JPS604131A - Blood sugar lowering agent - Google Patents

Abstract

PURPOSE:A blood sugar lowering agent containing a polysaccharide produced by Bacillus polymyxa as an active ingredient. CONSTITUTION:Bacillus polymyxa (e.g., Bacillus polymyxa 271 and S-4, etc.) is inoculated into a medium obtained by blending a solution containing 3-5wt% glucose, sucrose, lactose, any of molasses, as a carbon source with a nitrogen source such as pepton, corn steep liquor, yeast extract, urea, etc. and magnesium sulfate, other salts, etc. cultivated under aerobic conditions, and a high-viscosity polysaccharide is obtained by a means such as precipitation with ethanol, methanol, etc. or ultrafiltration. A blood sugar lowering agent containing it as an active ingredient is obtained. The agent has a tablet, granule, capsule, etc. as the dosage form. A dose is 20-40mg/kg/day, it is administered daily three- four times dividedly.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to the use of Bacillus polymixer.
The present invention relates to a hypoglycemic agent whose active ingredient is a polysaccharide produced by lyo+yxa).

It has been known that natural polysaccharides have a cholesterol-improving effect. Among these natural polysaccharides,
Bacillus polymyx
Research has been conducted on the polysaccharide produced by a), and in particular the structure of the polysaccharide produced by Bacillus polymyxa 271 has been studied in Journal of the Japanese Society of Agricultural Chemistry 51 (10) 591-596 (
1977) and others.

It is also known that these polysaccharides have cholesterol-lowering effects. For example, Nutrition Journal Kasa (6) 27
3-281 (1975) etc.

In addition, the polysaccharide produced by Bacillus polymyxa S-4 was developed by the present inventors from Bacillus polymyxa S-4 (FERMBP-
This is a new polysaccharide produced by a new bacterial strain of No. 164), and it was discovered that this polysaccharide has a remarkable effect of suppressing the increase in serum cholesterol levels, and was previously filed in Japanese Patent Application No. 14945-1983.
I applied as No. 2.

The present inventors further studied the physiologically active properties of the polysaccharides produced by Bacillus polymyxa, and found that these polysaccharides significantly suppress the rise in blood sugar levels after meals, and completed the present invention. did.

The present invention is a hypoglycemic agent containing poly-YMs produced by Bacillus polymyxa as an active ingredient.

The polysaccharide that is the active ingredient of the present invention is Bacillus polymixa mixed with a liquid containing 3 to 5% of glucose, sucrose, lactose, and various molasses as a carbon source, and nitrogen such as peptone, cornstarch liquor, yeast extract, urea, etc. iI? by adding source and magnesium sulfate and other salts. The highly viscous polysaccharide obtained by inoculating the prepared medium and culturing it under aerobic conditions is purified by means such as precipitation with ethanol, methanol, etc. or ultrafiltration.

And Bacillus Polymixer 271 (FERM-P
The polysaccharide obtained from the culture of 1824) consists of the constituent sugars nigerose: mannose: galactose: glucuronate in a ratio of 3:3:1:2, and has a specific optical rotation [α] p=+
92° (water) and a molecular weight of 200,000 or more.

In addition, Bacillus Polymixer S-4 (FERM BP-1
The polysaccharide obtained from the culture of 64) has constituent sugars nigercose: mannose: galactose: glucuronic acid: mannuronic acid of 2.4 to 3.27 2.4 to 3. o: 1:
1.5 to 2.4: Consists of 0.8 to 1.4, specific optical rotation [α): '-+64 to +90' (C = 0.3%,
Il-5cmH20), molecular weight 200,000 to 1,600,000, as shown in infrared absorption spectrum in Figure 1, solubility is easily soluble in water, formaldehyde, formic acid, caustic soda (>10M), slightly soluble in dimethyl sulfoxide, benzene, and ethanol.

Insoluble in acetone, color reaction is reddish-purple by Molish method,
It is a polysaccharide that has a blue-green color when determined by the Anthrone method and a yellow-orange color when determined by the phenol-sulfuric acid method.

Bacillus polymixa S-4, which produces this polysaccharide, is a new strain and has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry as FERM BP-164.

Its mycological properties are as follows.

A, Morphology 1) Durham staining: Variable 2) Cell shape: Shape Cylindrical Size 0.6-1. OX 2.5-6.0μm array Single or short chain 3) Spore formation: Shape Oval Size 1.0-2.0X 2.0-3.0μm Position Center (parallel) Spore light Forms.

4) Flagella (motile) octoflagellate (+) B, Physiological properties 1) Colony on nutrient agar medium: mucilaginous 2) Colony on glucose agar medium: mucilaginous 3) Sabouraud agar medium (
Colonies on glucose): slimy 4) Growth conditions: 4°C - 25°C - 37°C - 55°C - 5% Naα - 7% Naα - Anaerobic conditions 15) Utilization of citrate - 6) Pigment production - C1 Biochemical properties 1) Catalase production 12) Oxidase production - 3) Methyl red reaction (glucose) + 4) Acetoin production from glucose + 5) Glucose 0/F test F 6) Acid/gas production Glucose + Xylose + Galactose + fructose + mannose + arabinose + lactose + sucrose + starch + mannitol + 7) Reduction of nitrate 18) Reduction of methylene blue 19) Production of indole - 10) Production of hydrogen sulfide - ■) Milk coagulation Decapeptonization 12) Casein decomposition 113) Liquefaction of gelatin In order to obtain the novel polysaccharide of the present invention from this new strain, Bacillus polymyxa S-4, a well-known culture medium containing a known nutrient source that can be used by ordinary microorganisms is used. Cultivate by method. For example, carbon sources can be glucose, glycerin, sucrose, starch, maltose, animal and vegetable oils, etc., and nitrogen sources can be, for example, soybean flour, meat extract, yeast extract, peptone, cornstarch liquor, cottonseed meal, etc. Organic matter and ammonium sulfate, ammonium chloride, ammonium nitrate.

Inorganic nitrogen such as ammonium phosphate can be used. Further, inorganic salts such as common salt, potassium chloride, phosphate, and other heavy metal salts, and vitamins are added as necessary. In addition, in order to suppress foaming during fermentation, an antifoaming agent such as silicone (trademark manufactured by Shin-Etsu Chemical Al) may be added as appropriate.

Bacillus polymixa S-4 was inoculated into the above medium, and temperature +
P) (+ Fermentation conditions such as aeration agitation and fermentation time are selected to accumulate the maximum amount of the novel polysaccharide of the invention. For example, fermentation conditions are 20-40°C, preferably 28°C
~32°C, FJ″15-9, preferably 6.0-7.0
It is advantageous to carry out the treatment for 1 to 6 days, preferably for 3 to 4 days.

In order to isolate the polysaccharide of the present invention from the culture fluid obtained as described above, the polysaccharide of the present invention can be isolated by a conventional method, for example, by filtering the culture fluid, separating the bacterial cells from the filtrate, and precipitating with ethanol or methanol. It is obtained by isolation and purification by appropriate means after external filtration.

Next, an example of producing this polysaccharide will be shown.

Manufacturer 1 Crystalline glucose 30g, peptone 2g, urea 0.5g.

MgSO4・7H201g, KH2PO41g, M
nSO4・71120 0.06g, CaC0:i
After adjusting the pH of a medium consisting of 5g and 1β of water to 7.0, 1
Sterilize at 20°C for 15 minutes and use this medium 12! ! at 28℃,
Bacillus polymixa S-4 in a medium with the same composition as the above medium.
600 ml of seedlings cultured for 24 hours was inoculated, and 12
Incubate for 6 days while supplying sterile air at β/min.

Dilute the cultured solution with 10 times more water and add diatomaceous earth (
It was filtered through a 0.45μ membrane filter (Toyo Roshi Department), and then ultrafiltrated with a molecular weight of 50,000 (Bio Engineering Co., Ltd. (7)). IF-
5WM85V-1) to filter out those having a molecular weight of 50,000 or more to obtain liquid 801 with a solid content of 1.5%.

This was vacuum freeze-dried at 40° C. for 20 hours and then pulverized to obtain 120 g of the powdered polysaccharide of the present invention.

In addition, since the polysaccharide of the present invention contains glucuronic acid and mannuronic acid, alkali metal salts of these acids such as sodium and potassium, and calcium.

Salts such as alkaline earth metal salts such as magnesium may also be used.

Many of the polysaccharides of the present invention produced by the above method have a molecular weight of 1 million or more.

Therefore, in order to mainly produce polysaccharides with a low molecular weight of 4 million or less, if the culture solution obtained by the above method is treated with an acid such as hydrochloric acid, polysaccharides with an arbitrary molecular weight of about 200,000 to 1,000,000 can be produced. A polysaccharide of the invention is obtained.

Thus obtained multi-tl! The class has a molecular weight of 200,000 to 1,600,000,
Specific Mi luminous intensity (α) F = +64 ~ + 90 (C =
0.3%, 1=5an H2O), elemental analysis value C:
43.5%, H: 8.25%, and the infrared absorption spectrum is as shown in FIG.

This substance was hydrolyzed with 2N sulfuric acid at 100°C for 6 hours, and when separated and qualitatively analyzed by filter paper chromatography, it was found to consist of glucose, mannose, galactose, and uronic acid. When these monosaccharides were quantified as acetyl derivatives by gas chromatography, it was found that the composition ratio of glucose: mannose: galactose was approximately 3:3:1. After reducing the carboxyl group of uronic acid to alcohol, it was hydrolyzed and acetylated in the same way as for quantifying monosaccharides, and then quantitatively determined by gas chromatography. From the increased amount of glucose and mannose, it was determined that it was glucuronic acid or mannuronic acid. was found, and the composition ratio of glucuronic acid and mannuronic acid was calculated by subtracting the amounts of glucose and mannose measured previously. As a result, this polysaccharide is glucose: mannose: galactose:
Glucuronic acid: Mannuronic acid is 2.4-3.27 2.
4-3. o: 1: 1.5~2.4: 0.8~1
．． It was found that it consists of 4.

The chemical properties of the polysaccharide of the present invention were soluble, easily soluble in water, formaldehyde, formic acid, and caustic soda (10M), insoluble in ethanol and acetone, and sparingly soluble in dimethyl sulfoxide and benzene. The color reaction shows reddish-purple with the Molish method, blue-green with the Anthrone method, and yellow-orange with the phenol-sulfuric acid method.

A test is shown in which the polysaccharide produced by Bacillus polymyxa has a hypoglycemic effect.

Effects of multifamily II produced by Bacillus polymyxa on blood glucose levels and plasma insulin concentrations.

Test method: SD male white rat, 7 weeks old (body weight: 160 g to 18
0g) was fasted overnight and subjected to the test. The animals were divided into two groups of 5 animals per group so that the average body weight was approximately equal, blood was collected from the tails of the rats, and each group was given 25 mj! of glucose (anhydrous) dissolved in distilled water. of 20% glucose solution (w/v) per kg of animal body weight.
A control group was prepared by dissolving 5 g of glucose (anhydrous) and 750 μg of polysaccharide (BP) produced by Bacillus polymyxa S-4 in distilled water.
A prepared solution containing ml of 20% glucose and 5% BP was orally administered through a stomatological tube at the same level as the glucose dose in the control group.
0 minutes.

20 minutes, 30 minutes, 60 minutes, 90 minutes, ``120 minutes,
Blood was collected from the tail over time every 180 minutes and the blood sugar level was measured.

Similarly, two groups were orally administered two types of samples for 15 minutes.

Blood was collected from the tail over time every 30, 60, 120, and 180 minutes, and the plasma insulin concentration was measured.

To measure the blood sugar level and plasma insulin concentration, blood was centrifuged according to a conventional method, and then plasma was collected and subjected to analysis. Blood sugar levels were measured by an enzyme method, and plasma insulin concentrations were measured by an enzyme immunoassay. The results are shown in the table below.

Table 1 Blood sugar level (Numbers in the table are standard error above the mean) If this table is shown in the drawing, the second. It is expressed as shown in FIG.

As is clear from the above results, when the polysaccharide produced by the Bacillus polymyxa of the present invention is added, increases in blood sugar level and plasma insulin concentration are significantly suppressed.

In acute toxicity tests on mice, these polysaccharides
．． Even when administered orally at 818 g/kg, no problems occur and it is a safe substance.

The hypoglycemic agent of the present invention is prepared by blending the above-mentioned polysaccharide or pharmaceutically acceptable salt with a suitable pharmaceutical carrier and preparing it mainly in the form of an orally administered drug, such as a tablet, granule, or capsule.

The dosage of the polysaccharide, which is the active ingredient of the present invention, varies depending on the symptoms, but for adults, it is administered at 20 to 40 μ per day per 1 kg of body weight, divided into 3 to 4 times a day.

Next, examples of the present invention will be shown.

Example I BPo, 5 parts, lactose 20 parts, cornstarch 20 parts.

Mix 2 parts of magnesium stearate and use a tablet machine to make 1 part
The tablets are made into 500 square tablets.

[Brief explanation of drawings]

FIG. 1 shows an infrared absorption spectrum of a polysaccharide produced by Bacillus polymyxa S-4, measured by the KBr method. Figure 2 shows the active ingredient of the present invention (Bacillus Subo IJ Mixer S).
FIG. FIG. 3 is a graph showing the state in which the polysaccharide suppresses the increase in plasma insulin concentration. Second. The solid line in Figure 3 is glucose 2.5g/kg bd
When a wt sample is given, the dashed line represents 2.5 g of glucose/
These are the blood glucose level and plasma insulin concentration when a sample of kg bd iwt + 3% BP was given. Patent applicant: Showa Sangyo Co., Ltd. agent Riki Arai (and 2 others)

Claims (1)

[Claims] 1. Bacillus polymixer
A hypoglycemic agent characterized by containing as an active ingredient a polysaccharide produced by A. myxa). 2. Bacillus Polymixer is Bacillus Polymixer 271
and S-4, the hypoglycemic agent according to claim 1.