
Each of the 5 experimental fermentations, each containing a liter, was inoculated with 120 ml of a preculture of strain SE50 (obtained according to Example 1) and incubated for 65 hours at 28°C with stirring and aeration. , when the fermentation mixture is combined and the mycelium is separated and removed, 13000 AIU/
ml and 24 liters of culture containing 6 SIU/ml were obtained. The fermentation liquid was vacuumed at 20 mmHg and
It was concentrated to 1 liter by evaporation at a bath temperature of 100°C. 15000 rpm of the black colored concentrate
After centrifugation for 60 minutes _at 20 ml of fraction was collected in a fraction collector. Pale yellow fraction containing inhibitor (total amount
300 = 6 liters) and 1
It was concentrated to a liter and added dropwise to 10 liters of absolute alcohol with vigorous stirring, whereby a feathery, almost white precipitate separated out. The precipitate was separated, washed with absolute alcohol, then ether and dried in vacuo. Yield: 60 g of white powder containing 3 x 10 ⁶ AIU/g and 400 SIU/g Example 37 The majority of the inert turbid substances, especially the dark colored substances, are also separated off by precipitation with an equal volume of methanol. I was able to do it. For this purpose, 26 liters of centrifuged culture fluid [produced according to Example 36 but with 0.02% by volume of Bayer silicone E antifoam] (43000 AIU/ml) were collected. 20mmHg
and concentrated to 1.2 liters by vacuum evaporation at a bath temperature of 100°. The deep black solution was treated with an equal volume (1.2 liters) of methanol while stirring, which produced a feathery black precipitate. It was passed through a vertically slotted filter and the brown liquid (2.2 liters) was added dropwise into 10 liters of dry spirit with vigorous stirring. A light brown precipitate formed which was separated, washed with acetone and ether and dried in vacuo. Yield: 80g white powder containing 3700AIU/mg. Example 38 2 ml of a preculture of strain SE55 (prepared according to Example 4) in a 1 liter Erlenmeyer flask containing 120 ml of culture according to Example 4
After inoculating and culturing the culture on a rotary shaker at 28°C for 6 days to separate and remove the mycelium,
30AIU/ml and 0.15MIU/ml and 0.5SIU/ml
A culture fluid containing the following was obtained. When the mycelium was extracted according to Example 19,
An extract containing 15 AIU/ml, 0.17 MIU/ml and 0.58 SIU/ml was obtained. Example 39 When operating according to Example 39 using a culture medium according to Example 1, a culture medium containing 60 AIU/ml, 0.16 MIU/ml and 0.65 SIU/ml and 30 AIU/ml
and a mycelial extract containing 0.16 MIU/ml and 0.33 SIU/ml was obtained. Example 40 If a culture according to Example 38 is inoculated with a slant tube culture of strain SS26 on oatmill agar and the culture is grown for 5 days according to Example 39, then Example 20 Therefore, after extracting the mycelia and separating the culture fluid, an extract containing 0.14 MIU/ml was obtained. Example 41 When working according to Example 40 using a culture of strain SS45, a mycelial extract containing 0.1 MIU/ml was obtained. Example 42 When the operation according to Example 40 was carried out using a culture of strain SS53, a culture solution containing 0.05 MIU/ml and a mycelial extract containing 0.07 MIU/ml were obtained. Example 43 0.5% bactopeptone, 0.5% meat extract, 0.2% yeast extract, 0.03% casein hydrolyzate, 1% i-
Inositol, 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. When 120 ml of the culture was placed in a 1 liter Erlenmeyer flask and inoculated with a slanted agar culture of strain SS51 (prepared according to Example 1), it was incubated on a rotary shaker for 7 days at 28°C. 0.072 MIU/ml after culturing with
A culture fluid containing 0.054 MIU/ml (prepared according to Example 20) was obtained. Example 44 Experimental method to demonstrate the effects of glycoside-hydrolase inhibitors in mice and humans Mice (n=
6), starch, maltose, or sucrose was orally administered at 2.5 g/kg 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 the blood glucose level from the 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 six groups of 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. Glycoside-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.

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Claims (1)

[Claims] 1. Glycoside-hydrolase inhibition, which is characterized by culturing glycoside-hydrolase inhibitor-producing bacteria belonging to the family Actinoplanaceae, and extracting the glycoside-hydrolase inhibitor from the obtained culture. Method for manufacturing the agent.