JPH01191694A - Substance having antitumor activity - Google Patents

Abstract

NEW MATERIAL:A substance SATS 2542 having an antitumor activity and the following physicochemical properties and produced by an microorganism belonging to the genus Alteromonas. Decomposition point: decomposes at 213-245 deg.C into a brown color; specific rotation: [alpha]<20>D=+25 deg.(c=0.02, water); an aqueous solution of 1.0mg/ml is weekly acidic - neutral: solubility:soluble in water and slightly soluble or insoluble in organic solvents such as alcohols. acetone, ethyl ether, hexane, chloroform and ethyl acetate, mol. wt.:>=100,000 (gel filtration method); coloring reaction: positive to biuret reaction. Folin's reaction, anthrone sulfuric acid reaction and cysteine sulfuric acid reaction and negative to ninhydrin reaction, etc. USE:An antitumor agent. PREPARATION:Alteromonas sp.SCRC-2542 (FERM P-9819) is cultured.

Description

[Detailed description of the invention] [Industrial application field] The present substance is derived from Alteromonas
A novel antitumor active substance SA produced by microorganisms belonging to the genus
TS2542, a microorganism that produces the substance, and a method for producing the substance.

[Conventional technology]

Although there has been remarkable progress in chemotherapeutic agents in the field of cancer treatment, there are still many unsatisfactory points. At this stage, isolation of antitumor active substances from nature has become an important issue with the aim of developing cancer therapeutic agents.

Although many antitumor active substances found from natural sources including microorganisms are known, very few antitumor active substances isolated from marine microorganisms are known, especially antitumor active substances produced by microorganisms of the genus Alteromonas. No active substances are known.

[Problem to be solved by the invention]

Therefore, the present invention seeks to obtain a new type of antitumor active substance from marine microorganisms, which have not been studied in the past as sources of antitumor active substances.

The present inventors solved various difficulties often encountered when dealing with marine microorganisms, and as a result of extensive research on marine microorganisms, a new microorganism belonging to the genus Alteromonas has a new type of antitumor active substance. They discovered that it can be produced and completed the present invention.

Therefore, the present invention provides a novel anti-tumor active substance SATS2542 produced by a microorganism of the genus Alteromonas, a new microorganism of the genus Alteromonas that produces the substance, and the method of culturing the microorganism and collecting the anti-tumor active substance from this culture. The present invention aims to provide a method for producing the antitumor active substance, which is characterized by the following.

The antitumor active substance SATS2542 of the present invention has the following properties.

1. Decomposition point: browns and decomposes between 213°C and 245°C.

2. Specific optical rotation: [α] 0 = +25° (c = 0.
02°H2O) 3. Ultraviolet absorption spectrum: Q. The spectrum of the 02% aqueous solution shows no characteristic absorption in the range of 220 to 360 nm.

This chart is shown in FIG.

4. Infrared absorption spectrum: In measurement using a KBr disk, absorption due to peptides is shown at around 3300 and 1640 cm-', and absorption due to sugar is shown at around 840 cm-'. This chart is shown in FIG.

5. Distinction between basic, acidic, and neutral: 1. Omg/mf aqueous solution exhibits weak acidity to neutrality.

6. Color of substance: White with a slight brownish tinge.

7. Solubility in solvents: Soluble in water, poorly soluble or insoluble in organic solvents such as alcohols, acetone, ether, hexane, chloroform, and ethyl acetate.

8. Molecular weight: 100,000 when measured by Nigel filtration method
The above is shown.

9. Color reaction Buret reaction Juninhydrin reaction - Folin reaction Tenanthrone sulfuric acid reaction Cysteine sulfuric acid reaction Based on more than ten findings, it is estimated that the substance SATS2542 is composed of sugar and protein.

As specifically shown later, this substance SATS2542 was produced in vitro using mouse lymphocytic leukemia cells P388.
tr.

In the activity test, 50% inhibitory concentration IC.

1,8 x 10-3n/m! It is a powerful antitumor agent.

The microorganisms used in the present invention may belong to the genus Alteromonas and can produce the substance SATS2542, and such microorganisms can be newly isolated from nature.

As an example of a microorganism belonging to Alteromonas, the substance SAT
The present inventors newly isolated a microorganism capable of producing S2542, Alteromonas.
We can mention 5CRC-2542, which was identified as a new microorganism belonging to the genus. This new strain of microorganism 5CRC-2542 belonging to the genus Alteromonas has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microbiological Laboratory No. 9819.

This new bacterial strain was isolated as follows.

A medium having the composition shown in the table below was prepared.

Meat extract 1.0% Peptone 160% NaCl 0.5% Agar 1.5% Tap water
pl+7.0 This agar plate medium was inoculated with marine samples collected from various oceans, diluted appropriately with sterilized physiological saline, and cultured at 25° C. for 1 to 2 days.

Colonies that appeared on this agar plate were plated onto a slanted medium with the same medium composition.

In this way, a large number of bacterial strains were isolated from marine samples collected from oceans around the world. Next, the agar was removed from the medium shown in the table, and 5 mJ each was dispensed into test tubes, which were sterilized in the same manner. Each strain was cultured in this medium at 25°C for 21 to 2 days. In this way, the following bacterial strain producing the substance SATS2542 was obtained from a marine sample collected from Sagami Bay.

This new strain has the following mycological properties.

Observation items a) Morphology ■ Cell shape Short bacterium size
1. OX 2.0 μm2 Presence or absence of polymorphism No 3 Presence or absence of motility Flagellated epiphytic status Monopolar 4 Presence or absence of spores No 5 Gram staining Negative 6 Acid-fast negative b) Growth status in each medium 1 Broth agar plate culture (25℃, 2 days) A) Colony shape (diameter) 1.5-2.01 O) Colony shape
Circular C) Colony surface shape Smooth 2) Protruding state of the colony Tablet-shaped C) Peripheral edge of the colony To the golden edge) Color tone of the colony Pale yellow G) Transparency of the colony Translucent C) Gloss of the colony Gloss) Production of soluble pigment None 2 Meat juice agar slant culture (25℃, 2 days) a) Quality of growth Good ■) Colony gloss Gloss 3 Meat juice liquid medium (25℃, 2 days) a) Surface growth No o) Turbidity Cloudy c) Precipitation Powder 2) Gas generation None 4 Meat juice gelatin (25℃, 2 days) Gelatin liquefaction Liquefaction 5 Lidomus milk Reduction/coagulation/liquefaction C) Physiological properties 1 Nitrate reduction +2 Denitrification
-3MR- 4VP -5 Indole production - 6 Hydrogen sulfide production - 7 Starch hydrolysis - 8 Citric acid utilization a) Koser
-0) Christensen
+9 Use of nitrates -1
0 pigment production a) King 8 medium
-n) King B medium
-1) Urease -12 Oxidase +13 Catalase
+14 Growth range a) p) I
6-100) Temperature
5°C to 30°C15 Attitude towards oxygen Aerobic 160-F test (glucose) - Acid and gas production from 17 sugars 1, L-arabinose - 2, D-gysylose - 3, D-glucose + 4, D-mannose - 5, D-Fructose ± 6, D-Galactose - 7, Maltose - 8, Sea! Sugar -9, lactose
-10, trehalose
-1), 0-Sorbit -12
, D-Mannitol-13, Inoshift-14, Glycerin
- 15, Starch - (However, gas production - all items -) d) Other properties Growth on SS agar medium Growth on Ten Pine Conkey agar medium + 6.5% NaCl salt tolerance Ten DNase
Decomposition of 10 ornithine - Decomposition of arginine - C+C content 46.3% Decomposition of gelatin 10 NaCj! Requirements From the ten or more properties, this strain 5CRC-2542 has the following major characteristics.

■ Durham negative ■ Motile ■ Non-spore-forming aerobic bacterium ■ O-F test negative ■ Positive for dithalase and oxidase ■ - has book polar flagella ■ Gelatin, has DNA-degrading ability ■ Acid from glucose Production (+), gas production (-) ■
NaCl or seawater auxotrophy [phase] G+C content 46.3% ■ Has nitrate reducing ability 0 Ornithine degrading ability (-) The taxonomic position of this strain 5CRC-2542, which has these properties, can be found in the Verge's Manual ·of·
Bergey'
s Manual of Determinative
Bacteriology, 8th edition, 1974, and Bergey's Manual of Systematic Bacteriology.
f Systematic Bacteriology
) Volume 1, pp. 343-352, 1984 classification criteria,
Furthermore, Manual of Clinical Microbiology (Manual of C1) clinical Mi
crobiology) 3rd edition - 1980 and Journal of General Microbiology (Jou
rnal of General Microbiol
ogy) 1293057-3074 (1983), it was identified as follows.

Regarding the items from ■ to [phase] above, Alteromonas pyurefaciens (A l teromon
It matched with (as to take a look at). However, this strain was identified as a new microorganism belonging to the genus Alteromonas because it has the ability to reduce nitrate and does not have the ability to degrade ornithine or utilize citric acid, maltose, or sucrose.

Although this new bacterial strain isolated from nature has been described in detail above, it is also possible to obtain a strain with even higher productivity by causing mutations in this bacteria.

The strain of this invention can be stored according to conventional methods,
For example, it can be stored on an agar slant medium or by freeze-drying. As the agar slant medium, a medium commonly used for preserving Alteromonas bacteria, such as the medium described above for bacterial isolation, can be used. Furthermore, freeze-drying preservation can be carried out according to conventional methods.

The substance of the present invention SATS2542 is obtained by culturing the above-mentioned microorganism.
When producing a microorganism, any basic nutrient medium may be used as long as the microorganism of the present invention can grow therein.

This medium contains one or more nitrogen sources, such as yeast extract, peptone, meat extract, and the like. In addition, this medium may contain various types of 1! as a carbon source if necessary. You can add types. This medium contains sodium chloride,
Alternatively, it is necessary to add natural seawater or artificial seawater.

Either a solid medium or a liquid medium may be used for culture, but in order to obtain a large amount of the target substance SATS2542, a liquid medium is used and aerobic culture is performed using static culture, shaking culture, aeration/agitation culture, etc. It is preferable to carry out the culturing under these conditions. The culture temperature is such that the bacteria grow and the substance SATS2542
Any temperature is acceptable as long as it is produced within the temperature range.
Preferably 5-30"C, more preferably 15"C
~25°C. The pH ranges from 6 to 9, preferably from 7 to 8. The incubation time is the amount of material that can be collected.SATS25
Just choose the time when 42 is produced, preferably 8 to 4
It is 8 hours.

Next, the substance SATS2542 of the present invention is collected from the obtained culture, for example, in the following manner.

The above culture solution was first centrifuged to submerge the bacterial cells, and the supernatant was passed through a 0.2 ttm sterilization filter to remove the bacterial cells. The active substance was then concentrated by ultrafiltration.

This was fractionated using ammonium sulfate, and the precipitated portion was collected with a 50% saturated ammonium sulfate solution. Then, gel filtration was performed using Sephacryl S-300 to obtain a 0.
2 M NaCj! It was eluted with an aqueous solution, and a portion showing activity in an activity test using P2S5 was collected. After desalting using a semipermeable membrane, the product was freeze-dried to obtain SATS2542.

The method will be explained in more detail below using Examples and Test Examples, but is not limited to this method.

Meat extract 1.0%, peptone 1.0%, NaCj 20.
Medium 101 containing 5% and adjusted to pH 7.0 was added to 12
After heat sterilization at 1°C for 15 minutes, a new microorganism belonging to the genus Alteromonas, 5CRC-2542 (?A Koken Bacterial Collection No. 9819), was inoculated and cultured aerobically at 25°C for 24 hours. After culturing, the supernatant was collected using a centrifuge.

The culture supernatant (101) thus obtained was filtered through a sterile filter (0.2 m) and concentrated to 150 + nl using an ultrafiltration membrane with a molecular weight cutoff of 30,000 (Amicon). Ammonium sulfate was added to this concentrated solution, and both fractions that precipitated at 50% saturation were separated by centrifugation. This was made into an aqueous solution of about 3-3%, placed on a Sephacryl S-300 column (IX 37 cm), and 0.2M NaC1 column was added. Elute and culture cells P388
Both fractions (A) having growth-inhibitory activity against . The elution curve is shown in FIG. The active fraction eluate of A was desalted with a dialysis membrane and then lyophilized to form the antitumor substance SMAS254.
I got 7■ of 2.

The antitumor activity of SATS2542, the substance of the present invention, was confirmed as follows.

Mouse lymphocytic leukemia cells (P2S5) were added to RPMI-1640 culture medium containing 10% tallow serum, and the cultured cells were adjusted to 5 x 10' cells/ml. The substance SATS2542 obtained in 1 was added to a predetermined concentration and cultured at 37°C for 2 days. The number of floating cells was counted using a Coulter counter, and the 50% cell growth inhibition concentration (IC5°) was determined from the growth inhibition rate relative to the control group.

The results are shown in the table below.

SATS2542
1.8 x 10 Bow ADM (known substance)
1.3X105-FU (known substance>
4.9X10-2

[Brief explanation of the drawing]

FIG. 1 shows Example 1 of the present invention. The substance obtained in SATS2
542 is an ultraviolet absorption spectrum diagram. FIG. 2 shows Example 1 of the present invention. The substance obtained in SATS2
542 is an infrared absorption spectrum diagram. FIG. 3 shows Example 1 of the present invention in chromatography using a Sephacryl S-300 column. 2 is a graph showing the state of elution of the substance SATS2542 obtained in FIG.

Claims (1)

[Claims] 1. SATS2542, an antitumor active substance produced by microorganisms belonging to the genus Alteromonas and having the following physical and chemical properties: (1) Decomposition point: browns and decomposes between 213°C and 245°C. (2) Specific optical rotation: [α]＾2＾0_D=+25°(c=
(0.02, H_2O); (3) Ultraviolet absorption spectrum: The spectrum of a 0.02% aqueous solution shows no characteristic absorption in the range of 220 nm to 360 nm; (4) Infrared absorption spectrum: 3300 cm^ when measured with a KBr disk. -^1 and 1640cm^-
Absorption due to peptide is shown near ^1, 840 cm^-
Absorption by sugars occurs near ^1; (5) Basic, acidic, and neutral: 1.0 mg/ml aqueous solution is weakly acidic to neutral; (6) Solubility in solvents: in water It is soluble and poorly soluble or insoluble in organic solvents such as alcohols, acetone, ethyl ether, hexane, chloroform, and ethyl acetate; (7) Molecular weight: 100 when measured by gel filtration method.
. 2. Antitumor active substance SATS2542 according to claim 1
Alteromonas produces
Genus Microorganisms. 3. The microorganism is Alteromonas sp. SCRC-25
3. The microorganism according to claim 2, which is 42 (Feikoken Bibori No. 9819). 4. Antitumor active substance SATS254, characterized in that the microorganism according to claim 2 is cultured, and the antitumor active substance SATS2542 according to claim 1 is collected from the culture solution.
2. Manufacturing method.