JPS6149958B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a new physiologically active substance having amylase inhibitory activity and a method for producing the same.

In recent years, various diseases caused by overnutrition have been increasing in Japan as well. In particular, excessive intake of sugars that are easily digested and absorbed, particularly starch and sucrose, has led to an increase in blood sugar and associated metabolic diseases such as diabetes, obesity, and arteriosclerosis.
In addition, a diet that is biased towards sugars can lead to diarrhea,
It also contributes to various digestive system diseases such as gastrointestinal catarrh and abnormal fermentation in the intestines. However, no satisfactory preventive or therapeutic methods for these diseases have been established at present.

Therefore, based on the idea that by inhibiting the action of amylase, which digests starch, the digestion and absorption of sugar can be suppressed and, as a result, the rise in blood sugar can be prevented, the present inventor has developed an amylase inhibitor in microbial cultures. Searched for. As a result, a new physiologically active substance was discovered from the culture solution of the genus Cladosporium, and the present invention was completed. That is, the present invention provides a hypoglycemic agent produced by microorganisms.

This substance was shown to have a blood sugar-lowering effect in animal experiments using rats. This substance is hereinafter referred to as "Tomastatin."

The present invention relates to a new physiologically active substance "tomastatin" produced by mold, and a method for culturing mold and collecting tomastatin from the culture, particularly a method for culturing Cladosporium bacteria and collecting tomastatin from the culture. be.

The microorganism that can be used in the present invention is a tomastatin-producing bacterium belonging to the genus Cladosporium, and a strain recognized by the present inventor to be particularly effective is, for example, Cladosporium Cladosporioides strain M3573. be. This strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, with accession number 5892 (FERM-
P No. 5892).

It goes without saying that Cladosporium species other than those mentioned above can also be used, regardless of their variants or mutant strains, as long as they have the ability to produce tomastatin.

Cladosporium cladosporioides isolated from soil in Suginami Ward, Tokyo by the present inventor
The mycological properties of strain M3573 are as follows.

Characteristics of colonies Growth rate: When cultured at 25°C, colony diameters on potato dextrose, cornmeal agar, and cornmeal agar media were 4.8, 3.7, and 4.8 cm, respectively.
It grows quite quickly. It does not grow in any medium at 37°C, and the suitable temperature for growth is 24-28°C. Although it grows in the above medium at pH 2-8, the preferred growth range is pH 3-7.

Growth pattern: On all three types of media, the colonies are flat (slightly barrel-shaped in Tsapetsukudochus), olive-gray to dark olive-gray, felt-like to slightly woolly, with numerous pale hyphae within the network of aerial hyphae. Also, no colored conidial heads or colored secretions were observed, and the underside of the colony was dark green to black.

Microscopic properties Conidiophore: unbranched, 27-250 x 2.6-4.0μ,
Smooth conidia: Budding type conidia, conidial heads are branched or seemingly tufted, diameter of conidial heads is 30-100μ,
The unbranched part of the conidial chain has 3 to 4 conidia.
Conidia are smooth, pale and colored, lemon-shaped to oval with constrictions at both ends, 5-10 x 4-5μ, and large cylindrical conidia (13-16 x 4-6μ) are observed at the base. .

Based on the above-mentioned colonies and microscopic characteristics, this bacterial strain was identified as Cladosporium Cladosporioides.
The mycological characteristics of Cladosporium cladosporioides are described in the following literature: That is,
Shunichi Udagawa, Keisuke Tsubaki et al.: Illustrated encyclopedia of fungi, Volume 2, 859~
860 pages, 1978 (Kodansha Scientific),
GAde Vries: Contribution to the Knowledge
of the Genus Cladosporium Link ex Fr，57
Page, Thesis, Baarn, 1952.

Tomastatin is produced in a culture by aerobically culturing a tomastatin-producing bacterial strain by a culture method known as a mold culture method. For example, a tomastatin-producing strain is subcultured on a potato-dextrose-agar medium manufactured by Difco, and for the production of tomastatin, the cells grown on this agar medium can be directly inoculated into the production medium and cultured. In addition, tomastatin can be produced by culturing the bacterial cells grown in the production medium in a new production medium.

Tomastatin-producing bacteria grow at temperatures between 7 and 35 degrees Celsius, but
A temperature of 20 to 30°C is usually preferred for tomastatin production. In order to culture Cladosporium bacteria that produce tomastatin, known nutrient sources for culturing molds and other microorganisms can be used. For example, glucose, maltose, dextrin, starch, lactose, sutucarose, glycerin, etc. can be used as carbon sources. Among these carbon sources, glucose and glycerin are the preferred carbon sources for tomastatin production.

To produce tomastatin, all known nitrogen sources for mold and other microbial growth can be used. For example, peptone, meat extract, yeast, yeast extract, soybean flour, peanut flour, corn staple liquor, rice bran, inorganic nitrogen sources, etc. can be used.

When producing tomastatin by culturing tomastatin-producing bacteria, inorganic salts and metal salts are added if necessary. Also, trace amounts of heavy metals can be added if necessary.

Tomastatin can be obtained by culturing its producing bacteria aerobically, but it can be obtained by aerobic culture methods commonly used, such as
A solid state culture method, a shaking culture method, and an aerated agitation culture method are used. When antifoaming is required during culture or medium sterilization, antifoaming agents such as silicone oil and surfactants can be used. The culture temperature is preferably 20 to 30°C.

The physiological activity of tomastatin can be assayed in a conventional manner by the amylase activity measurement method that measures the liquefying ability of animal α-amylase (pig pancreas) to starch [Nobujuro Namiki, Kunio Kamigun, Takatoshi Nagate, Kazuhiko Sugita,
Hiroshi Hara, Etsuko Mori, Sadafumi Omura, Masahiro Ozeki, Starch Science, Vol. 26, pp. 134-144, 1979].

Cultivation is continued until tomastatin is substantially accumulated, and the substance is extracted and separated from the culture using various methods based on the properties of the substance revealed by the present invention, as shown in the Examples below. This can be done by a suitable combination of methods. Namely, salting-out methods using ammonium sulfate, etc., chromatography methods using various ion exchangers, gel filtration methods using various carriers, various electrophoresis methods including focused electrophoresis, ultrafiltration methods, freeze-drying methods, and dialysis methods. methods, treatment methods using various organic solvents, etc. By appropriately combining these means,
Moreover, tomastatin can be isolated from the culture by repeated use.

Next, the physicochemical and physiological properties of tomastatin will be shown.

(1) Molecular weight is approximately 25,000 (gel filtration method).

(2) Ultraviolet absorption spectrum: Figure 1.

(3) It has the properties of a protein.

(4) Soluble in water and salt solutions, about 60% ammonium sulfate saturation
It is salted out and becomes a precipitate. Also insoluble in acetone, ethanol, ethyl acetate, and benzene.

(5) Adsorbed by anion exchangers such as DEAE and cellulose.

(6) The isoelectric point is 3.8 by focal electrophoresis.

(7) Elemental analysis values C: approx. 44%, H: approx. 7%, N: approx. 9% (8) Color reaction Buillet reaction (blue), phenol sulfuric acid reaction (orange), phorin color reaction (blue) ( 9) Sugar (phenol, sulfuric acid method) / protein (Lowry method) is approximately 0.5 (10) Infrared absorption spectrum: Figure 2 (11) α-amylase action of pig pancreas is approximately 0.4μ
50% inhibition at g (protein)/ml.

(12) Acute oral toxicity (LD ₅₀ ) for mice is 1g/
Kg or more.

The physiological activity of tomastatin was investigated through various animal experiments, and its effectiveness was confirmed. For example, male Wistar rats weighing 150 to 170 g were fasted for 24 hours, and heated to dissolve 1 starch.
g/Kg was administered orally. At the same time, 5 mg/Kg of tomastatin was orally administered, and blood was collected from the rat's tail vein 30 minutes and 1 hour later, and the blood sugar level (blood glucose level) was measured by a conventional method. As a result, animals treated with tomastatin had a 31% lower weight after 30 minutes and 1 hour compared to the control group fed only starch.
% and 19% blood sugar levels were lower.

As described above, tomastatin has a blood sugar lowering effect and can be used in medicine as a preventive/therapeutic agent for, for example, diabetes, obesity, arteriosclerosis, constipation, diarrhea, and the like.

Tomastatin can be administered orally or parenterally, for example, in the form of capsules, tablets, injections, and the like. Oral preparations are usually preferred. The dosage varies depending on age, symptoms, body weight, etc., but it is usually administered to adults at about 1 to 1000 mg per day in 1 to 4 divided doses. However, larger amounts or more times can be administered if necessary.

Next, examples of the present invention will be shown. Since the above-mentioned properties have been clarified by the present invention, based on these findings, various modifications may be made to the collection of tomastatin from culture or related materials. Means are possible. The present invention is not limited to the examples,
It includes all methods that can be easily extrapolated from the findings already described.

Example 1 Glucose 3%, glycerin 7%, soybean meal 3%, peptone 0.8%, sodium nitrate 0.2%,
Cladosporium cladosporioides M3573 strain was inoculated into a liquid medium containing 0.1% magnesium sulfate (MgSO ₄ .7H ₂ O) and cultured aerobically at 25° C. for 80 hours. The culture filtrate (8 liters) was saturated to 60% with ammonium sulfate, and the resulting precipitate was collected and thoroughly dialyzed against 5 mM phosphate buffer (PH6.9). The dialysis fluid was placed on a DEAE/cellulose column equilibrated with the same buffer to adsorb tomastatin. Next, the same column was developed with the same buffer containing salt (0 to 0.4M), and the active fraction was collected. After concentrating the active fraction, place it on a Sephadex G-100 column.
It was developed with 5mM phosphate buffer (PH6.9) containing sodium chloride. As a result, tomastatin has approximately 25,000 molecules
It was eluted in the following fractions. When this fraction was further subjected to focal electrophoresis, an activity peak was observed at an isoelectric point of 3.8. After thoroughly dialyzing this active fraction against distilled water, purified tomastatin 6.0 was purified by freeze-drying.
mg of light brown powder was obtained.

[Brief explanation of the drawing]

Figure 1 shows the ultraviolet absorption spectrum of tomastatin, and Figure 2 shows the infrared absorption spectrum of the same substance.

Claims (1)

[Claims] 1. Tomastatin, a new physiologically active substance having the following physicochemical properties. (1) Molecular weight is approximately 25,000 (gel filtration method). (2) Ultraviolet absorption spectrum: Figure 1. (3) It has the properties of a protein. (4) Soluble in water and salt solutions, about 60% ammonium sulfate saturation
It is salted out and becomes a precipitate. Also insoluble in acetone, ethanol, ethyl acetate, and benzene. (5) Adsorbed by anion exchangers such as DEAE and cellulose. (6) The isoelectric point is 3.8 by focal electrophoresis. (7) Elemental analysis values C: approx. 44%, H: approx. 7%, N: approx. 9% (8) Color reaction Biuretz reaction (blue), phenol sulfuric acid reaction (orange), phorin color reaction (blue) ( 9) Sugar (phenol/sulfuric acid method)/protein (Lowry method) is approximately 0.5 (10) Infrared absorption spectrum: Figure 2 2 Cultivate tomastatin-producing bacteria belonging to the genus Cladosporium and collect tomastatin from the culture solution A method for producing tomastatin, a new physiologically active substance, characterized by: 3. The manufacturing method according to claim 2, wherein the tomastatin-producing bacterium is Cladosporium cladosporioides M3573.