JPH05284977A - New antibiotic leucoplastin and its production - Google Patents

Abstract

(57) [Summary] (Modified) [Purpose] It has an antitumor activity and also has an effect of enhancing the production of interleukin-3 in mammalian splenocytes, and has a medical effect on leukopenia and thrombocytopenia. A novel antibiotic leukoplastin having the following chemicals and a method for producing the same. [Structure] Leukoplastin-producing bacterium belonging to the genus Streptomyces, for example, MI696-AF3 strain
No. 738), and a novel antileptic substance leukoplastin collected from the culture and a method for producing the same.

Description

Detailed Description of the Invention

[0001]

INDUSTRIAL APPLICABILITY The present invention has a novel antitumor activity, an activity to enhance the production of interleukin-3 in mammalian splenocytes, and a medical activity to leukopenia and thrombocytopenia. The present invention relates to the antibiotic leukoplastin, and also the present invention relates to a method for producing leukoplastin.

[0002]

2. Description of the Related Art About 5,000 kinds of antibiotics have been reported so far as antibiotics produced by microorganisms.
These antibiotics are widely used for the treatment of tumors, cancer and bacterial or other infectious diseases or for other purposes. Among them, it is known that microorganisms belonging to the genus Streptomyces produce antibacterial antibiotics such as kanamycins and antitumor antibiotics such as adriamycin and aclacinomycin.

[0003]

However, there is a demand for an antibiotic which has a stronger antitumor activity than that of the conventionally known antitumor antibiotics and has a low toxicity and which is effective in treating human cancer.

Further, there is a need to obtain new antibiotics having biological activity useful in the treatment of various diseases.

[0005]

[Means for Solving the Problems] The present inventors have conducted various researches for obtaining novel antibiotics having useful biological activity. As one of the results, one strain of actinomycetes isolated from a soil sample collected in Zushi City, Kanagawa Prefecture, was used to cultivate a strain with a strain number of MI696-AF3, and mouse spleen was added to the resulting culture solution. It was found that an antibiotic having an activity of enhancing the ability of cells to produce interleukin-3 (IL-3) is produced. We have
We proceeded with research, succeeded in isolating this antibiotic from the culture medium, studied the physicochemical properties of this antibiotic, and as a result confirmed that this antibiotic is a novel substance and confirmed it. Leukoplastin
I named it.

As a result of further research, a novel antibiotic, leukoplastin, is used as an anti-cancer agent (carcinostati) in vivo against mouse Ehrlich solid tumors transplanted in mice.
c) It was found to exhibit activity. Further, it was found that leukoplastin has a therapeutic effect on leukopenia and thrombocytopenia in blood experimentally induced in mice.

Therefore, in the first aspect of the present invention, it is a colorless powder which exhibits the following characteristics, that is, the behavior of amphotericity, and the elemental analysis values are carbon 58.96%, hydrogen 6.81%, nitrogen 6.
46%, oxygen 27.44%, specific rotation is [α] _D
²⁸ = + 210.0 ° (c 0.75, methanol), and the ultraviolet absorption spectrum (in methanol) is log
ε: 296.0 nm (5.15), 233.6 n
m (5.03), and infrared absorption spectrum (KBr
Tablets are as shown in FIG. 1 of the accompanying drawings, and are easily soluble in methanol, soluble in water, sparingly soluble in chloroform, and positive for the ninhydrin reaction. Salt is provided.

In the second aspect of the present invention, a leukoplastin-producing bacterium belonging to the genus Streptomyces is cultured, and an antibiotic leucoplastin having the above-mentioned characteristics is collected from the culture. A method of making leukoplastin is provided.

The leukoplastin according to the present invention is an antibiotic discovered using as an index that it has an activity of enhancing IL-3 production of mouse splenocytes, and its acute toxicity to mammals is low. In addition, leukoplastin is an antibiotic that has anti-tumor activity against mouse Ehrlich solid tumor in vivo and has a medical effect to restore leukopenia and thrombocytopenia.

Next, the physicochemical properties of leukoplastin will be described.

Shape: colorless powder Elemental analysis: carbon 58.96% hydrogen 6.81%, nitrogen 6.46%, oxygen 27.44% SIMS: 421 (M + H) ⁺ molecular formula: C ₂₁ H ₂₈ N ₂ O ₇ melting point: 181-183 ° C specific optical rotation Degree: [α] _D ²⁸ + 210.0 ° (c 0.75, methanol) Solubility: Easily soluble in methanol, soluble in water, poorly soluble in chloroform.

Ultraviolet absorption spectrum (in methanol): l
ogε: 296.0 nm (5.15), 233.6 nm (5.03)

Infrared absorption spectrum: FIG. 1 of the accompanying drawings shows the infrared absorption spectrum of leukoplastin obtained by the potassium bromide tablet method. In Fig. 1, the horizontal axis is the wave number (cm ^-1 )
And the vertical axis represents the transmittance (%).

Proton Nuclear Magnetic Resonance Spectra: The proton nuclear magnetic resonance spectrum of leukoplastin in heavy water is shown in FIG. 2 of the accompanying drawings. The horizontal axis of FIG. 2 represents ppm. The internal standard is tetramethylsilane.

¹³ C nuclear magnetic resonance spectrum (in heavy dimethyl sulfoxide): The main peaks are shown below. The internal standard is tetramethylsilane.

191.4s, 175.8s, 171.9s, 170.2s, 136.5
s, 129.4d, 128.4d (2), 126.7d (2), 93.4s, 76.2d
, 68.3d, 65.6t, 65.1s, 56.0d, 42.4d, 37.8t
, 22.2t, 20.1q, 8.8q (Note): s is a singlet, d is a doublet, t is a triplet, and q is a quartet.

Since leukoplastin has the above-mentioned physicochemical properties and nuclear magnetic resonance spectrum, and no known substance corresponding to this property has been reported, leukoplastin was determined to be a novel antibiotic. ..

Leukoplastin is an amphoteric substance, and is a pharmaceutically acceptable inorganic acid such as hydrochloric acid, sulfuric acid or phosphoric acid,
Alternatively, it reacts with a pharmaceutically acceptable organic acid such as acetic acid or methanesulfonic acid to form an acid addition salt. Also, leukoplastin is reacted with an alkali metal or alkaline earth metal hydroxide to form a metal salt, such as a sodium salt or a potassium salt. It also forms a salt with an alkylamine.

The biological properties of leukoplastin are described below.

(A) The effect of leukoplastin on mouse splenocyte interleukin-3 (IL-3) -producing ability was examined by the following test.

After removing the spleen from a 15-week-old female CDF ₁ mouse and dissociating the spleen cells, the cell concentration was 5 in a minimum essential medium containing 10% fetal bovine serum [MEM, manufactured by Nissui Pharmaceutical Co., Ltd.]. The cells were suspended so that the density was × 10 ⁶ cells / ml. 100, 10, 1 and 0.1 μg / m 2 respectively in this cell suspension
Leukoplastin was added at a concentration of 1 and cultured in a carbon dioxide gas incubator (CO ₂ concentration 5%) at 37 ° C. for 5 days. 100 μl of this culture supernatant was added to a suspension of IL-3 dependent IC-2 cells adjusted to a cell concentration of 2 × 10 ⁵ cells / ml at a volume ratio of 1: 1 and further added for 24 hours. Cultured.

Radiolabeled thymidine (10 nCi, manufactured by Amersham) was added 8 hours before the end of the culture. The cultured cells were fixed on the filter paper by a cell harvester, and the radioactivity value of the whole cultured cells was measured by a liquid scintillation counter. The radioactivity (C) of the cells of the control group, which was the cells cultured without addition of leucoplastin, was 100.
Assuming that and compared with this, the percentage value (T / C,%) of the ratio of the radioactivity value (T) of the cells of the leukoplastin treatment group
Was evaluated as the increase rate of IL-3 production, and the results are shown in Table 1 below.

As is clear from Table 1, leukoplastin significantly enhanced IL-3 production in mouse splenocytes over a wide range of leukoplastin concentrations.

[0024]

(B) The effect of leukoplastin on peripheral blood leukocyte count and platelet count in mammals was examined in the following test.

50 mg / kg of cyclophosphamide, which is known as an antitumor agent having an inhibitory activity on blood cells, was intraperitoneally administered twice on the first and fifth days of the test.
Leukoplastin was added to F ₁ mice from the first day of the test.
Each of them was intraperitoneally administered at a dose of 50.5, 0.5 and 0.05 mg / kg once a day for a total of 6 times for 6 consecutive days (treatment group, 5 animals per group). The control group (5 animals per group) received cyclophosphamide twice, but not leukoplastin.

On the 7th day of the test, blood was collected from the orbital vein of each individual, and the white blood cell count and platelet count were measured with a Coulter counter. In order to examine the effect of leukoplastin, the rate of increase in the number of leukocytes or the number of platelets (%) in the treated group that received leukoplastin administration compared to the control group was calculated from the following formula.

[0028]

The leukocyte count increase rate by leukoplastin administration is shown in Table 2 below, and the platelet count increase rate by leukoplastin administration is shown in Table 3.

Table 2 Effect of increasing leukoplastin on white blood cell count Leukoplastin dose (mg / kg) White blood cell count increase rate (%) 50 145 5 185 0.5 197 0.05 0.05 133

Table 3 Effect of increasing leukoplastin on platelet count Leukoplastin dose (mg / kg) Platelet count increase rate (%) 50 338 5 538 0.5 502 0.05 109

As is clear from Tables 2 and 3 above, leukoplastin showed a significant increasing effect on the white blood cell count and platelet count in a wide range of doses. From this result, it is considered that this substance is useful for recovery of various diseases showing leukocyte and thrombocytopenia, that is, anemia, leukemia, and deficiency of hematopoietic function such as leukocyte and thrombocytopenia caused by the use of carcinostatic substances.

(C) Ehrlich solid tumor-bearing mice were treated with leukoplastin for a treatment experiment by the following test.

A suspension of Ehrlich's ascites cancer cells was transplanted into ICR mice subcutaneously in the groin so that the number of transplanted cancer cells was 2 × 10 ⁶ cells / mouse. From the 7th day after transplantation of cancer cells, leukoplastin was added to each of 12.5, 6.25, 3.1.
Oral doses of 1.56 and 0.78 mg / kg were orally administered once a day for a total of 7 consecutive 7 days (leukoplastin administration group, 4 animals per group). Leukoplastin was not administered to the control group (8 animals per group). From transplantation of cancer cells 1
On the 5th day, the solid tumor was taken out and weighed, and the decrease in the solid tumor weight (T) of the leukoplastin-administered group relative to the solid tumor weight (C) of the control mouse was taken as the inhibition rate to It was calculated by the calculation formula.

[0035]

The results are summarized in Table 4 below. As is clear from Table 4, leukoplastin showed a remarkable growth inhibitory activity against mouse Ehrlich solid tumors.

Table 4 Leukoplastin dose (mg / kg) Inhibition rate (%) 12.5 48 6.25 41 3.1 49 1.56 20 0.78 16

(D) Acute toxicity of leukoplastin When leukoplastin was intraperitoneally administered to female ICR mice and evaluated, the LD ₅₀ of leukoplastin was 500 m.
It was more than g / kg.

To produce leukoplastin in the second method of the present invention, leukoplastin-producing bacteria belonging to the genus Streptomyces are cultured in a medium containing a nutrient source.
An example of a leukoplastin-producing bacterium that can be used in this method is an actinomycete isolated from a soil sample collected in Zushi City, Kanagawa Prefecture in February 1988, MI696-AF3.
There are strains with the strain numbers. MI696- above
The mycological properties of the AF3 strain are as follows.

1. Form MI696-AF3 strain forms straight aerial hyphae which are relatively longer than the basal hyphae branched under the microscope, but no helix formation or limbus is observed. 5 at the tip of the aerial mycelium
A chain of 0 or more spores is recognized, and the size is 0.6 ×
It is about 1.0 to 1.2 microns. The surface of spores is smooth.

2. Regarding the state of growth in various culture media The standard shown in [] is the Color Harmony Manual (Container Corpo) of the Continer Corporation of America.
relation of America Color h
armonymanual) was used.

(1) Sucrose / nitrate agar medium (2
Culture at 7 ° C) White to pink-white aerial hyphae are slightly settled on colorless growth, and soluble dye is not observed.

(2) Glucose / asparagine agar medium (27 ° C. culture) Growth light yellow [2ca, Lt Ivory-2ea,
Lt White], aerial mycelium white to pink white [3ca,
[Pearl pink], soluble dyes are not observed.

(3) Glycerin / asparagine agar medium (ISP-medium 5,27 ° C. culture) light yellow tea [3ie, Camel] to yellow tea [3le, Ci]
nnanmon] has a pink white to light pink [4]
ca, Fresh Pink] aerial hyphae are settled, and the soluble pigment exhibits yellow tea.

(4) Starch / inorganic salt agar medium (ISP
-Culture at 4,27 ° C) Light yellow [2ea, Lt What] to light yellow tea [2
ic, HoneyGold] develops aerial hyphae of light pink [4ca, Fresh Pink], and no soluble pigment is observed.

(5) Tyrosine agar medium (ISP-medium 7, 27 ° C. culture) Growth light yellow [2ea, Lt wheat] to light yellow tea [2ic, Honey Gold], aerial mycelia brown-white [3ca, Pearl Pink] ], Soluble dyes are not observed.

(6) Nutrient agar medium (27 ° C. culture) Growth is colorless, no aerial hyphae do not grow, and no soluble pigment is observed.

(7) Yeast-malt agar medium (ISP-
Medium 2, 27 ° C culture) Light yellow tea [2ic, Honey Gold ~ 3le,
Cinnamon] develops light pink [4ca,
Fresh Pink-5ca, FreshPin
K] aerial hyphae are settled and no soluble pigment is observed.

(8) Oat mill agar medium (ISP-medium at 3,27 ° C.) Growth is colorless, and aerial mycelium is pink white to light pink [4ca,
Fresh Pink-5ca, Fresh Pin
k], no soluble dye is observed.

(9) Glycerin / nitrate agar medium (27
℃ solubilization) For the development of light yellow [1 1/2 ea, Lt Yellow] to yellow tea [3 ng, Yellow Maple],
An aerial mycelium of brown-white [3ca, Peal Pink] was settled, and a soluble dye of yellow tea was slightly observed.

(10) Starch agar medium (27 ° C. culture) Growth light yellow to light yellow tea [2 gc, Bamboo] Aerial mycelium light pink [4 ca, Fresh Pink]
No soluble dye is found.

(11) Lime malate agar medium (27 ° C. culture) White aerial mycelium grows on colorless growth and no soluble pigment is observed.

(12) Cellulose (synthetic solution containing filter paper pieces,
(27 ° C. culture) White aerial mycelium grows faintly on colorless growth, and no soluble pigment is observed.

(13) Gelatin stab culture Both 15% simple gelatin medium (20 ° C. culture) and glucose / peptone / gelatin medium (27 ° C. culture) showed colorless growth, no aerial mycelium growth, and soluble dye. I can't.

(14) Skim milk (cultured at 37 ° C.) Growth is colorless, aerial hyphae do not grow, and soluble dyes are slightly brownish.

3. Physiological properties (1) Growth temperature range Starch yeast agar medium (soluble starch 1.0%, yeast extract [Nippon Pharmaceutical Co., Ltd.] 0.2%, string agar 3.0%, pH 7.0 to 7.2) ) At 20 ° C., 24
As a result of the test at each temperature of ℃, 27 ℃, 30 ℃, 37 ℃, 50 ℃, MI69 at any temperature except 50 ℃
Although the 6-AF3 strain developed, the optimum growth temperature of this strain seems to be around 30 ° C to 37 ° C.

(2) Liquefaction of gelatin (15% simple gelatin, 20 ° C culture; glucose peptone gelatin, 2
Cultivation at 7 ° C) In a simple gelatin medium, liquefaction was observed around the 7th day after culturing, and its action was moderate to strong. glucose·
In the peptone-gelatin medium, liquefaction was observed around the 11th day after culturing, and the rate thereof was slow and moderate.

(3) Hydrolysis of starch (starch / inorganic salt agar medium and starch agar medium, both were cultured at 27 ° C.) Both starch / inorganic salt agar medium and starch agar medium were found to be hydrolyzable from about 3 days after the culture. The action is strong.

(4) Coagulation / peptonization of defatted milk (defatted milk, 37 ° C. culture) Without coagulation, peptonization started from the 8th day after culturing, and the rate slowly progressed to the 3rd week. Complete.

(5) Production of melanin-like pigment (tryptone yeast broth, ISP-medium 1; peptone yeast iron agar medium, ISP-medium 6; tyrosine agar medium, ISP-medium 7, both at 27 ° C. ) Trypton East Bros, Peptone East
Negative on iron agar and tyrosine agar.

(6) Utilization of carbon source (Pridham-Gotrieve agar medium, ISP-medium 9, 27 ° C. culture) D-glucose, L-arabinose, D-xylose,
It grows well using rhamnose and lactose, and does not use inositol, D-fructose, sucrose, D-mannitol and raffinose.

(7) Dissolution of lime malate (lime malate agar medium, 27 ° C. culture) Positive.

(8) Nitrate reduction reaction (0.1% potassium nitrate-containing peptone water, ISP-medium 8, 27 ° C. culture) is positive.

(9) Decomposition of cellulose (synthesis solution with filter paper pieces added, culture at 27 ° C.) Negative.

Summarizing the above properties, MI696-A
The aerial hyphae of strain F3 are linear, no helix formation or limb branching is observed, and the surface of spores is smooth. M in various media
The I696-AF3 strain grows colorless or light yellow to light yellow tea with pink-white to light-pink aerial mycelium growing, and no soluble pigment is observed, or slightly brownish. The production of melanin-like pigment is negative in any of tryptone yeast broth, peptone yeast iron ferment agar, and tyrosine agar. Proteolytic power is moderate to
He is a strong person and has a strong water degradability of starch. The 2.6-diaminopimelic acid contained in the cell wall was LL-type.

From these properties, it is considered that the MI696-AF3 strain belongs to the genus Streptomyces . Furthermore, a search for closely related known strains revealed that Streptomyces roseofulbus [ Strre
ptomyces roseofulbus , reference:
1) "International Journal
f Systematic Bacteria
y "18, p. 165 (1968); and 2)" Int.
international Journal of System
"Electric Bacteriology", Volume 30, 3
99 (1980)] and Streptomyces roseospors [ Streptomyces roseosespo]
rus , reference: “International Jou
rnal of Systematic Bacter
"Iology", Vol. 18, 370 (1968)].

Therefore, the MI696-AF3 strain and these strains were compared and examined in practice. The results are summarized in Table 5 below.

Table 5

As is clear from Table 5, MI696-A
The F3 strain is similar to both Streptomyces roseosporus and Streptomyces roseofurbus. However, the MI696-AF3 strain does not show a soluble pigment of a pH indicator (oxidation-reduction indicator) possessed by the latter, which is greatly different from the latter in the utilization of D-fructose, sucrose, and raffinose. On the other hand, Streptomyces roseosporus and MI696-AF3
The strain performed very similar in all respects.

Therefore, the MI696-AF3 strain was treated with Streptomyces roseosporus ( Streptomyces).
roseosporus ) MI696-AF3.

The MI696-AF3 strain was applied for deposit at the Institute of Microbial Science and Technology, Institute of Industrial Science and Technology, Tsukuba City, Ibaraki Prefecture, and on March 2, 1989, it was entrusted as Microbiology Research Institute No. 10598, and in 1990 22:00 on March 22
No. (FERM BP-2738), which has been deposited under the terms of the Budapest Treaty.

Cultivation of the MI696-AF3 strain also produces an antitumor antibiotic, conagenin (JP-A-2-306953 and European Patent Application No. 0399).
444 specification).

In the second method of the present invention, leukoplastin is produced as follows. That is, the production of leukoplastin according to the present invention is carried out by culturing a leucoplastin-producing bacterium in a medium containing a nutrient that can normally be used by microorganisms, and separating leucoplastin from the culture.

The carbon source, nitrogen source, and inorganic substance used in the medium may be any types as long as the strain used can assimilate. For example, carbon sources include mannose, glucose,
Galactose, maltose, glycerol, dextrin, starch and molasses can be used. As the nitrogen source, soybean flour, meat extract, peptone, yeast extract, corn steep liquor, fish meal, nitrogen-containing organic substances such as wheat germ and ammonium chloride, ammonium phosphate,
Various ammonium salts such as ammonium sulfate and ammonium nitrate can be used. As the inorganic substance, in addition to calcium carbonate, potassium chloride, sodium chloride, phosphate, calcium carbonate, magnesium sulfate, iron sulfate, manganese chloride, zinc sulfate and the like, trace amounts of heavy metal salts are appropriately used. Animal and vegetable oils can also be added as an antifoaming agent.

Although the culture conditions vary depending on the medium composition, etc.,
For example, the culture is performed under aerobic conditions such as shaking culture or aeration-agitation culture. Aerobic deep culture can also be performed.

MI69 was used as a leukoplastin-producing bacterium.
When the 6-AF3 strain is used, the culture of this strain is 25-
It may be carried out at a temperature of 40 ° C, but preferably at 28 to 37 ° C. Leukoplastin is produced and accumulated in the obtained culture, particularly in the culture solution.

In order to isolate and purify the leukoplastin of the present invention from the culture thus obtained, the usual methods for isolating antibiotics from the culture solution of microorganisms such as solvent extraction, adsorption or partitioning are used. The chromatography method, gel filtration method, dialysis method, precipitation method and the like can be used alone or in combination, or can be used repeatedly. For example, leukoplastin is adsorbed on the activated carbon from the culture filtrate and extracted from the activated carbon with 50% acetone water.

To further purify leukoplastin, it is advisable to carry out chromatography using an adsorbent such as silica gel or alumina. The active portion can be confirmed by silica gel thin layer chromatography (TLC).

[0079]

EXAMPLES The present invention will now be described with reference to examples, but the present invention is not limited thereto.

Example 1 Streptomyces roseospor cultivated on an agar slant medium
us) MI696-AF3 strain (Microtech Lab.
No.), galactose 2.0%, dextrin 2.0
%, Soy peptone (Difco Bact Soyton) 1.
0%, corn steep liquor [Nippon Shokuhin Kako Co., Ltd.] 0.5%, ammonium sulfate 0.2%, calcium carbonate 0.2%, defoaming silicone oil [Shin-Etsu Chemical Co., Ltd. Silicon KM70] One platinum loop was inoculated into two Erlenmeyer flasks with waffles each containing 110 ml of a liquid medium (pH 7.4) consisting of 0.03%, and the mixture was shake-cultured at 27 ° C. for 3 days.

The obtained culture was used as a seed culture. Maltose (HAYASHIBARABIOCHEMIC
AL) 2.0%, bacterial meat extract [Kyokuto Pharmaceutical Co., Ltd.] 0.5%, peptone [Nippon Pharmaceutical Co., Ltd. polypeptone] 0.5%, powdered yeast extract S [Nippon Pharmaceutical ( Co., Ltd.] 0.3%, sodium chloride 0.3%, magnesium sulfate (heptahydrate) 0.1% in a liquid medium containing copper sulfate (pentahydrate) as an inorganic salt. 8
50 g of iron sulfate (heptahydrate) 0.4 g, manganese chloride (tetrahydrate) 3.2 g, zinc sulfate (heptahydrate) 0.8 g
A liquid medium prepared by adding an aqueous solution dissolved in 0 ml of distilled water at a rate of 1.25 ml / l was inoculated with 91 ml of the waffle Erlenmeyer flask into which 110 ml was dispensed, and 3 ml of the seed culture was inoculated at 4 ° C at 27 ° C. The cells were shake-cultured for one day.

The cells were separated by filtering the obtained culture broth, and 200 g was added to 8100 ml of the obtained culture filtrate.
Activated carbon was added and filtered off. The active ingredient adsorbed on the activated carbon was extracted with 4 l of 50% acetone water, and this extract was concentrated under reduced pressure.

The obtained solid residue was dissolved in 2 l of distilled water and adjusted to pH 3 with hydrochloric acid, and then the solution was extracted with an equal amount of butanol. PH of butanol phase after extraction
After returning to 8, the mixture was concentrated under reduced pressure to obtain 7.5 g of a brown oily substance containing leukoplastin.

The above brown oily substance was applied to a 150 ml silica gel column packed with a mixed solvent of butyl acetate-butanol-acetic acid-water (6: 4: 1: 1), and elution chromatography was performed with the same mixed solvent. Merck "Ar
t. Rf values 0.50 to 0.55 by thin layer silica gel chromatography using 5715 "silica gel plates.
Active fractions containing a substance giving a ninhydrin-coloring spot were collected in [developing solution; butanol-acetic acid-water (4: 1: 1)] and concentrated under reduced pressure to obtain 1.2 g of a crude leukoplastin substance. ..

This crude material was subjected to high performance liquid chromatography [Senshou Scientific Co., Ltd., Senshupack Nucleosil 5C ₁₈ , 20φ × 300.
mm] at a flow rate of 5 ml / min.
Elute with gradient from 10% methanol to 100% methanol in 0 minutes, then 10 minutes in 20 minutes
Elution was performed with 0% methanol. Fractions showing a peak with a retention time of 23 minutes were collected and concentrated under reduced pressure to obtain 22.4 mg of pure leukoplastin as a colorless powder.
Melting point 181-183 [deg.] C. A solution of this colorless powder in methanol was subjected to Merck "Art. 5715" silica gel thin layer chromatography [butanol-acetic acid-water (4: 1: 1).
It was found that single leukoplastin was obtained by giving a single spot.

[0086]

INDUSTRIAL APPLICABILITY As described in detail above, the present invention provides leukoplastin as a novel antibiotic and a method for producing the same. The novel antibiotic, leukoplastin according to the present invention exhibits antitumor activity, and also has an effect of enhancing the production of interleukin-3 by splenocytes, further recovery of hematopoietic dysfunction in mammals, and the use of anticancer agents. It is also effective in recovering from the suppression of hematopoietic function due to radiation therapy of cancer.

[Brief description of drawings]

FIG. 1 is an infrared absorption spectrum of leukoplastin of the present invention.

FIG. 2 is a proton nuclear magnetic resonance spectrum diagram.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Internal reference number FI technical display location C12R 1: 465) (72) Inventor Hiroshi Naganawa 3-17 Denenchofuhonmachi, Ota-ku, Tokyo (72) ) Inventor Masa Hamada 26, No.405, Naitocho, Shinjuku-ku, Tokyo 405 (72) Inventor Kenji Maeda 2-46-11, Gobongi, Meguro-ku, Tokyo (72) Inventor Tomio Takeuchi Higashi-gotanda, Shinagawa-ku, Tokyo 5th-11th No. FUJI MANSION 701

Claims (2)

[Claims] 1. A colorless powder exhibiting the following characteristics, that is, an amphoteric behavior, and the elemental analysis values are 58.96% carbon, 6.81% hydrogen, 6.46% nitrogen, and 27.44% oxygen. The specific rotation is [α] _D ²⁸ = + 210.0 ° (c 0.
75, methanol) and the ultraviolet absorption spectrum (in methanol) has a log ε: 296.0 nm (5.
15) 233.6 nm (5.03), and the infrared absorption spectrum (KBr tablet) is as shown in FIG. 1 of the attached drawings. The antibiotic leukoplastin or a salt thereof, which is positive for the ninhydrin reaction. 2. A method for producing leukoplastin, which comprises culturing a leukoplastin-producing bacterium belonging to the genus Streptomyces and collecting the antibiotic leukoplastin having the characteristics of claim 1 from the culture. Law.