JPH0156074B2 - - Google Patents

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, hemolytic streptococcus (hereinafter referred to as streptococcus)
Attenuated viable bacterial cells have been prepared and are already being used as anticancer drugs. In addition, after crushing the cells of hemolytic streptococcus, the active ingredients are extracted with water or a saline solution, an organic solvent is added, and the antitumor ingredients are precipitated and recovered.
1647), a method of lysing hemolytic streptococci with the lytic enzyme lysozyme, cellulase or proteolytic enzyme, and fractionating the active fraction as a water-soluble fraction (British patent
1163865), and a method in which water-insoluble substances are collected after crushing the cells of hemolytic streptococcus and treated with a nuclease and a protease (Japanese Patent Application Laid-Open No. 7014-1982). As described above, it is widely known that Streptococcus bacteria themselves or their bacterial cell components have antitumor activity. It was nothing more than a constituent substance. If you try to isolate the active ingredient from the bacterial body or inside the bacterial body,
It was necessary to fractionate the entire bacterial body by lysing it or mechanically crushing it. Such treatments complicate purification and isolation of active ingredients is extremely difficult. In cases where the active ingredient was actually separated and measured, the molecular weight
200,000 proteins (Special Publication No. 43841, No. 43841, No. 43841, No. 43841)
44,617) and a glycoprotein with a molecular weight of 150,000 (Unexamined Japanese Patent Publication No. 1983
−22026) 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 the culture, the antitumor components could be eluted. They discovered that SPF-1 is eluted into the culture solution (Japanese Patent Application Laid-Open No. 60-92218), and were able to isolate the physiologically active substance SPF-1 from the culture solution. (Japanese Patent Application Laid-Open No. 60-30689) The present inventors further conducted intensive research with the aim of isolating a more effective component from the culture filtrate of hemolytic streptococcus.
We have isolated the antitumor component SPF-10, which inhibits the growth of L1210 (hereinafter referred to as cultured cell L1210) and is characterized by being a positive color reaction fraction by the Anthrone sulfuric acid method. The antitumor component SPF-10 of the present invention is determined by elemental analysis,
Although it is thought to be a peptide-like substance containing sugar based on the color reaction, etc., the ultraviolet absorption spectrum indicates that it is a new substance that is different from known antitumor substances. The present invention relates to the production of antitumor component SPF-10, which is characterized by culturing antitumor component SPF-10-producing bacteria belonging to the genus Streptococcus and collecting antitumor component SPF-10 from the culture. It encompasses the law. In the present invention, bacteria that produce the antitumor component SPF-10 belonging to the genus Streptococcus can be widely used. Next, the bacteria producing the antitumor component SPF-10 will be described. Streptococcus pyogenes ATCC 21060 Streptococcus sp. ATCC 21597 Streptococcus pyogenes ATCC 21546 Streptococcus pyogenes ATCC 21547 Streptococcus pyogenes ATCC 21548 The culture medium is meat extract Medium, Yeast Extract Medium, Brain Heart Infusion Medium (BHI
A natural medium such as Streptococcus spp. is often used, but any medium can be used as long as it is suitable for the growth of Streptococcus bacteria. The culture is performed at a pH of 5.0 to 8.0, preferably 6.1 to 7.2, and a culture temperature of 30 to 40°C, preferably 35 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
After culturing at 35-37℃ and reaching the logarithmic growth phase,
A period between ˜20 hours, especially 5 to 10 hours is preferred. After that, by continuing the culture for 1 to 20 hours, preferably 3 to 15 hours, the antitumor component SPF-10 is added during the culture.
can be accumulated in large amounts. 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 penicillin G.
A concentration of 100 to 7000 units/ml, preferably 300 to 5000 units/ml of culture solution is sufficient. The culture solution obtained by culturing Streptococcus bacteria with the addition of penicillin or related substances is centrifuged to remove the bacterial cells, and ammonium sulfate is added to the filtrate. Separate the amount.
The obtained ammonium sulfate precipitate containing the antitumor component SPF-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, this aqueous solution is adsorbed on a DEAE cellulose column, and it is eluted stepwise using a phosphate buffer solution. , cultured cells L
Collect the active fraction that inhibits the growth of 1210, adsorb this active fraction onto a DEAE Sephadex A-25 column, and elute it by linearly increasing the sodium chloride concentration in the phosphate buffer. It can be obtained by separating the non-buffer-adsorbed portion and the low sodium chloride concentration portion, desalting them, and freeze-drying them. Next, the antitumor component SPF obtained in Example 1
The 10 freeze-dried specimens exhibit the following physical and chemical properties. 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] ²⁰ _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. Absorption is observed from 250nm to 270nm. 5 Infrared absorption spectrum Shown in Figure 2. Around 3270cm ^-1 , 2900cm ^-1 , 1670cm ^-1 , 1535cm
^-1 , 1450cm ^-1 , 1400cm ^-1 , 1330cm ^-1 , 1260cm ^-1 ,
Absorption is observed at 1160cm ^-1 , 1090cm ^-1 and 1030cm ^-1 . 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 Pale yellow 8 Color reaction Lowry reaction + Bieurett reaction + Ninhydrin reaction + Anthrone sulfuric acid reaction + Mauritsch reaction + Cysteine sulfuric acid reaction + Orsine hydrochloric acid reaction - 9 Molecular weight Molecular weight approximately 1000 ~ as measured by gel filtration method
500000. 10 Solubility in solvents Soluble in water, methanol, ethanol,
n-propanol, acetone, ethyl ether,
It is sparingly soluble or insoluble in solvents such as n-hexane, chloroform, and ethyl acetate. Next, a method for measuring antitumor activity against cultured cells L 1210 in vitro and a color reaction method using the Anthrone sulfuric acid 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 were suspended in RPMI 1640 medium (containing 5 mg/kanamycin) supplemented with 10% FBS. 0.5 ml of this culture solution was poured into a Falcon 2058 tube so that the number of cells was 1×10 ⁵ cells/tube. Next, 0.5 ml of a predetermined amount of the standard product (anti-tumor component SPF-10) dissolved in the culture solution to the desired concentration was added to this culture solution, and cultured at 37°C in the presence of 5% _CO2 . did. 48 hours after adding the standard sample, staining with trypan blue was performed using the following formula.
IR (%) was calculated. IR (%) = (A) - Number of viable cells in each experimental group/Number of viable cells in control group x 100 Here, (A) indicates the number of viable cells in the control group. A control was performed at the same time using 0.5 ml of culture solution that did not contain the standard. Color reaction using the Anthrone sulfuric acid method Add 2 ml of Anthrone reagent (0.20 gr of Anthrone to 1 ml of the sample dissolved in deionized water to the desired concentration).
(dissolved in 100ml of concentrated sulfuric acid) and mix.
After 30 minutes, replace 1 ml of standard with 2 ml in 1 ml of deionized water.
The solution containing Anthrone reagent was used as a control.
Measure absorption at 620nm. 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 ATCC 21060
The seed culture solution obtained by inoculating 100ml of BHI medium and statically culturing at 37℃ for 8 hours is used as the medium shown in Table 1.
A1 was inoculated and precultured anaerobically under the same conditions as the seed culture. 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 10 Pour medium A8 into a jar fermenter and heat at 120℃ for 10 minutes After heat sterilization, cool to 37℃, inoculate with preculture solution 1, and incubate at 37℃ for 15.5 hours at PH.
6.8, replace with nitrogen while stirring at 300 rpm and culture anaerobically. Then penicillin G1000 units/
ml culture solution, and culture was continued for an additional 5 hours. The resulting culture solution was centrifuged to remove bacterial cells. Add ammonium sulfate to the culture filtrate and
A fraction precipitated at 90% saturation was collected. This ammonium sulfate salting-out sample was dissolved in 300 ml of phosphate buffer (KH ₂ PO ₄ -Na ₂ HPO ₄ ) of 1 × 10 ^-2 M.PH7.0, and this aqueous solution was adsorbed on a DEAE cellulose column (5 × 70 cm). After that, the cells were eluted stepwise using the above phosphate buffer containing 0.3M sodium chloride, and if the color reaction by the Anthrone sulfuric acid method was positive and the cultured cells were
An active fraction that inhibits the growth of L 1210 was collected. This active fraction was added to DEAE Sephadex A-25.
Adsorb onto a column (2.6 x 70 cm), then elute by linearly increasing the sodium chloride concentration in the phosphate buffer, and the color reaction by the Anthrone sulfuric acid method is positive and the growth of cultured cells L 1210 is inhibited. The active fraction 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 growth inhibition activity of polymer-permeable E. coli mutant strain MP-2 (JP-A-60-30689). The eluate of the active fraction of A was treated with ammonium sulfate.
The precipitated fraction was dissolved in the above phosphate buffer, desalted using a dialysis membrane, and then lyophilized to obtain 6.5 gr of SPF-10. An antitumor activity test using this antitumor component SPF-10 as a standard is shown in Experimental Examples 1 and 2. Example 2 Streptococcus pyogenes ATCC 21060
were 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/ml culture solution, and the culture was further increased by 10
Lasted for an hour. This culture filtrate was purified in the same manner as in Example 1 to obtain 4.1 gr of antitumor component SPF-10. Example 3 Streptococcus biogenes ATCC 21060
were cultured in the same manner as in Example 1 using medium A shown in Table 1. In this case, penicillin G is 3000 units/
ml culture solution, and culture was continued for an additional 3 hours. This culture filtrate was purified in the same manner as in Example 1 to obtain 6.8 gr of antitumor component SPF-10. Example 4 Streptococcus pyogenes ATCC 21060
were cultured in the same manner as in Example 1 using medium B shown in Table 2. In this case, penicillin G is 1000 units/ml.
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 5.6 gr of antitumor component SPF-10. 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 ATCC 21060
were cultured at 35° C. in the same manner as in Example 1 using medium C shown in Table 3. In this case, penicillin G
The mixture was added at a concentration of 1000 units/ml 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 5.5 gr of antitumor component SPF-10. 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 ATCC 21060
were cultured in the same manner as in Example 1 using medium D shown in Table 4. In this case, penicillin G is 1000 units/ml.
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 5.9 gr of antitumor component SPF-10. Table 4 Medium D Maltose 0.25% Casamino acids 0.3% Yeast extract 1.0% Acidic monobasic potassium phosphate 0.1% Magnesium sulfate 0.05% PH=6.9 Example 7 Streptococcus sp. ATCC 21597 was grown in medium A shown in Table 1. The cells were cultured in the same manner as in Example 1. In this case, penicillin G is 1000 units/ml.
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 3.2 gr of antitumor component SPF-10. Example 8 Streptococcus pyogenes ATCC 21546
were cultured in the same manner as in Example 1 using medium A shown in Table 1. In this case, penicillin G is 1000 units/
ml culture solution, and culture was continued for an additional 5 hours. This culture filtrate was purified in the same manner as in Example 1 to obtain 3.8 gr of antitumor component SPF-10. Example 9 Streptococcus pyogenes ATCC 21547
were cultured in the same manner as in Example 1 using medium A shown in Table 1. In this case, penicillin G is added at 1000 units/ml culture solution, and the culture is further
Lasted for an hour. This culture filtrate was purified in the same manner as in Example 1 to obtain 4.6 gr of antitumor component SPF-10. Example 10 Streptococcus pyogenes ATCC 21548
were cultured in the same manner as in Example 1 using medium A shown in Table 1. In this case, penicillin G is added at 1000 units/ml culture solution, and the culture is further
Lasted for an hour. This culture filtrate was purified in the same manner as in Example 1 to obtain 2.3 gr of antitumor component SPF-10. Example 11 Streptococcus pyogenes ATCC 21060
were cultured in the same manner as in Example 1 using medium A shown in Table 1. In this case, cephalosporin C is
The mixture was added at a concentration of 600 μg/ml 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 2.4 gr of antitumor component SPF-10. Experimental Example 1 The in vitro antitumor activity test of the antitumor component SPF-10 obtained in Example 1 was conducted using the method for measuring antitumor activity described in the text, and the results are shown in Table 5. Shown below. Table 5 Results of antitumor activity of SPF-10 SPF-10 (mg/ml) IR% 2.0 100 1.0 98 0.5 89 Experimental example 2 In vivo ) antitumor activity test in
The experiment was carried out using CRJ-CD-1 (ICR strain, female, 7 weeks old) mice. Sarcoma-180 ascites cancer cells were used as tumor cells, suspended in physiological saline, and inoculated intraperitoneally into mice at 5×10 ⁵ cells/mouse. Starting 24 hours after cancer cell inoculation, SPF-10 was administered intraperitoneally once a day for 5 consecutive days, and the number of survivors was observed. The results are shown in Table 6. 【table】

[Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption spectrum of the antitumor component SPF-10, and Figure 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.

Claims (1)

[Claims] 1. Antitumor component having the following physicochemical properties:
SPF-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] ²⁰ _D = -65.0° to -85.0° (C = 1.00) 4 Ultraviolet absorption spectrum In the ultraviolet absorption spectrum of a 0.1% aqueous solution of this substance, absorption is observed from 250 nm to 270 nm. 5 Infrared absorption spectrum around 3270cm ^-1 , 2900cm ^-1 , 1670cm ^-1 , 1535cm
^-1 , 1450cm ^-1 , 1400cm ^-1 , 1330cm ^-1 , 1260cm ^-1 ,
Absorption is observed at 1160cm ^-1 , 1090cm ^-1 and 1030cm ^-1 . 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 + Bieurett reaction + Ninhydrin reaction + Anthrone sulfuric acid reaction + Moritzhu reaction + Cysteine sulfuric acid reaction + Orsine hydrochloric acid reaction - 9 Molecular weight When measured by gel filtration method, the molecular weight is about 1000 ~
500000. 10 Solubility in solvents Soluble in water, methanol, ethanol,
n-propanol, acetone, ethyl ether,
It is sparingly soluble or insoluble in solvents such as n-hexane, chloroform, and ethyl acetate. 2 Antitumor component belonging to the genus Streptococcus
A method for producing an antitumor substance SPF-10, which is characterized by culturing SPF-10 producing bacteria and collecting the antitumor substance SPF-10 from the culture. 3 Antitumor component belonging to the genus Streptococcus
Antitumor component SPF-10 according to claim 2, characterized in that when culturing the SPF-10 producing bacteria, penicillin or a related substance is added at an appropriate time during the culture. manufacturing method.