KR830001418B1 - Method for preparing a molarine derivative - Google Patents

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Description

Method for preparing a molarine derivative

The history of the discovery of cyclodextrin glycosyl transferase (EC 2,4,1,19) has been around for a long time, and the enzyme was first discovered in Bacillus macerans, so Bacillus maserance amylase macerans amylase).

To date, the enzyme is, for example, Japanese Patent Application Laid-Open No. 47-20373, Japanese Patent Application Laid-Open No. 50-60189, Japanese Patent Application Laid-Open No. 50-88290, and Hanchis Bender Archives of Microbiology. Bacillus. Maserance, Bacillus megaterium, as shown in Aggri Cultural and Biological Chemistry, Vol. 40, pp. 753, 1976, 1977, pp. 27-282. , Bacillus Circulans, Bacillus polymyxa, Bacillus steadermophillus, Klebsiella pneumoniae, Bacillus no. 38-2 (good alkaline bacteria) etc. are known.

Moranotin, represented by the structural formula [II] as a starting material, was isolated from the first herb, sangbaekpi (Japanese Yak, etc., 50 agricultural chemical papers, 571 pages, 1976, and Japanese Patent Laid-Open No. 52). -83951).

Then, the inventors made it possible to manufacture [I] by the fermentation method by the bacteria belonging to Streptomicros. (Japanese Patent Application Laid-Open No. 54-84094)

Melting of cyclodextrin glycosyl transferase has been shown in many patents and literature. For example

(A) Preparation of Hesperetine Dihydrochalcon-7-Maltoligoside as Preparation of Sweeteners (Amylase Symposium, vol. 4, p. 61, 1972)

(B) Method for preparing origosaccharides in which lactose is bound to terminals (Japanese Patent Application Laid-Open Nos. 47-20273 and 54-119092)

(C) Preparation of cyclic dextrins (Japanese Patent Application Laid-Open No. 50-88290)

(D) Production method of α-glycosyl stevioside as a sweetener (Japanese Patent Laid-Open No. 54-5070)

These inventions have three characteristics of cyclodextrin glycosyl transferase, namely

① starch → cyclodextrin

② cyclodextrin + glucose → oligoglucose (coupling)

(Glucose) n + (glucose) m → (glucose) n + x + (glucose) m − x. (Equalization) (m, n, x is an integer) (see Amylase Symposium, vol. 7, p. 61, 1972).

The substrate specificity of the cyclodextrin glycosyl transferase in the coupling reaction has been discovered in recent years. [E.g. Aggregal and Biological Chemistry Vol. 42, p. 2369, 1978]

The present invention is based on the novel finding that the molarin represented by Structural Formula [II] serves as a substrate for the coupling reaction.

Next, the present invention will be described in detail. The cyclodextrin glycosyl transferase used in the present invention can be easily obtained by the following method. That is, microorganisms having a production capacity of the enzyme, for example, bacteria belonging to the genus Bacillus and Klebsiella, carbon sources such as starch, bran, corn source photokernel, pleptone, coon gluten myil, yeast extract, nitrogen sources Cyclodextrin glycosyl transferase, which is grown on a nutrient medium containing inorganic substances such as ammonium and magnesium chloride and other substances suitable for the production of the present enzyme, is produced by known purification methods such as salting out and dialysis. (Iv), adsorption to starch, desorption, gel filtration, ion exchange chromatography and the like may be used for purification. In the reaction, the culture filtrate may be used as a coenzyme solution, but in general, partial purified enzyme, purified enzyme, and immobilized enzyme are used.

In the presence of the cyclodextrin glycosyl transferase obtained in this manner, the reaction of moranorin and cyclodextrin (which may be starch or dextrin) produces origo glucosyl morarin. The reaction conditions vary depending on the origin of the cyclodextrin glycosyl transferase used, but usually Origo glucosyl molarine is prepared at a reaction temperature of about 40 ° C., pH 5.0-8.5, and reaction time 1 day-3 days.

In order to isolate and purify originol glucosyl molarin in the reaction solution, a known separation and purification method may be used. For example, First, the reaction solution is passed through a column of strong acidic ion exchange resin, the basic material is adsorbed, eluted with 0.5 N ammonia water, and the eluate is concentrated under reduced pressure. The concentrated solution is suspended on a Sephadix column, the required fractions are collected, and freeze-dried to obtain a white powder. Identification of each fraction is possible by high performance liquid chromatography, for example. Using high-performance liquid chromatography (ALC / GPC-244 type) manufactured by Waters Co., Ltd., a column for sugar analysis (μ-Bondapack CH), acetonitrile: water = 70: 30, flow rate 1.5cc / min, The retention time of the component is as follows.

The elemental analysis value, molecular weight, and specific lightness (aqueous solution) of origo glucosyl molarine obtained in this manner are as follows. Molecular weight was measured using the Hitachi molecular weight measuring apparatus 115 type.

In parentheses, the theoretical or non-luminescent concentrations are measured.

Origo glucosyl molarine obtained in this way is a novel substance not described in the literature, and has a significantly higher blood glucose elevation suppressing effect than the starting substance as shown below, and is extremely useful as a medicine.

<Blood sugar rise inhibitory effect>

Four male 5 week-old SD mice (weight 100-120 g) were orally administered 10 mg / kg of the test compound with 2 g / kg sucrose and blood collected from the tail vein over 180 minutes. Measure Next, the area increase value (ΔAUC) under the time-glycemic curve is obtained. When the value when only water is administered is basal and the value when only sucrose is administered as a control, the rate of blood glucose increase inhibition of the administration group of the test compound is calculated by the following equation.

The suppression rate of blood sugar rise thus calculated is as shown in the following table.

Next, the Example regarding the manufacturing method of this invention is listed.

EXAMPLE

(A) Cultivation of Bacillus maserans

neutralised lived digest) 한천 1.5%의 경사면 배지위에서 충분히 성육되어 있는 바실루스 마세란스 IFO 3490주를 3백금이(白金耳) 접종하고 37℃에서 3일간 배양한다. 이 배양액 300ml를 같은 배지조성 9ℓ의 자아 퍼멘터(jar fermenter)에 접종하고, 37℃, 3일간, 충분히 통기, 교반하면서 배양하면, 원심 상징액(上澄液)으로서 약 130-150단위(단위의 정의는 (나)에 표시한다)의 효소액을 얻는다.(1) 150 ml of culture medium was added to 500 ml of Erlenmeyer flask, and 1% of Coon's photocar, 1% of soluble starch (NH ₄ ) ₂ SO _4, 0.5% of CaCO ₃ , and pH 7 was added, and heated at 120 ° for 15 minutes. Sterilize. Peptone 1%, yeast 0.5%, glucose 0.3%, glycerol 1.5%, NaCl 0.3%, lever powder (OXOID

Neutralized lived digest 3390 strains of Bacillus maserans IFO, fully grown on 1.5% agar inclined medium, are inoculated with platinum and incubated at 37 ° C for 3 days. When 300 ml of this culture solution is inoculated into a 9 liter jar fermenter of the same medium composition, and cultured with sufficient aeration and stirring at 37 ° C. for 3 days, it is about 130-150 units (centrifugal supernatant). The definition is shown in (b)).

② 4% Bran, 0.5% CoonStyrika, 0.5% Soluble Starch, (NH ₄ ) ₂ SO ₄ 0.5%, CaCO ₃ 0.5% pH 6.7 When the mixture is inoculated with 3 ml of Bacillus maserans IFO 3490 and shaken for 4 days at 37 ° C., an enzyme solution of about 90 units is obtained as a centrifugal supernatant.

(B) units of cyclodextrin glycosyl transferase activity

Dissolve 0.7% of soluble starch (for Japanese Hangi Chemical Biochemistry Research) in 0.05M acetic acid buffer pH 5.5 to prepare a substrate solution. 50 µl of the enzyme solution is added to 950 µl of the substrate solution, the reaction is carried out for 10 minutes at 40 ° C, and 0.5 ml of 0.5 N acetic acid is added to stop the reaction. After the reaction, 100 µl was taken, 0.8 ml of iodine solution in which iodine was dissolved in 0.01 MKI aqueous solution and 3 ml of water were added, and the absorbance was measured at 660 nm after stirring. (A _T )

In the same manner, 100 µl of a solution obtained by adding 50 µl of water and 0.5 ml of 0.5 N acetic acid to 950 µl of the substrate solution is added, and an iodine solution is added, and the absorbance is measured at 660 nm. (A _R ) at this time,

This is an activity in which 1 ml of enzyme solution causes a decrease in absorbance of 1% at 40 ° C. for 1 minute.

(C) Preparation of coenzyme solution

The supernatant is obtained by centrifuging Bacillus maserans IFO 3490 culture. It is lyophilized and dissolved in a small amount of water to obtain a concentrated solution of the enzyme. At 5 ° C, the outer liquid is sufficiently dialyzed with water, and the inner liquid from which the low molecular weight is removed is used as the crude enzyme liquid.

(D) Reaction

8 g of hydrochloride hydrochloride and 8 g of α-cyclodextrin are dissolved in 400 ml of 870 units of coenzyme solution (348,000 units), and the mixture is reacted by shaking culture at 38 ° C. for 3 days at pH 5.7. The reaction mixture was partially taken, and the basic substance was collected and analyzed by high-performance liquid chromatography (ALC / GPC-244 type bile column for water analysis μ-Bondapack CH, acetonitrile: water = 70: 30, 1.5 ml / min). The area ratio of the differential refractive system of each fraction was determined as follows using the Shimazu Chromatograph Pack C-RIA.

Unreacted Moranoline 43%

4- (α-D-glucosyl) -moranoline 16%

4- (α-D-maltosyl) -moranoline 16%

4- (α-D-maltotriosyl) -moranoline 12%

4- (α-D-maltotetrasil) -moranoline 8%

(E) isolation and purification

About 400 ml of the reaction solution is heated at 120 ° C. for 10 minutes. 160 ml of trichloroethylene is added, it stirs vigorously, and the lower layer is discarded. 80 ml of trichloroethylene was added to the supernatant again, the mixture was stirred, and the lower layer was discarded. The supernatant is centrifuged to give a transparent supernatant, which is passed through a 80 ml column of strong acidic ion exchange resin (Dowex 50w x 2 (H ⁺ )) to adsorb the basic substance. Elution is performed with 0.5 N ammonia water, and the eluate is freeze-dried. Sepadic G-15 while identifying the fractions obtained by high-performance liquid chromatography (ALC / GPC-244 manufactured by Waters, a column for sugar analysis, acetonitrile: water = 70: 30, 1.5 ml / min). (48 mm x 850 mm), Sepadix G-25 (55 mm x 470 mm) and Sepadix G-10 (40 mm x 820 mm) to obtain the desired Origo glucosyl moranoline.

Claims (1)

Cyclodextrin glycosyltransferase was applied to a mixed aqueous solution of moranorin and cyclodextrin or soluble starch to obtain a mixed solution containing origo glucosylmoranoline represented by the following general formula (I), which was then isolated to each component. Method for producing origo glucosyl molarine, characterized in that.

However, n represents an integer from 0 to 3.