FI58158B - FOERFARANDE FOER SEPARERING OCH RENGOERING AV MIKROBIOLOGISKT FRAMSTAELLDA AMINOSOCKERDERIVAT - Google Patents

Description

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Pmi »och rcgiltwrstyrtlMn 'Amakan uttajd odi utl ^ kriftan puMfctrad 29 · 00. ου (32) (33) (31) atuolkws — 8 «fM prtorlt« t 22.09 * 73 26.lO.73, 21.II.73 Federal Republic of Germany-Forbundsrepubliken Tyskland (DE) P 23U7782.9 p 23U7782.9, p 23U7782.9 (71) Bayer Aktiengesellschaft, Leverkusen, Federal Republic of Germany-Förbunds-republiken TiysklandiDE) (72) Werner Frommer, Wuppertal-Elberfeld, Bodo Junge, Wuppertal-Barmen,

Uwe Keup, Wuppertal-Elberfeld, Lutz Muller, Wuppertal-Elberfeld,

Walter Puls, Wuppertal-Elberfeld, Delf Schmidt, Wuppertal-Vohvin-kel, Federal Republic of Germany-Forbundsrepubliken iyskland (DE) (7U) Oy Kolster Ab (5U) Method for the separation and purification of microbiologically prepared amino-sugar derivatives - Förfarande för separering microbiological framställda aminosockerderivat (62) Divided separated from the application 27UO / 7U (patent 56698) -Avdelad fr & n trap 27UO / 7U (patent 56698)

It is already known that some actinomycetes, in particular microorganisms of the family Actinoplanaceae, develop inhibitors of glycoside hydrolases, mainly enzymes that degrade gastrointestinal carbohydrates. One such group of inhibitors is relatively temperature resistant and resistant to acids and alkalis at room temperature. These inhibitors are amino sugars in chemical structure.

Most of the inhibitors in this group are very potent amylase inhibitors, but as sucrase inhibitors only medium or weak (see Finnish Patent Publication No. 49,774).

The invention relates to a process for the separation and purification of microbiologically prepared amino-sugar derivatives with which amino sugars have the general formula 58158 □ n

HQ OH OL

B

HO - (y N H VR

> == / H) - (CH 2 OH h oh oh where R is a saccharide chain with 1-7 hexose units.

The process according to the invention is characterized in that microorganisms belonging to the genus Actinoplanes are aerobically separated from the mycelia obtained by culturing the mycelium under aerobic conditions and in an aqueous nutrient medium and possibly with activated carbon at pH 1-3. b) the filtrate bound to the ion exchanger is either desorbed in its entirety with ammonia and then fractionated into the individual components in a manner known per se using gel filtration or cellulose column chromatography eluted, and eluted separately from the solvent, eluted with dilute acid,

As mentioned above, these new amino sugar derivatives are potent inhibitors of gastrointestinal glycoside hydrolases. In this case, the lower members, where R is a saccharide chain with 1 or 2 hexose units, have a predominantly strong sucrase inhibitory effect, while the higher members strongly inhibit predominantly amylase.

The amino sugar derivatives prepared by the process of the invention have a strong specific sucrase and / or amylase inhibitory effect.

Some new amino sugar derivatives have a much greater in vivo inhibitory effect than their in vitro inhibitory effect.

58158

It is also particularly surprising that the lower members of the homologous series prepared according to the invention inhibit glucose resorption in vivo. Based on these surprising properties, the preparation of new amino sugar derivatives is important from a pharmacological point of view.

Strains of the genus Actinoplanes belonging to the family Actinoplanaceae belonging to the actinomycetes are used to carry out the method according to the invention. Examples are the strains Actinoplanes spec. SE 50 (CES 961 70), SB 10 (CBS 957 70) and SE 82 (CBS 615 71). These microorganisms have already been described in German Application Publication No. 2,064,092 and have been deposited with numbers in parentheses at Centralbureau vor Schirmnelcultures (Baarn / HoHanti).

Strains SE 50/13 (CBS 614 71) and SE 50/110 (CBS 674 73) proved to be particularly suitable for the overall yield of the amino sugar derivatives prepared according to the invention and / or for the size of the residue R of the amino sugar derivatives formed as a mixture during fermentation. The descriptions of both strains are quite similar to the parent strain SE 50. These strains were obtained without the use of mutagens by natural selection from strain SE 50.

The culture broth to be treated in the process of the invention is obtained by culturing microorganisms belonging to the genus Actinoplanes in an aqueous nutrient medium which, in addition to the carbon source, contains a nitrogen source, salts and antifoams at conventional concentrations which can vary within wide limits.

Carbon sources are mainly carbohydrates, especially starch, maltose, glucose and mixtures of two or three such substances, and even more complex mixtures, such as malt extract.

Nitrogen sources which can be used in microbiology are the usual complex mixtures, such as, for example, casein hydrolyzate, yeast extract, peptone, fishmeal, fish extracts, corn steepwater, meat extract and also mixtures thereof as well as amino acids and / or ammonium salts.

Cultivation is performed with aerobically conditioned vibration cultures or tank cultures. The quality and concentration of the carbon source and the fermentation strain 58158 affect the size of the residue R in the final product to such an extent that individual members of the homologous series or at least members of a very narrow region of the homology series can be produced almost selectively. In this case, it has been found that nutrient solutions with a starch content of more than 2% form primarily amino-derivative derivatives with 4 to 7 hexose units, and that strain SE 50/13 (CBS 614 71) is particularly suitable for this production. However, in some cases it is sufficient to add 0.1 to 3%, mainly 0.5 to 2% of starch to nutrient solutions containing, for example, 3.5% glucose as the basic carbon source, in order to obtain mixtures of amino sugar derivatives containing 4 to 7 hexose units. It has further been shown that in starch-free nutrient solutions and in particular by the addition of maltose to the nutrient solution, in particular strain SE 50 (CBS 961 70) - amino sugar derivatives containing mainly 2-3 hexose units can be obtained. Particularly suitable in the process according to the invention for the preparation of an amino sugar derivative in which R is glucose are nutrient solutions in which only glucose is the carbon source. There is an excess of glucose in the 3rd nutrient solution, more long-chain amino sugar derivatives are also formed during long fermentation. With certain limitations, this can be attributed to the fact that during fermentation, the depletion of nitrogen sources occurs simultaneously with the depletion of glucose.

If only glucose is to be used in the nutrient solutions and maltose is added as the carbon source, a predominantly amino sugar derivative with 2 hexose units is obtained. Pure maltose can then also be replaced by cheaper mixtures, such as Maltzin, with a naturally occurring malt extract produced by Diamalt AG, Munich. Corresponding to the maltotriose content, the following higher homology is also formed. Strain SE 50/110 proved to be particularly suitable for the preparation of lower homologues, which in optimal nutrient solutions gives lower homologues in approximately twice as high yields as strain SE 50/13.

s 58158

The other composition of the nutrient solution, in particular the concentrations of nitrogen sources and the salt composition, can vary widely during fermentation.

The nutrient solutions are sterilized in the usual way. The pH values of the nutrient solution are between 5.0 and 8.5, mainly between 6.0 and 7.8. Incubation temperatures are between 15 and 45 ° C, mainly between 24-32 ° C. When preparing higher homologues using strains SE 50 and SE 50/13, a higher temperature, e.g.

28 ° C, is more preferred; when preparing lower homologues using strains SE 50 and SE 50/110, a lower temperature, e.g. 24 ° C, is more preferred. The cultivation time is 1-8 days, mainly 2-6 days. A longer culture time favors the formation of higher homologues, especially when carbohydrates are present in excess. The end point of fermentation is determined by determining the inhibitory activity in the enzymatic inhibition assay and the composition by plate chromatography.

The resulting amino sugar derivatives are isolated and purified from microbiological culture broths.

If the starting solutions are very dark colored, they are decolorized with activated carbon at acidic pH (pH = 1-3] with an adsorption resin, e.g. Lewapoi® Ca 9221 (nonspecific polystyrene-based resin], grain size 0.35 AGr, before adsorption of the substances according to the invention. ], at a pH between 2 and 7, mainly between pH 2 and 3. In the acidic region, activated carbon binds primarily dyes, whereas Lewapol does not adsorb the amino sugar derivatives according to the invention in a neutral or acidic environment.

—Separation of small-molecule amino sugar derivatives with inhibitory activity from neutral saccharides can also be achieved by chromatography on cellulose-based ion exchangers, mainly with phosphocellulose (Phospho-Cellulose, Fos. in particular 5-10 mM, with a pH between 2.5 and 8, mainly between pH 5 and 6. A prerequisite for efficient fractionation is that the salt contents of the preparations to be fractionated 58158 are as low as possible, while the dyes interfere with the reduction.

To prepare individual members of the homologous series of inhibitory amino sugar derivatives of the invention as pure products, the prepurified preparations are chromatographed on a suitable molecular sieve, e.g. Bio-Gel®P-2 (polyacrylamide, Bio-Rad, Munich), and the eluate fractions are examined by plate chromatography. Fractions containing pure amino sugar derivatives are combined, rechromatographed and lyophilized after concentration or precipitated with organic solvents.

According to their chemical nature, the substances prepared by the process of the invention belong to carbohydrates. They form a series of homologues which - are based on the following amino sugar derivative 9 ^ 33 ^ 13 * ^ ^ ** hexose), CH2OH h oh oh

The higher members thus differ from the basic body in that they each contain one monosaccharide unit, mainly a hexose unit, in particular a glucose unit, more than in the previous member of the series.

An example of the structural principle of a homologous series is the case where the saccharide chain is formed by 1-7 glucose units. (The formula then shows a saccharide chain with n = 1-7 glucose units):

OH, 0H CH3 OLOH

CH 2 OH H OH OH V OH OH n

The basic body of the series corresponds to the case where n = 1 and is referred to as "Component II". Each member of the series differs from the next lower or next higher member in that it has one more or less glucose unit. One compound that is 7 58158 glucose units higher than "Component II" is then called "Component III", a compound larger than 2 glucose units is called "Component IV", etc.

All of these compounds are known to form glucose in complete acid hydrolysis and an unspecified so-called "Component I".

"Component II" can be characterized in terms of its physicochemical properties, structure and reactivity as follows: Component II is a colorless, amorphous solid which is highly soluble in water, absorbed in dimethyl sulfoxide and methanol.

- (a) Plate chromatography

Propellant: ethyl acetate / MeOH / h 2 O 10: 6: 4 (DC-Fertigfolien = finished membranes for plate chromatography).

I. DC-Fertigf olien F 1 500 Kieselgel, Schleicher & Schiill

Rf values: II 0.46 maltose: 0.50 Gluose: 0.65 II. DC-Fertigplatten F 254 Kieselgel, Merck

Rf values: II 0.47 maltose: 0.54 Gluose: 0.66

Component II is made visible on silica gel plates, most preferably AglN 2 / NaOH spray reagent 11a. The brownish-black color develops even at room temperature or when slightly warmed.

(b) Gas chromatography:

For gas chromatographic analysis, component II is silylated with <X- and β-D-glucose or also using sucrose as an internal standard in a mixture of pyridine M), trimethylchlorosilane (0.5) and N-methyl-trimethylsilyl- trifluoroacetamide (1).

Column: 1Θ0 cm, glass, packing 3% SE 30 / Chromosorb WAW

Temperatures: Mouth piece 3 00 ° C

Detectors 300 ° C

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Oven 220 ° C isothermal until <X and / or VD glucose peaks have eluted; then a temperature program of 15 ° C / min up to 300 ° C.

Detectors; F I D (Flammen Ionisations Detector)

Gases: Carrier gas: N2 40 ml / min

Fuel gas: Air 80 ml / min H2 20 ml / min

Retention times: α-D-glucose 3 min / VD-glucose 4 min

Sucrose 12-13 min II 16-17 min c) Rotation angle

The non-crystallized sample II obtained by concentrating the methanol solution of - II was dissolved in water and the specific rotation was determined: / <* 70 = + 134.3 °.

d) IR spectrum Low-spoken, poorly structured IR spectrum when using KBr. The main absorption bands at the valence oscillation sites of 0-H and C-0.

e) NMR spectrum: CD 3 OD / 220 MHz: (Figure 1) £>, ppm Multiplicity relative intensity

1.3 doublets} 0 = 6.5 Hz 3 H

2.3 "triplets"; 0lU.J2 8-10 Hz 1 H

3.15 "triplet"; u-J2 7-9 Hz 1H

3.3-3.9 signals cannot be distinguished by 12 H

4.13 AB system; 0 = 12 Hz 2 H _

4.48 ^ doublets; 0 = 7 Hz ^ ^ H

and 5.1J doublets; 0 = 2.5 Hz j 4.9 singlet 11 H protons exchanged against deuterium

5.0 doublets; 0 = 2-3 Hz 1 H

(weakly distinguished)

5.8 doublets; 0 = 3-4 Hz 1 H

(weakly distinguished) _

33 H

9 58158 f] Acetylation of component II:

Component II is reacted in a 1: 1 mixture of acetic anhydride and pyridine at room temperature to give the deca-acetyl derivative (molecular weight: 903). If the reaction is carried out in a 1: 1 mixture of glacial acetic acid / acetic anhydride using catalytic amounts of h 2 SO 4, it can be shown by mass spectroscopy that, in addition to the decaacetyl derivative, an undecaacetyl derivative has also been formed (molecular weight: 945).

□ NNR spectrum of the first acetyl derivative: (Figure 2)

MS spectrum of the deca-acetyl derivative:

Molecular peak: 903 (relative intensity 2.5%)

Base peak: 843

Important fragment peaks in the above measurement range: 844 (55% rel. Intensity) 784 (36% rel. Intensity) 783 (34% rel. Intensity) 759 (35% rel. Intensity) 556 (36% rel. Intensity) 496 (37 I rel. Intensity) 436 (29% rel. Intensity) g) Methylation of component II

Methylation of component II with a CH 2 Cl 2 / NaH combination in dimethyl sulfoxide according to Hakomori gives the decamethyl derivative of component II as a major product, also a small amount of the undecamethyl derivative.

Mass Spectrum:

Molecular peak: 623 (relative intensity 6.1%)

Base peak: 535 2nd molecular peak: 637 (relative intensity 0.2%)

Based on the spectroscopic values and chemical properties, the following structural formula is obtained for component II: 10 581 58

OH OH CH 2 MgOH

HO / \ N - / Λ O / \ —OH

CHgOH H OH 'OH OH OH

Component II is a mixture of the o (and?) Forms, as shown in particular by NMR spectra.

The next member of the homologous series, "Component ^ II Im ^ 25H43 ° 1 θ1 ^ 00 structural formula

OH OH CH3 CH2OH CH2OH

-Q- ° ° -Q- “

CHjOH H OH OH OH OH OH OH OH

Component III has the following physical and chemical properties:

It is a highly water-soluble amorphous solid product, a) Plate chromatography

Driving fluid: Ethic ester / MeOH / H 2 O 10: 6: 4 I. DC-Fertigfolien F 1500 Kieselgel (Schleicher & Schull): _

Rf values: III 0.35 maltose: 0.50 II. DC-Fertigplatten F 254 Kieselgel (Merck):

Rf values: III 0.33 maltose: 0.54

Component III is obtained with AgNO 2 / NaOH oral reagent already at room temperature or by slightly heating the plates with a brownish-black color spot.

11 58158 b) IR spectrum Slightly telling, poorly separated IR spectrum when using KBr, main absorption bands in the valence oscillation range of 0-H and C-0 (3,700 to 3,100 cm -1 or 1,180 to 950 cm -1) ).

c) Rotation angle fo 7 in H 2 O: + 147.2 ° d) NMR spectrum of III in D 2 O / 220 MHz: (Figure 3) e) Methylation according to Hakomori:

Methylation of III with CH 2 Cl 2 and NaH in DMSO as the main product also gives a 14-fold methylated product.

f) Mass spectrum of the methylation product

The molecular peak at Θ27 (1.5% relative intensity) corresponds to the gross formula ^ g ^ gNO ^ g. (At a relative intensity of 0.1, Θ41 has a second molecular peak.) The major fragment peaks are: 739 (27% relative intensity) 592 (3.7% relative intensity) 535 (30% relative intensity) 388 (9% relative intensity) relative intensity) 386 (13% relative intensity) 284 (13% relative intensity) 187 (12% relative intensity) 171 (40% relative intensity) 101 (34% relative intensity) 88 (25% relative intensity) intensity) 75 base peak.

Component IV is the next higher member of the homologous series. The compound decomposes in acid partial hydrolysis to components II and glucose in a molar ratio of 1: 2.

Thin layer chromatography:

Propellant: n-butanol / ethanol / water = 50:30:20 DC-Fertigplatten F 1500 Kieselgel (Schleicher & Schull): R,. .values: IV: 0.41-0.46 glucose, 2 5S158

The inhibitory effect of higher homologues, especially components V-VIII, on sucrase is very much lower and on pancreatic Οξ-amylase very much stronger than that of the lower members of the series (components II-IV).

When hydrolyzing higher components, lower homologues formed as intermediates can always be shown among the products; cleavage products also include glucose and maltose.

Thin layer chromatography:

Driving fluid: n-butanol / ethanol / water = 50:30:20 DC-Fertigplatten F 1500 Kieselgel (Schleicher S Schull):

Glucose_number °: V: ° '30 '° '34 VI: 0.21 - 0.23 VII: 0.14 - 0.16 VIII: 0.09 - 0.11

The specific inhibitory activities of inhibitory amino sugar derivatives are determined by in vitro enzyme inhibition assays.

Amylase test

An amylase inhibitor unit (1 AIY) is defined as the amount of inhibitor capable of inhibiting two amylase units by 50%. One amylase unit (AY) is the amount of enzyme which, in one minute under the experimental conditions mentioned below, cleaves 1 to 1 equivalent of glucosidic bonds in starch. The JuVal of the cleaved bonds is determined colorimetrically as the YuVal of the reducing sugars formed with dinitrosalicylic acid and is expressed as pVal maltose equivalents using the maltose reference curve. To perform the experiment, to 0.1 ml of amylase solution (20-22 AY / ml), add 0-10 μg of inhibitor or 0-20 μl of test solution in 0.4 ml of 0.02 molar mixture of sodium glycerophosphate buffer. / 0.001 mole of CaCl 2 at pH 6.9, and the mixture is allowed to equilibrate in a water bath at 35 ° C for about 10-20 minutes. It is then incubated for five minutes with 0.5 ml of a 1% starch solution preheated to 35 ° C (Stärke, löslich.

Merck, Darmstadt, No. 1252) at 35 ° C and then add 1 ml of dinitrosalicylic acid reagent (according to P. Bernfeld,

Co lowi c k-Kap lan, Meth. Enzymol., Band 1, page 149). To color the batch, heat the batch with a boiling water bath for five minutes, then cool and add 10 ml of distilled water. The extinction at 540 nm is measured against an amylase-free blank prepared in a similar manner. To interpret the test result, the amylase reference curve prepared previously is read after the addition of inhibitor, the amylase activity still effective, and from this the percentage inhibitory effect of the amylase used is calculated. The percentage inhibition is given by the equation jug inhibitor + AY + + + calculated on a dry matter basis ++ as a function of the AY of the same series of uninhibited batches, the 50% inhibition point is read from the curve and converted to AIY / mg inhibitor.

Sakkaraasikoe

The sucrase inhibitor unit (SIY) is defined as the amount of inhibitor that inhibits two sucrase units by 50%. One unit of sucrase (SY) is the amount of enzyme which, under the experimental conditions specified below, breaks down 1 μmol of sucrose into glucose and fructose in one minute. The molar amount μ of glucose formed is determined by the glucose oxidase reaction under conditions in which sucrose is no longer degraded by sucrase. To perform the assay, 0-20 pg of inhibitor or 0-20 μl of test solution and 0.1 molar sodium maleate buffer pH 6.0 are added to 0.05 L of sucrase solution adjusted to 0.12. the volume is 0.1 ml. Equilibrate for 10 minutes at 35 ° C and then add 0.1 ml of a 0.05 molar solution of sucrase preheated to 35 ° C in 0.1 molar sodium maleate buffer pH 6. Incubate for 20 minutes at 35 ° C and stop the sucrase reaction by adding 1 ml of glucose. -

1) Sucrase dissolved from porcine small intestinal mucosa (B. Borgström, A. Dahlquist. Acta Chem. Scan. 12, (1958), page 1997). Diluted with 0.1 M sodium maleate buffer pH

6.0 with a corresponding SY concentration.

Incubation of 1 to 5,815 8 oxidase reagents is continued for 30 minutes at 35 ° C.

1 ml of 50% H2SO4 is then added and the measurement is carried out at 545 nm against the corresponding test value. To interpret the test results, the percentage inhibition of the sucrase used is calculated and the value obtained from the 50% inhibition point is converted to SIY / g or SIY / liter by means of a glucose comparison curve.

I test your East

The Maltase Inhibitor Unit (MIY) is defined as the amount of inhibitor that inhibits two maltase units by 50%.

One unit of maltase (MY) is the amount of enzyme which, in one minute, under the experimental conditions mentioned below, degrades 1 μmol of maltose into 2 μmol of glucose. The molar amount μ of glucose formed is determined by the glucose oxidase reaction under conditions in which maltose is no longer degraded by maltase. To perform the experiment, 0.05 ml of 2) a maltase solution with a MY adjusted to 0.060 to 0.070, 0-20 mg of inhibitor or 0-20 ml of test solution is added, and then 0.1 mol of sodium maleate buffer pH 6.0 is added until the volume is 0. , 1 ml. Equilibrate for 10 minutes at 35 ° C and then add 0.1 ml of a 0.05 molar maltose solution preheated to 35 ° C in 0.1 molar sodium maleate buffer (pH 6.0). Incubate for 20 minutes at 35 ° C and stop the maltase reaction by adding 1 ml of glucose oxidase reagent, and continue germination for 30 minutes at 35 ° C. Then 1 ml of 50% H2SO4 is added and the measurement is performed at 545 nm against the corresponding blank value.

1) Glucose oxidase reagent is prepared by dissolving 2 mg of Gluose oxidase (Boehringer No. 15423) in 100 ml of 0.565 moles of Tris-HCl buffer pH 7.0 and then adding 1 ml of detergent solution (2 g Triton X 100 + Θ g 95 % ethanol pa), 1 ml of a solution of dianisidine (260 mg of o-dianisidine HCl in 20 ml of H 2 O) and 0.5 ml of a 0.1% aqueous solution of peroxidase (Boehringer No. 15302 ).

2) Maltase dissolved from porcine small intestinal mucosa (B. Borgström, A. Oahlquist, Acta Chem. Scand 12, (1958), page 1997). Diluted with 0.1 mol of sodium maleate buffer (pH 6.0) to a corresponding MY concentration.

3) Preparation of glucose oxidase reagent: as described in the sucrase test (p. 19, footnote 2).

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To interpret the test results, the inhibitory effect of the maltase used is calculated as a percentage and the value obtained from the 50% inhibitory site is converted to MIY / g or MIY / liter by means of a glucose comparison curve.

The results of in vitro enzyme inhibition experiments using private homologues of the amino sugar derivatives of the invention and certain homologous mixtures are summarized in the following table, in which the hexose units represent glucose units: n = number of hexose units AIY / g SIY / g MIY / g

Amino-sugar derivative with n = 1 300 000 30 000 5 000 n = 2 300 000 6Θ 000 15 000 n = 3 1 400 000 21 000 5 000 n = 4-6 17 500 000 Θ 500 n = 5-7 30 000 000 2 500

The following tables compare the above in vitro effect of glucose units (n) on sucrase and β-amylase inhibition in vivo.

In vitro sucrase inhibition SIY / mg ED50 mg / kg 1 30 3.0 2 68 0.21 3 21 2.65 4-6 6.5 15.30 5- 7 2.5 - 52.00 58158 16 C-amylase inhibition in vitro in vivo AIY / mg ED50 mg / kg 1,300 0.76 2,300 1.60 3 1400 1.61 4-6 17500 1.42 5-7 30000 1.00

Glucose units «C-amylase inhibition Number of sucrase inhibition (n) AIY / mg SIY / mg 4 67,000 7.0 5 57,000 3.5 6 42,000 1.2 7 24,000 0.06

As can be seen from the tables, as the molecular weight of the homologous series increases, the specific inhibitory activity against pancreatic β-amylase increases sharply; thus, for compounds with n = 5-7, the in vitro inhibitory activity of the enzyme is 100 times stronger than that of an amino sugar derivative with n = 1 or n = 2. On the other hand, sucrase inhibition is most pronounced for derivatives with n 2. 1, where n * 1, the contraceptive effect is only half that, and the specific sucrose inhibitory effect of higher derivatives drops further from this.

In an in vivo sucrose loading assay, the activity has been found to be approximately parallel to the specific inhibitory activity observed in the in vitro assays. In contrast, in vivo starch loading experiments showed an increase of 10-40-fold in the activity of amino sugar derivatives with n = 1, 2 and 3 compared to the amylase inhibitory capacity in in vitro experiments.

It is known that animals and humans experience hyperglycemia after ingestion of carbohydrate-rich nutrients and nutrients (e.g., cereals, potato starch, fruit, fruit juices, beer, chocolate) due to the rapid breakdown of carbohydrates by glucose and fungal glycosylphenyl. pancreatic amylases, maltases, sucrases) according to the following diagram: Starch or glycogen amylase yt your taste y J j & 'maltose' glucose sucrase y

Sucrose glucose + fructose This hyperglycemia is particularly pronounced and persistent in diabetics. In obese people, dietary hyperglycemia often results in particularly high insulin secretion, which in turn increases fat production and reduces fat breakdown. In the presence of such hyperglycaemia, hypoglycaemia is frequently observed in insulin-dependent and obese individuals. It is known that both hyperglycemia and ingestion in the stomach promote the formation of gastric fluid, which in turn causes or promotes the formation of gastric ulcer, gastric ulcer or duodenal ulcer.

It has now been found that inhibitors of g 1coside hydrolases obtained and isolated by the methods of the invention significantly reduce dietary hyperglycemia, hypo-insulin hypo-g 1 y after exposure to rat and / or human metabolism in wheat starch or sucrose or sucrose. , and accelerate the passage of these said carbohydrates through the stomach.

In addition, they prevent the absorption of glucose from the intestine. Furthermore, the conversion of carbohydrates into adipose tissue lipids and the storage of dietary fat in adipose tissue is reduced or slowed.

It is further known that microorganisms cleave carbohydrates in the oral cavity, especially sucrose, and thus promote the formation of rot rot.

Glycoside hydrolase inhibitors are therefore suitable as therapeutic agents for the following diseases: obesity, blood obesity (arterial embrittlement), diabetes, pre-diabetes, gastritis, gastric ulcer, duodenal ulcer and cataract.

Clarifications of some of the reagents and excipients used: Amberlite IRA 410 Cl (anion exchanger); Amberlite IRC 120 (Form H) strongly acidic cation exchanger; Amberlite (HCO 2 'form) (anion exchanger); Amberlite IRA 410 OH (strongly basic anion exchanger); Amberlite IRC 50 H (strongly acidic Kati onion exchanger); / ftohm and Haas Company and Dowex 50 WX 4H (strongly acidic cation exchanger). Dow Chemical Co. Midland, Michigan, USA trademark.

As the cellulose-based anion exchanger, for example, DE AE-Zellulose, Firma Schleicher und SchQll.

As the polyacrylamide gel, for example, Biogel P-2 (trademark of Bio-Rad Laboratories, Richmond, California, USA) can be used. _

Maltzin is a natural malt extract from Oiamalt AG, Munich (BRD).

Lewapol® is a non-specified poly-styrene adsorption resin from Bayer AG, Leverkusen (BRD).

Clarcel is a filter aid (diatomaceous earth / cellulose) from CECA, Velizy-Villacoublay, France.

SE 30 is a silicone elastomer from Hewlett Packard.

The hexose units in the following examples represent glucose units.

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Plate Chromatography Study To evaluate the composition of the final fermentation product and preparations by plate chromatography, 1 μl of fermentation broth or 1 μg of preparations are placed on silica gel pre-prepared membrane plates (Schleicher & Schull, Dassel. Typ F 1500) and developed twice with n-butanol / ethanol / 20 / water.

To obtain a sucrase-inhibitory dye, an enzyme gel (20 ml / 20 x 20 cm plate) is sprayed onto a developed and well-dried plate and the gel is allowed to solidify. It is then pre-incubated for five minutes in a humidity chamber at room temperature and then sprayed with sufficient substrate gel. After this second gel layer has solidified, the plate is placed in a humid chamber and germinated at 40 ° C. Inhibition color (light spots, reddish brown background) develops in 60-90 minutes. The development of color formation is stopped at the optimum moment, and the plate with its agar layers is dried in a ventilation dryer with warm air11a.

Preparation of gels

Enzyme gel: 1.5 g of Agarose ((-'Industrie Biologique Francais) is suspended in 100 ml of 0.2 M Na-maleate buffer, pH 6.0, and then dissolved by boiling. The clear Agarose solution is frozen at 50 ° C. 250 μl of Triton X-100 solution (2 g of Triton X-100 + Θ g of ethanol pa) and 0.5 ml of dianisidine solution (20 mg _ dianisidine / 1 ml acetone) are added to the vessel. 1 ml GOD / POD reagent (12.5 mg Glucoseoxidase, Reinheitsgrad I, Boehringer, Best. No. 15423 and 2.5 mg Peroxidase, Reinheitsgrad 20 58158 II, Boehringer, Best. No. 15302, dissolved 5 ml of maleinate buffer) and 4-5 units of sucrose obtained from the small intestine of the pig.1 The gel must be kept at 50 ° C for injection, otherwise it will solidify in the nozzles during injection.

Substrate gel: 0.5 g of agarose is suspended in 100 ml of Na-maleate buffer, pH 6.0 and dissolved by boiling. The enzyme preparation prepared as described on page 16, footnote 1) is then cooled to 50 ° C, 100 l of

Triton (2 g Triton X-100 + 8 g ethanol, p.a.) and 1 g sucrose (Serva No. 35579). After the sucrose has dissolved, the gel is ready for use.

To obtain an amylase inhibitory dye, an amylase gel (20 ml / 20 x 20 cm plate) is sprayed onto a developed and dried chromatographic plate and allowed to solidify. After pre-incubation for 5 minutes at room temperature, the plate with gel membranes is placed in a 0.5% starch solution (1 g of starch, Merck No. 1252, dissolved by boiling in 200 ml of a buffer solution of 0.2 m glycerophosphate / 0, 01 m CaCl 2, pH 6.9) and kept there for two minutes at 40 ° C and inverting the solution. The plate is then rinsed with well-distilled water and immersed in a dilute solution of colorless starch (4 ml of J2 ~ stock solution in 500 ml of H2O; J2 "PBus solution: 2.2 g + 4.4 g of KJ dissolved To 100 mL of H 2 O). The optimum color is formed after about one minute. The disc is photographed immediately as the blue spots fade quickly.

Preparation of amylase gel: 1 g of agarose is dissolved in 100 ml of a buffer solution at 100 ° C in 0,2 m sodium glycerophosphate / 0,01 m CaCl 2 pH 6.9 and, after cooling to 50 ° C, 100 μl of amylase crystal pension (10 mg porcine amylase / ml saturated NH 4 sulphate solution, Bo. Boehringer, No. 15017).

See. footnote 1) 2 ’58158 on page 13

Example 1

A 3-day-old shake culture of Actinoplanes strain SE 50/13 (CBS 61l 71) was inoculated into a glass fermentation vessel containing 8 liters of nutrient solution containing 5.0% starch, 1.0% yeast extract, 0.2% K 2 HPO 2. and incubated with efficient stirring and conditioned for three days at 28 ° C to give a culture broth of 105,000 Al / ml. The pH of a six-liter batch of fermentation broth taken from this was adjusted, after cooling to 20 ° C, to 2.5 with semi-concentrated HNO 2, 30 g of Carboraffin activated carbon were added and stirred for 10 minutes. It was then centrifuged for 15 minutes at 10,000 rpm, and the clear, pale yellow surface phase was concentrated to 500 ml after neutralization with NH 4. 500 ml of concentrate was mixed for 1 * 5 minutes with 200 g of Amberlite IRA 1 + 10 cl, filtered with suction, and U / 5 parts by volume = 1 * 00 ml of methanol was added to the filtrate to separate the major part of the higher molecular weight starch cleavage products (together with with the remaining activated carbon residue). After centrifugation for 5 minutes at 5,000 rpm, an 850 ml overhead phase was added dropwise with vigorous stirring to 1 liter of absolute ethanol. The white fluffy precipitate was filtered off with suction, washed three times with abs. ethanol and twice with ether and dried in vacuo at 50 ° C. Yield 36 g of a white powder with 10 x 10 AIY / g.

30 g of the product obtained were dissolved in 250 ml of H 2 O. The conductivity of this solution was 10 mS, pH 5.5 · To remove the salt, 60 g of Amberlite IRC 50 H (a weakly acidic cation exchanger that binds the amino sugar derivative from the aqueous solution only slightly) and 20 g of Amberlite IRA 1 * 10 OH were added to the solution and stirred for 20 minutes. The pH of the filtrate (conductivity 0.5 mS, pH 3.5) was adjusted to 1-norm. HCl to 3.0 (conductivity 0.6 mS). This solution was pumped at a rate of 1 * 2 ml / h through a column packed with Dowex 50 WX 1+ H +, 0.071+ - 0.037 mm (2.5 cm, height 1 * 0 cm, equilibrated in 0.001 N HCl). and then washed with 2 liters of 0.001 norm. HCl. After washing the column, it was eluted with 1.2% aqueous ammonia and 10 ml fractions were collected. The inhibitor-active fractions were combined, the ammonia was sucked off in vacuo and then the solution was concentrated in vacuo to 30 ml. The product was precipitated by dropwise addition of the solution to 600 ml of dry alcohol, the precipitate was filtered off with suction and washed after washing with alcohol and ether in vacuo. Yield 1 *, 1 * g of product with 26.5 x 10 4 Al / g.

Fine purification was performed by gel filtration of the product in 0.5 g portions (dissolved in 10 ml of water) on a column packed with Biogel P-2 (0.07 * + - 0.037 mm, Bio Rad) (diameter 5 cm, length 95 cm ). Development was performed with water at a flow rate of 80 ml / hour. 12 ml fractions were collected. The total carbohydrate content (anthrone test, extinction E 620) 22 581 58 and the sucrase inhibitor and amylase inhibitor concentrations were determined from all fractions. Aminosugar derivative fractions with n = 5-7 detected by plate chromatography (amylase inhibition dye) were pooled, concentrated in vacuo and precipitated as described above. ethanol. Yield from 0.5 g of crude product: 0.2 g of an amino sugar derivative with n = 5 ~ 7 and having 30 x 10 ^ AlI / g and 2 5000 SIY / g.

Example 2

A 120 ml nutrient solution in a 1 liter Erlenmeyer flask containing 3.5% glucose, 2% starch, 0.5% casein hydrolyzate, 1.3% yeast extract, 0.3% CaCO 2 and 0.3% K 2 HPO 2 and adjusted to pH 7.8 and sterilized for 30 min / 121 C. It was inoculated with 6 ml of a preculture of Actinoplanes strain SE 50/110 - in a nutrient solution containing 3% soya powder, 3% glycerol and 0.2% CaCO 3. and the culture was incubated for 3-1 + days on a rotary shaker at 2i + ° C to obtain a culture solution of 153,000 AIY / ml and 12,200 SIY / l.

Mycelium was separated from the culture broth (2 liters) prepared as described above by centrifugation (13,000 rpm), and the resulting culture filtrate having an activity of 13,000 SIY / g was stirred to reduce the salinity (conductivity of the culture filtrate: about 10 mS) for 500 hours. g of a mixture of 2.5 parts Amberlite IRC-50 (H + form) and 1 part Amberlite IRA-1 + 10 (OH' form).

The ion exchanger was separated, the solution was concentrated to just under 100 ml and centrifuged to remove insolubles at 20,000 rpm. For 15 minutes. The volume of the upper phase was made up to 100 ml; its conductivity was now 3.5 mS and was applied for further purification to a column (55 x 1 + 00 mm) containing phosphocellulose (P-Cellulose, SERVA Kr. U5130; pretreated by known methods and equilibrated with 5 mmol in ammonium phosphate buffer, pH 5.5) · Said phosphate buffer was used as driving fluid; the flow rate was 90 ml / h, and fractions with a volume of 18 ml were collected during the run. -

After examining the carbohydrate content of the eluate fractions (by means of the anthrone test) and the sucrasease inhibiting components (by means of the sucrase inhibition test), the fractions which had been shown to be nearly carbohydrate towers and which at the same time proved to be particularly effective in the sucrase inhibition test were pooled. -170), concentrated to 150 mL and filtered through a column (50 x 300 mL) of Amberlite IRA-U10 (HCO 3 'form). To improve the control of deionization, the eluate was collected fractionally (10 ml fraction over 20 minutes) and examined for carbohydrate content (by anthrone test: almost negative all the time), phosphate content (by ascorbic acid-molybdenum reagent: all time negative) and sucrose -bit ability (using enzyme inhibition test). The inhibitory fractions (3-30) were combined, concentrated and lyophilized, redissolved and lyophilized to give 280 mg of crude inhibitor.

581 58 23

For further purification, the crude inhibitor was fractionated using Bio-Gel-P-2 as described in Example 1. Fractions containing pure amino sugar derivative with n = 1 were isolated after lyophilization with 30 mg of product with 0.3 x 10 6 Al / g and 35,000 SIY / g.

Example 3 In a 200 ml Erlenmeyer flask containing 25 ml of a nutrient solution containing 0.1% starch, 0.02% yeast extract, 0.1%, K 2 HPO 2, 0.2% (NH 4 , 0.05%

MgSO 4, 0.05% KCl, 0.01% FeSO 4, was inoculated with strain Asp.niger ATCC 1139 * + spore- ^ 4 o '..... ...

the suspension and culture were incubated at 28 ° C on a rotary shaker, the AIY tutor dropped from 210,000 AIY / ml to 53,000 AIY / ml in six days and to 21,300 AIY / ml in 10 days. At the same time, the SIY / ml concentration increased from 7.0 to 72.

20 ml of the nutrient solution incubated with the spore suspension for 10 days was centrifuged to isolate the mycelium for 30 minutes at 3,000 rpm. The remaining 15 ml batch (72,000 SIY / l) was desalted by stirring for 30 minutes with 2 g of Amberlite IRC 50 H + and 1 g of Amberlite IRA ^ 10 OH (conductivity less than 2 mS). Filtered and the filtrate was passed through a column (0 1 cm x 10 cm) with a Dovex H + equilibrated at 0.001 norm at a rate of 5 ml / h. HCI. It was then washed with 200 ml of 0.001 norm. HCl. To extract the product through the column, a 0.6 / 6 NH 4 solution was pumped and the extract was collected in 5 ml fractions. The fractions having sucrase inhibitory activity were combined, concentrated to 2 ml on a rotary evaporator and 2 ml of methanol were added. The pH of this solution was adjusted to 3 ~ * + and mixed by dropwise addition to 100 ml of acetone. The precipitate was filtered off with suction, washed with acetone and ether and dried in vacuo. Yield: 26 mg, 28,000 SIY / g of amino sugar derivatives with n -2 and 3. For further purification * t, 0 g of inhibitor was gel filtered in 0.5 g portions through Biogel P-2. To this end, each 0.5 g preparation was dissolved in 10 ml of HgO 2 and applied to a Biogel P-2 column (0.07 * + - 0.037 mm, FA. Bio-RAd) with a diameter of 5 cm. Development was performed with water at a flow rate of 80 ml / h. 12 ml fractions were collected. The total carbohydrate concentration (anthrone test, extinction E 620) as well as the sucrase inhibitor and Amylase inhibitor concentrations were determined from all fractions. In addition, fractions were examined by plate chromatography (enzyme inhibition dye: see "Plate Chromatography Study"). 7 mg of an amino sugar derivative with n = 2 and 60,000 SIY / g were obtained.

Therapeutic Experiments I Experimental Arrangements to Demonstrate the Effect and Glucose Resorption Inhibition of Glycoside Hydrolase Inhibitors in Rats and Humans.

To induce dietary hyperglycemia and hyperinsulinemia, fasted rats (6) or fasted subjects are orally administered either 2.5 g of sucrose or 2.5 g of maltose or 1 g of cooked 2ί + 58158 starch or 2.5 g of glucose per kilogram, or 50 g of sucrose or 50 g of maltose or 50 g of cooked starch or 50 g of glucose per person orally as an aqueous solution or suspension. The other rats, six, receive the same amounts of said carbohydrates and in addition one of the active ingredients mentioned in the examples at the indicated doses. Healthy subjects are administered alternately one of said carbohydrates every other day or less frequently with or without any of said active ingredients. Blood glucose and serum insulin are determined both before the administration of the carbohydrate active ingredient and at short intervals of five minutes to three hours from blood taken from the retro-orbital venous plexus of rats or from blood taken from the subjects' elbow or capillaries. Blood glucose assays are performed on an Auto-Analyzer (Technicon, Hoffman: J. Biol Chem. 120, 51 (1937) or enzymatically with glucose oxidase and o-dianisidine hydrochloride), serum insulin assays by the method of Hales and Rande: Biochem. J. 88, 137, (19 ^ 3) -II Experimental set-up to demonstrate the inhibition of carbohydrate conversion of adipose tissue lipids.

Rats (6) receive by gavage one of said carbohydrates in inactive form together with a suitable dose of the same carbohydrate but 1U + labeled with radioactive carbon (C) - the active ingredient mentioned in the mentioned examples. At short intervals from the start of the experiment, the animals are sacrificed and the adipose and / or kidney surrounding adipose tissue is sampled, the lipids are extracted with appropriate fat solvents, e.g. chloroform / methanol (2: 1), and the lipid-free metabolic metabolites are washed away.

Lipids isolated in this way are assayed for radioactivity on a Liquid Scintillation Counter. Activity is expressed in dpm / 1 g adipose tissue.

In the tables, P denotes the probability of error and S the standard deviation.

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Claims (1)

58158 28 Claim: A process for the separation and purification of microbiologically prepared amino sugar derivatives of the general formula HO OH C with 1 to 7 hexose units, characterized in that microorganisms belonging to the genus Actinoplanes under aerobic conditions and in an aqueous nutrient medium are separated from the mycelium obtained by culturing and the resulting culture filtrate, possibly with activated carbon at pH 1-3, b) the filtrate bound to the ion exchanger is either desorbed entirely with ammonia and then fractionated into individual components in a manner known per se using gel filtration or cellulose column chromatography, or eluted with an acidically dilute acid, and the individual amino sugars are isolated from the eluates thus obtained.