JPH01181792A - Physiogically active substance ci-010 - Google Patents

Abstract

NEW MATERIAL:A physiologically active substance CI-101 having the following physicochemical properties. Appearance: pale yellow powder; melting point: 97-99 deg.C; elementary analysis (%): C 68.0 and H 9.7; mol wt.: 368; molecular formula; C21H36O5; specific rotation: [alpha] =+57 deg. (C=1%, CHCl3); solubility; insoluble in water and soluble in methanol, chloroform and ethyl acetate; color reaction: negative to ninhydrin reaction, anthrone reaction and Molisch test and positive to iodine reaction; acidic, etc. USE:The substance inhibits collagenase originated from a human being and is useful as an inhibitor to the decomposition of the cartilages of chronic articular rheumatism and osteoarthitis patients and as a metastasis inhibitor for cancer. PREPARATION:Streptomyces.sp-A4457 is cultured in a medium containing nutrivtive substances under an aerobic condition preferably at 6-7pH and 28-30 deg.C for 4 days.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a novel physiologically active substance Cl-010.

[Prior Art] The physiologically active substance Cl-010 of the present invention has a molecular weight of 368,
The existence of a substance having the molecular formula Cx s Hs -On and having the same physicochemical properties and physiological activity has not been reported to date.

[Problems to be Solved by the Invention] Collagenase causes cartilage destruction in rheumatism patients [Cawston'IF + Arthritis & Rheum patients].
atism), Volume 27, Pages 285-290 (1
984), periodontal ligament disease [Golub et al.
Journal, Periodonta) L-Research (J
, Periodontal Re5ear-ch),
Vol. 20, pp. 12-23 (1985).
Page 8 (1980)] One of the enzymes that plays an important role
It is thought that there is one. Therefore, providing an inhibitor specific to human collagenase is useful for preventing or treating diseases involving these collagenases.

[Means for Solving the Problems] As a result of intensive searches for compounds having such properties, the present inventors found that Streptomyces sp.
The present invention was accomplished by discovering that S. tomyces sp.) A 4457 produces a substance that specifically inhibits human collagenase.

The strain that produces the target substance of the present invention is a strain newly isolated by the present inventors from the soil of Hachioji City, Tokyo, and the microorganism name is Streptomyces sp.
tomyces sp, ) A 4457 J, Microorganism Deposit No. 1, Microbial Research Institute No. 9705 (FERM P-
9705) J and has been deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology, and the target substance of the present invention obtained by culturing this strain was named physiologically active substance Cl-010.

The mycological properties of this strain are shown below.

1) Morphotrophic hyphae are well developed and irregularly branched on synthetic and natural agar media, but no septa are observed. Spores can be formed well on starch inorganic salt agar, yeast/malt agar, oatmeal agar, etc. When observed under a microscope, the spore-forming hyphae are simply branched and the spores are formed in a straight line, but in rare cases, a spiral shape with 2 or 3 turns is also observed. Spores are usually found in chains of 10 or more and have a smooth surface. The shape of the spore is oval and its size is 0.43~0.60X0.93~1.185
It is m.

No sclerotia, sporangia, or flagellar spores are observed.

2) Growth status on culture media Table 1 shows the results of visual observation when cultured on various media at 30°C for 114 days.

3) Physiological properties (υGrowth temperature range) Grows well in the range of 20 to 37°C on oatmeal agar medium.Does not grow in the temperature range of 10°C or lower and 45°C or higher.

(Biochemical properties a) Distinction between aerobic and anaerobic aerobic b) Liquefaction of gelatin Positive (weak) C) Coagulation of skim milk
Negative d) Peptonization of skim milk Positive e) Hydrolysis of starch Positive f) Melanin-like pigment formation Negative 0) Utilization of carbon source (on Bridham-Godelive agar) = D-
Arabinose, D-xylose.

D-glucose, D-fructose.

Does not use raffinose, D-mannitol: It is clear that this strain belongs to actinomycetes from the properties of sucrose, inositol, and L-rhamnose. Manual of Determinative Batteriology, 8th edition (1974) by J. Bergier and Volume 2 of 1 The Actinomycetes by Waxman (1961)
As a result of comparison with many known bacterial species reported in
1891) Waxman & Henridge [St.
reptomy-ces albus (Ro26i-D
oria, 1891) Waksman and Hen
r-ici], but since it was not possible to determine for sure, this strain was compared to Streptomyces sp.
It was named 4457.

The production of the physiologically active substance Cl-010 is carried out by culturing Streptomyces sp. A 4457 under aerobic conditions in a medium containing various nutritional substances, roughly according to the method for producing general fermentation products. .

The medium is mainly a liquid medium, and the carbon source is glucose, molasses, starch, etc., either alone or in combination.

In addition, the physiologically active substance Cl-010 helps the growth of this strain.
Organic and inorganic salts that promote the production of can be added as appropriate. As the antifoaming agent, commonly used antifoaming agents such as Adekanol and silicone can be used.

As a culture method, aerobic culture such as shaking culture or aerated agitation culture is suitable, and the culture is carried out at pH 4-8.25-35°C for 3-6 days, preferably at pH 6-7.28-30JC for 4 days.

The physiologically active substance Cl-010 produced by this culture can be isolated according to a general method for collecting fermentation products.

Since the physiologically active substance Cl-010 is mainly accumulated within the bacterial cells, the following method is effective, for example.

That is, after completion of the culture, the physiologically active substance Cl-010 is extracted from the bacterial cells separated by centrifugation or filtration with an organic solvent such as a lower alcohol or acetone. After concentrating this extract, it is transferred to a water-insoluble organic solvent such as ethyl acetate or chloroform, and concentrated to form a syrup. Add this syrup again to ethyl acetate, acetone, chloroform,
Silica gel [Wako Gel C-200 (trade name, manufactured by Wako Pure Chemical Industries, Ltd.)] is dissolved in an organic solvent such as methanol.
The physiologically active substance Cl-010 can be purified and isolated by subjecting it to column chromatography using Sephadex LH-20 (trade name, manufactured by Pharmacia) and collecting the active fraction. .

Physiologically active substance Cl-010 isolated by the above purification method
has the following physical and chemical properties.

Physical and chemical properties a) Appearance: pale yellow powder b) Melting point: 97-99°C C) Elemental analysis values: C: 68.0%, H: 9.7% d) Molecular weight 2368 e) Molecular formula: C,, H ,,0°f) Specific optical rotation: [α] = +57°C (C = 1%, cHcI23)g
) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in an acidic methanol solution is shown in FIG.

h) Infrared absorption spectrum: The infrared absorption spectrum measured in the KBr tablet is shown in FIG.

i) H-NMR spectrum: H-NMR measured in dimethyl sulfoxide-α6
The spectrum is shown in FIG. -j) Solubility in solvents: Insoluble in water, soluble in methanol, chloroform, ethyl acetatek) Color reaction: Negative: Ninhydrin, Anthrone, Maurice Positive: Iodine reaction l) Distinction between basic, acidic, and neutral: No flagenase inhibitors with properties consistent with physical and chemical properties beyond acidity have been reported, and the physiologically active substance Cl-01
0 is a novel flagenase inhibitor.

[Effect of the invention] The physiologically active substance Cl-010, which is the target substance of the present invention, has the following properties:
Because it specifically inhibits human-derived collagenase, it is effective as an inhibitor of cartilage destruction in patients with rheumatoid arthritis and osteoarthritis.
Alternatively, it is useful as a cancer metastasis inhibitor.

[Example] Hereinafter, the present invention will be explained in more detail with reference to Examples and Test Examples.

(Example) (1) In a 500 m11 slope flask, 100 m11 medium
Contains 2g of Gurufus, 2g of oatmeal, and 0.0g of meat extract per serving.
3g 1 salt 0.3g, calcium carbonate 0.25g,
A sterile liquid medium of pH 7 consisting of 0.04 g of ferric sulfate and 0.04 g of manganese chloride was added, inoculated with Streptomyces sp. A 4457 strain, and incubated at 30°C for 7 days.
This was cultured with shaking for 2 hours, and used as a seed culture solution. Next, the content is 50
Using Jar Fermentor No. 1, 600 ml of the seed culture solution was inoculated into a sterile medium 301 having the same composition as the seed culture, and cultured with stirring and aeration at 30°C for 196 hours. The stirring number at this time was 350 r, p, m, and the aeration amount was 301. /minute.

After the culture was completed, the supernatant and the bacterial cells were separated using a centrifuge, and 2 kg (wet weight) of the resulting bacterial cells were extracted twice with 5i of methanol. The extracts were combined and concentrated to remove methanol, followed by extraction twice with ethyl acetate (11).

The ethyl acetate extracted fractions were combined and dehydrated with anhydrous sodium sulfate. - After dehydration at night, it was concentrated under reduced pressure, and 120ml of chloroform was added to the resulting brown syrup and dissolved.

Half of this chloroform solution was mixed with silica gel [Wafugel C-200 (trade name, manufactured by Wako Tsumugi Kogyo Co., Ltd.)] prepared with chloroform. 51 column. 31
After washing with chloroform, impurities were eluted with chloroform 31 containing 2% methanol, and then the fractions eluted with chloroform containing 10% methanol were collected and concentrated under reduced pressure to obtain 8 g of light brown syrup. Weigh out 2g of this syrup, add 20ml of methanol to dissolve it, and add 2j2 Sephadex LH-20 (trade name, manufactured by Pharmacia).
Gel filtration was performed using methanol using a column. After concentrating the obtained active fraction to dryness, it was dissolved in 50 ml of chloroform, and the chloroform solution was diluted twice with the same amount of diluted hydrochloric acid.
Subsequently, the mixture was washed twice with water and then concentrated to dryness. Obtained 1
, 3 g of the powder was dissolved in methanol, gel filtration was performed again using a Sephadex L H-20 column under the same conditions, and the active fraction was concentrated to dryness to obtain 1.1 g of a pale yellow powder. .

The melting point of this powder was 97 to 99°C, and the elemental analysis values were as follows.

Actual value; C: 68.0%, H: 9.7% Theoretical value; C
: 6g, 5%, H: 9.8% (test example) Test method Substrate: T, E,
-ff-Stone (T, E, Cawston) et al. [Methods φ in Enzymology (Meth
Acid-soluble flagens were extracted according to the method of ODs in Enzymology, Vol. 80, pp. 711-723 (1981).

Using this, an acetyl-labeled product with acetic anhydride was prepared and used as a substrate.Azocoll (manufactured by Calbiochem) was used as a substrate for bacterial collagenase.

Enzyme solution: Human collagenase was obtained by activating synovial fluid of a human rheumatoid arthritis patient by a method using trypsin [Inflammation, Vol. 4, pp. 247-254 (1984)]. In addition, as bacterial collagenase, Clostridium histolyticum type
stridium histolyticu+nryp
e■, manufactured by Sigma Corporation) was used.

Activity measurement method: The activity of human collagenase is measured using Nagai (N
[Biochim, Biophys, A
ct-a), Volume 497, Pages 190-199 (1
979)]. That is, the labeled flagen was dissolved in 5mM acetic acid to a concentration of 0.3%, and this solution was diluted with 200%
0.1 M Tris-HCl buffer pH 7.5, 1
After adding 0mM calcium chloride and sufficiently gelling at 37°C, 100μ of enzyme solution was added and reacted at 37°C for 18 hours.

After the reaction was completed, centrifugation was performed at 10.000 r, p, m for 10 minutes, and the radioactivity released in the supernatant was collected using a liquid scintillation counter (Minakisi, Tricarb 4.000).

(manufactured by Paracard Japan).

In addition, a method for measuring collagenase activity derived from bacteria is the method of A, J. Barrett et al. [Biochem L-Journal (Biochem, J.
), Volume 201, Pages 189-198 (1982
(2013)]. That is, 20 mg/yn of azocol
0.2M Tris-HCl buffer containing 0.5M sucrose and 20mM calcium chloride, pH 7.
5, and 1 ml of the enzyme solution was added to 1 mH of this solution, and the reaction was carried out at 37° C. for 30 minutes. The reaction is 1m
The mixture was stopped by adding 100 mM disodium ethylene diantetraacetate acid disodium (ED-'IA 2Na) solution, and after filtration with filter paper, the absorbance of the filtrate at 520 nm was measured.

Test results 50% of the physiologically active substance Cl-010 in the above measurement method
The activity inhibition concentration (IC1°) was 5 Xl0-'M for human collagenase and lXl0-'M for bacterial collagenase.

[Brief explanation of the drawing]

Figure 1 shows the ultraviolet absorption spectrum of the substance of the present invention, Figure 2 shows the infrared absorption spectrum of the substance of the invention, and Figure 3 shows the H-NMR spectrum of the substance of the invention.

Claims (1)

[Claims] (1) Physiologically active substance c I with the following physical and chemical properties
-010 a) Appearance: Pale yellow powder b) Melting point: 97-99°C c) Elemental analysis: C: 68.0%, H: 9.7% d) Molecular weight: 368 e) Molecular formula: C_2_1H_2_6O_5 f) Specific rotation Degree: [α]^2^6_D=+57℃ (C=1%, CHCl_
3) g) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in an acidic methanol solution is shown in FIG. h) Infrared absorption spectrum: The infrared absorption spectrum measured by the KBr tablet method is shown in FIG. i) ^1H-NMR spectrum: ^1H-N measured in dimethyl sulfoxide-α_6
The MR spectrum is shown in FIG. j) Solubility in solvents: Insoluble in water, soluble in methanol, chloroform, ethyl acetatek) Color reaction: Negative: Ninhydrin, Anthrone, Maurice Positive: Iodine reaction l) Distinction between basic, acidic, and neutral: Acidic