JPS6216426A - Antitumor component spf-10 and production thereof - Google Patents

Abstract

NEW MATERIAL:An antitumor consitutent component SPF-10. Elementary analysis value (%); C; 45.6-46.1; H, 6.5-5.6; N, 14.8-15.4; O, 29.8-32.2. Ash, 0.9-2.1. Decomposition point; Browns at 165 deg.C and decomposes black at 240 deg.C. [alpha]D<20>=-65.0--85.0 (C=1.00). Molecular weight (measured by the gel filtration method); About 1,000-500,000. Color reaction; Positive to the anthrone-sulfuric acid reaction, Lowry reaction, etc. Solubility; Soluble in water and insoluble and slightly soluble in methanol, acetone, etc. USE:An antitumor agent capable of inhibiting the growth of cancerated cultivated leukocyte cells L1210. PREPARATION:A microorganism of the genus Streptococcus and capable of producing SPF-10, e.g. Streptococcus pyogenes, is anaerobically cultivated at 35-37 deg.C by the spinner culture method, and a penicillin is added thereto 5-10hr after the logarithmic growth phase to accumulate a large amount of SPF-10, which is then separated and purified by salting out with ammonium sulfate and column chromatography to afford the aimed SPF-10.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel antitumor component SPF-10 and a method for producing the same.

Conventionally, preparations prepared by attenuating viable cells of hemolytic streptococcus (hereinafter referred to as hemolytic streptococci) have been used as anticancer agents.

In addition, there is a method in which the active ingredients of hemolytic streptococcus are extracted by crushing and draining the cells or using a saline solution, and an organic solvent is added to collect the antitumor ingredients as a precipitate (Japanese Patent Publication No. 38-1647). , a method in which cells are lysed using cellulase or proteolytic enzymes, and the active fraction is fractionated as a water-soluble fraction (British Patent No. 1163865); the cells of hemolytic streptococcus are crushed, water-insoluble substances are collected, and nucleolytic enzymes and proteases are used to decompose the cells. A method of treating with an enzyme (Japanese Unexamined Patent Publication No. 55-7014) is known.

As described above, it is widely known that Streptococcus bacteria themselves or their bacterial cell components have antitumor activity, but what was previously known was that Streptococcus bacteria themselves or their cell components have antitumor activity. It was just that. In order to isolate the active ingredient from the bacterial cells or inside the bacterial cells, the entire bacterial cell had to be fractionated by lysis or mechanical crushing.

Such treatments complicate purification and isolation of active ingredients is extremely difficult. In an example that was actually separated and measured as an active ingredient, a protein with a molecular weight of 200,000 (
Japanese Patent Publication No. 48-43841, Japanese Patent Publication No. 51-44617) and a glycoprotein with a molecular weight of 150,000 (Japanese Patent Publication No. 58-22
026) is known.

The present inventors previously conducted research to find a method for eluting antitumor components into the culture solution of hemolytic streptococcus, and found that by adding penicillin or related substances during culture, the antitumor component was released into the culture solution. Unexamined Japanese Patent Publication No. 6, which discovered that elution occurs in
0-92218), physiologically active substance SPF from the culture solution
-1 was isolated. (Unexamined Japanese Patent Publication No. 60-30
No. 689) The present inventors further conducted intensive research with the aim of isolating more effective components from the culture filtrate of hemolytic streptococcus. In the present invention, the cancerous white blood cell culture cell L1210 (
We have now isolated an antitumor component SPF-10, which is characterized by inhibiting the growth of cultured cells (hereinafter referred to as L 1210) and exhibiting a positive color reaction by the Anthrone sulfuric acid method.

The antitumor component SPF-10 of the present invention is considered to be a peptide-like substance containing sugar based on elemental analysis, color reaction, etc., but the ultraviolet absorption spectrum shows that it is a novel substance that is different from known antitumor substances. It is recognized as a substance.

The present invention provides a method for producing antitumor component SPF-10, which comprises culturing antitumor component SPF-10-producing bacteria belonging to the genus Streptococcus and collecting antitumor component SPF-10 from the culture. It is inclusive.

In the present invention, bacteria that produce the antitumor component SPF-10 belonging to the genus Streptococcus can be widely used. Next, bacteria producing the antitumor component SPF-10 will be described.

(Streptococcus pyogenes) Streptococcus pyogenes ATCC21
546 Streptococcus pyogenes AT
CC21547 Streptococcus pyogenes
Natural media such as meat extract medium, yeast extract medium, plain heart infunizimin medium (BHI medium) are often used as the ATCC21548 culture solution, but any medium suitable for the growth of Streptococcus bacteria may be used. can be used.

The culture is carried out at a pH of 5.0 to 8.0, preferably 6.1 to 7.0.
2, and the culture temperature is 30 to 40°C, preferably 3
The temperature is 5 to 37°C, and static culture or stirring culture can be performed anaerobically.

In the present invention, adding penicillin or its related substances at an appropriate time during culture plays an important role in collecting the antitumor component SPF-10.

The addition time of penicillin or related substances is 35-3
Cultivation at 7° C. is preferably carried out for 3 to 20 hours, particularly 5 to 10 hours after the logarithmic growth phase has taken place. By continuing the culture for 1 to 20 hours, preferably 3 to 15 hours, a large amount of the antitumor component SPF-10 can be accumulated during the culture.

Penicillin or its related substances may be any known related substances that have similar effects to penicillin, but penicillin G is commonly used.

The amount added is 100 to 7000 units/rd of penicillin G.
Preferably, about 300 to 5000 units/IIIQ culture solution is sufficient.

The culture solution obtained by culturing Streptococcus bacteria with the addition of penicillin or its related substances is centrifuged to remove bacterial cells, and ammonium sulfate is added to the filtrate.
Collect both fractions of % saturation. Obtained antitumor component SP
The ammonium sulfate precipitate containing F-10 can also be stored in a frozen state.

To extract the antitumor component SPF-10 from this ammonium sulfate salt precipitate, the salt precipitate is dissolved in a buffer solution, and this aqueous solution is mixed with DEA.
Adsorb onto a DEAE cellulose column, elute stepwise using phosphate buffer, collect the active fraction that inhibits the growth of cultured cells L 1210, and adsorb this active fraction onto a DEAE Sephadex A-25 column. This is then eluted by linearly increasing the sodium chloride concentration in the phosphate buffer, and the buffer-unadsorbed portion and the low sodium chloride concentration portion are fractionated, desalted, and freeze-dried. The freeze-dried specimen of the antitumor component SPF-10 obtained in Example 1 exhibits the following physical and chemical properties.

1. Elemental analysis' C45,6~46.1%H6,5~
6.6% N 14.8-15.4% 0 29.8-32.2% Ash 0.9-2.1% 2. Decomposition point This substance turns brown at 165°C and turns black at 240°C. Disassemble.

3. Specific optical rotation [α) fi”=-65,0 to -85,0
(C=1.0O) 4. Ultraviolet absorption spectrum The ultraviolet absorption spectrum of a 0.1% aqueous solution of this substance is shown in FIG.

5. Infrared absorption spectrum shown in Figure 2.

6. Distinction between basic, acidic, and neutral The pH of a 1.0% aqueous solution of this substance is 6.5.

7. Color of the substance Light brown 8. Color reaction Lowry reaction 10-Buret reaction 10-ninhydrin reaction 10-anthrone sulfuric acid reaction 10-Morisch reaction 10-cystine sulfuric acid reaction 10-orcine hydrochloric acid reaction - 9. Molecular weight When measured by gel filtration method, the molecular weight is approx. 1,
000 to 500,000.

10. Solubility in solvents It is soluble in water, but poorly soluble or insoluble in solvents such as methanol, ethanol, n-propanol, acetone, ethyl ether, n-hexane, chloroform, and ethyl acetate.

Next, a method for measuring antitumor activity against cultured cell L 1210 in vitro and a color reaction method using the anthrone sulfate method will be described.

Method for Measuring Antitumor Activity Antitumor activity was measured by measuring the growth inhibition rate (IR%) of cultured cells L1210.

L 1210 cells in RPMI 164 with 10% FBS
0 medium (containing 5 mg/n kanamycin). Pour 0.5 mg of this culture solution into a Falcon 2058 tube, and adjust the number of cells to 1 x 10'cell+/tube.
Next, a predetermined amount of the preparation (
Add 0.5 mg of the antitumor component SPF-10) dissolved in the culture medium to the desired concentration, and incubate at 37°C with 5% C.
Cultured in the presence of O□. 48 hours after adding the standard, staining with trypan blue was performed, and IR (
%) was calculated.

Here, (A) indicates the number of viable cells in the control group.

A control was performed at the same time using 0.5 mQ of culture solution containing no standard.

Color reaction by Anthrone sulfuric acid method 2+
nQ Anthrone reagent (0.20gr Anthrone
oom Q dissolved in concentrated sulfuric acid) and mix 3.
After 0 minutes, replace 1 mQ of the standard with 1 mfi of deionized water for 2 m
620n using the solution injected with Q's Anthrone reagent as a control.
Measure the absorption at m. The quantitative value is obtained from a calibration formula obtained by measuring in the same manner using glucose.

Next, examples of the present invention will be shown.

Example 1 Streptococcus pyogenes
The seed culture solution obtained by inoculating ATCC21060 into BHI medium 100+nQ and static culture at 37°C for 8 hours was inoculated into the medium AIQ shown in Table 1, and pre-cultured anaerobically under the same conditions as the seed culture. went.

Table 1 Medium A Maltose 0.25% Meat Extract
1.0% Polypeptone 1.0% Yeast extract 0.25% Acidic monobasic potassium phosphate 0.1% Magnesium sulfate 0.05% pH = 6.8 Pour medium A8Q into a 10Q jar fermentor and add 1
After heat sterilization at 20°C for 10 minutes, cooled to 37°C, inoculated with preculture solution IQ, incubated at 37°C for 15.5 hours, pH 6,
While stirring at 8,300 revolutions/min, the culture is replaced with nitrogen and cultured anaerobically. Then penicillin 01000 units/rrr
Q culture solution was added, and the culture was continued for an additional 5 hours. The resulting culture solution was centrifuged to remove bacterial cells.

Ammonium sulfate was added to the culture filtrate to give a concentration of 50 to 90%.
Both fractions which precipitated at saturation were separated. This ammonium sulfate salting out sample was mixed with IXl0-2M, pH 7.0 phosphate buffer (KHz
PO4-Na, HPO4) was dissolved in 300m+2, and this aqueous solution was applied to a DEAE cellulose column (5X 70cm
) after adsorption.

Stepwise elution was performed using the above phosphate buffer containing 0.3 M sodium chloride, and an active fraction that showed a positive color reaction by the Anthrone sulfuric acid method and inhibited the growth of cultured cells L 1210 was collected.

This active fraction was adsorbed on a DEAE Sephadex A-25 column (2.6 x 70 cm), then eluted by linearly increasing the sodium chloride concentration in the phosphate buffer, and colored using the Anthrone sulfuric acid method. An active fraction that showed a positive reaction and inhibited the growth of cultured cells L1210 was collected.

This elution curve is shown in FIG. In FIG. 3, the dotted line part is the part where the buffer solution is not adsorbed, and the solid line part is the part eluted by linearly increasing the sodium chloride concentration. A is a fraction with growth inhibition activity of cultured cell L 1210, and B is a fraction with polymer-permeable E. coli mutant strain MP-2 (Japanese Patent Application Laid-Open No. 60-306
89) Growth inhibiting activity fraction.

The eluate of the active fraction A was obtained by adding ammonium sulfate to 90% saturation, dissolving the precipitated fraction in the above phosphate buffer, desalting it using a dialysis membrane, and freeze-drying it to obtain S P F
-106.5 gr was obtained.

Antitumor activity tests using this antitumor component SPF-10 as a standard are shown in Examples 1 and 2.

Example 2 Streptococcus pyogenes ATCC21060 was cultured in the same manner as in Example 1 using medium A shown in Table 1. In this case, penicillin G was added at 300 units/IIIQ culture solution, and the culture was continued for an additional 10 hours. This culture filtrate was purified in the same manner as in Example 1 to obtain the antitumor component S PF-104, 1gr.

Example 3 Streptococcus pyogenes ATCC21060 was cultured in the same manner as in Example 1 using medium A shown in Table 1. In this case, penicillin G was added at a concentration of 3000 units/IIN culture solution, and the culture was continued for an additional 3 hours. This culture filtrate was purified in the same manner as in Example 1 to obtain antitumor component S.
PF-106.8 gr was obtained. Example 4 Streptococcus pyogenes ATCC21060 was cultured in the same manner as in Example 1 using medium B shown in Table 2. In this case, penicillin G was added at a concentration of 1000 units/mQ culture solution, and the culture was continued for an additional 5 hours. This culture filtrate was purified in the same manner as in Example 1 to obtain the antitumor component SPF.
-105.6 gr was obtained.

Table 2 Medium B Maltose 0.1% Meat extract 0.5% Polypeptone 1.0% Yeast extract 0.25% Sodium chloride 0.1% PH=7.2 Example 5 Streptococcus pyogenes ATCC21060 in Table 3 Using medium C as shown, 35°C in the same manner as in Example 1.
It was cultured in In this case, penicillin G is too.

Unit/mQ culture solution was added, and the culture was continued for further 5 hours. This culture filtrate was purified in the same manner as in Example 1 to obtain the antitumor component SPF-105.5gr.

Table 3 Medium C Maltose 0.1% Meat extract 0.5% Polypeptone 0.5% Casamino acids 0.3% Yeast extract 0.5% Acidic potassium monophosphate 0.1% Magnesium sulfate 0.05% pH= 6.5 Example 6 Streptococcus pyogenes ATCC21060 was cultured in the same manner as in Example 1 using the culture medium shown in Table 4. In this case, penicillin G was added in an amount of 1,000 units per 7 mL of culture solution, and the culture was further continued for 5 hours. This culture filtrate was purified in the same manner as in Example 1 to obtain the antitumor component SPF.
-105.9 gr was obtained.

Table 4 Medium D Maltose 0.25% Casamino Acid
0.3% Yeast extract 1.0% Acidic potassium monophosphate 0.1% Magnesium sulfate 0.05% pH=6.9 Example 7 Streptococcus sp. ATCC 21597 was tested using medium A shown in Table 1. Culture was carried out in the same manner as in Example 1. In this case, penicillin G was added at a concentration of 1000 units/mQ culture solution, and the culture was continued for an additional 5 hours. This culture filtrate was purified in the same manner as in Example 1 to obtain the antitumor component SPF.
-103Jgr was obtained.

Example 8 Streptococcus pyogenes ATCC21546 was cultured in the same manner as in Example 1 using medium A shown in Table 1. In this case, penicillin G was added at a concentration of 1000 units/laQ culture solution, and the culture was further continued for 5 hours. This culture filtrate was purified in the same manner as in Example 1 to obtain antitumor component S.
PF-103.8gr was obtained.

Example 9 Streptococcus pyogenes ATCC21547 was cultured in the same manner as in Example 1 using medium A shown in Table 1. In this case, penicillin G was added at a concentration of 1000 units/mQ culture solution, and the culture was continued for an additional 5 hours. This culture filtrate was purified in the same manner as in Example 1 to obtain antitumor component S.
PF-104.6gr was obtained.

Example 1 Streptococcus pyogenes ATCC 21548 was cultured in the same manner as in Example 1 using medium A shown in Table 1. In this case, penicillin G was added at a concentration of 1000 units/mQ culture solution, and the culture was continued for an additional 5 hours. This culture filtrate was purified in the same manner as in Example 1 to obtain antitumor component S.
PF-102.3gr was obtained.

Example 11 Streptococcus pyogenes ATCC21060 was cultured in the same manner as in Example 1 using medium A shown in Table 1. In this case, cephalosporin C is 600μg/mQ
The mixture was added to form a culture solution, and the culture was continued for an additional 5 hours. This culture filtrate was purified in the same manner as in Example 1 to obtain the antitumor component SPF-102.4gr.

Experimental Example 1 The in vitro antitumor activity test of the antitumor component SPF-10 obtained in Example 1 was as follows:
The antitumor activity was measured using the method described in the text, and the results are shown in Table 5.

Table 53 Results of antitumor activity of PF-10 SPF-10 (
mg/m12) IR%2・0
100 1.0 98 0.5 89 Experimental Example 2 The in vitro antitumor activity test of the antitumor component SPF-10 obtained in Example 1 was conducted using CRJ-CD-1 (ICR system). The experiment was carried out using female mice (7 weeks old).

Sarcoma-180 ascites cancer cells were used as the tumor cells, suspended in physiological saline, and intraperitoneally injected into the mouse.
X 10' cells/mouse were inoculated.

SP once a day for 5 consecutive days starting 24 hours after cancer cell inoculation
F-10 was administered intraperitoneally and the number of survivors was counted, and the results are shown in Table 6.

Table - Antitumor activity of 63PF - Days of survival of mice
0 10 15 20
25 30 control 7/7 2/
7 0/7 0/7 0/7 0/7SPF-10(5
■/Mouse) 7/7 7/7 7/7 4/7 4
/7 3/7

[Brief explanation of drawings]

1rM shows the ultraviolet absorption spectrum of the antitumor component SPF-10, and FIG. 2 also shows the infrared absorption spectrum. FIG. 3 is a diagram showing the elution curve of the active fraction in Example 1 on a DEAE Sephadex A-25 column. Agent Patent attorney Door 1) Parent Male No. 1 Diagram 0m Diagram 3 → F

Claims (3)

[Claims] (1) Antitumor component SPF with the following physical and chemical properties
-10. 1. Elemental analysis C 45.6-46.1% H 6.5-6.6% N 14.8-15.4% O 29.8-32.2% Ash 0.9-2.1% 2. Decomposition point This substance turns brown at 165°C and turns black at 240°C and decomposes. 3. Specific rotation [α] ^2^0_D = -65.0 to -85
．． 0 (C=1.00) 4. Ultraviolet absorption spectrum The ultraviolet absorption spectrum of a 0.1% aqueous solution of this substance is shown in FIG. 5. Infrared absorption spectrum shown in Figure 2. 6. Distinction between basic, acidic, and neutral The pH of a 1.0% aqueous solution of this substance is 6.5. 7. Color of substance Light brown 8. Color reaction Lowry reaction + Biuret reaction + Ninhydrin reaction + Anthrone sulfuric acid reaction + Molish reaction + Cysteine sulfuric acid reaction + Orsine hydrochloric acid reaction - 9. Molecular weight Molecular weight approximately 1 when measured by gel filtration method ,0
00 to 500,000. 10. Solubility in solvents It is soluble in water, but poorly soluble or insoluble in solvents such as methanol, ethanol, n-propanol, acetone, ethyl ether, n-hexane, chloroform, and ethyl acetate. (2) Antitumor component SP belonging to the genus Streptococcus
Cultivate F-10 producing bacteria and extract antitumor component SP from the culture.
Antitumor component SP characterized by collecting F-10
Manufacturing method of F-10. (3) Antitumor component SP belonging to the genus Streptococcus
The antitumor component SPF-10 according to claim 2, characterized in that when culturing the F-10 producing bacteria, penicillin or a related substance is added at an appropriate time during the culturing. manufacturing method.