KR810000552B1 - Method of manufacturing anti-malignant tumor agent - Google Patents

Abstract

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Description

Method of manufacturing anti-malignant tumor agent

1 through 4 show U.V. fractions of fractions obtained by the process shown in Example 2. Spectrum and I.R spectrum are shown.

1 shows U.V. of active ingredients having a molecular weight of 5,000 to 10,000. It shows the spectrum (dilution ratio of the original culture liquid and water is 1:10).

2 shows U.V. The spectrum is shown (original culture solution).

3 shows I.R. of active ingredients having a molecular weight of 5,000 to 10,000. Display the spectrum.

4 shows I.R. of active ingredients having a molecular weight of 1,000 to 5,000. Display the spectrum.

The present invention relates to a method for producing a novel anti-malignant tumor agent. In more detail, the present invention provides a novel active substance obtained by culturing Staphylococcus epidermidis ST (STC) No. 3706 (ATCC 31310) in a liquid medium. It relates to a manufacturing method.

The substance has an effect on preventing leukemia and malignancies. When the swelling occurs in a leukemia patient, the present invention unexpectedly reduced the number of leukemia cells and finally found that the leukocyte count of the patient was normal.

을 투여한 결과, 이 동물들의 백혈병 바이러스 양이 줄어들고 백혈병 세포의 수가 줄어들며, 백혈병이 걸린 기관의 회복, 수명의 연장 및 살아남은 동물의 수를 증가시킨 효과를 나타냈다.The present inventors isolated microbial strains from the boil of leukemia patients and cultured them to the animal infected with leukemia.

Administration of these animals resulted in a reduction in the amount of leukemia virus in these animals, a reduction in the number of leukemia cells, recovery of organs with leukemia, prolongation of lifespan, and an increase in the number of surviving animals.

Continuing the study, the inventors found that the filtrate is effective not only for leukemia, but generally for other malignancies.

The present inventors concentrated and purified the filtrate of the culture, separated the water-soluble substance considered to be the active ingredient into several components, and isolated and purified to demonstrate that this substance has the anti-bioactivity.

Next, the present invention will be described in detail.

[1] identification of strains

Strains of microorganisms used in the present invention are novel. This is the no. Deposited as 3706 and ATCC 31310 in unperforming and “ATCC” respectively.

This strain is freely available to anyone in these laboratories.

This strain was found to be a strain of Staphylococcus epidermides as follows.

From the test results, it was found that the pathogen belongs to Staphylococcus or Aerococcus.

Thus, the pathogen finally determined to be Staphylococcus epidermidis. The present inventors named this strain "STF".

[2] preparation of active ingredients:

Pathogens multiply well in general media, such as nutrient media containing carbon, nitrogen and inorganic salts.

In order to utilize useful active ingredients made by bacteria, aerobic culture in a liquid medium in a stationary state or shaking state is preferable. Suitable examples of media are as follows.

Although only liquid medium is illustrated above, a general solid medium may also be used. The fungus is incubated in the medium at -20 ° C to + 40 ° C for 24 hours-120 hours. The optimum temperature was about 37 ° C. The colony surface was white and round.

The obtained culture was centrifuged, the solid material was separated, and a solution was obtained (hereinafter referred to as "culture medium"). Various experiments were added to this culture.

[3] favorable active ingredients

The resulting culture was administered to animals infected with various diseases to achieve the following results:

(1) Inhibition of malignancy caused by inoculation of leukemia virus: 303 ICR mice were divided into three groups.

Two of the two groups were inoculated with Freind's leukemia virus, and one group of the culture solution was intraperitoneally administered at a rate of 0.ml/10g/day for 8 days, and the splenic abnormality was inhibited as follows.

(2) Malignant tumor suppression caused by inoculation of leukemia cells:

BDF ₁ mice were inoculated with mouse leukemia cell L-1210 to induce induced leukemia. Mice die after a limited time. L-1210 cells were inoculated into two groups of mice consisting of six mice in one group, and then the culture solution was continuously administered to the group for 5 days to obtain the following results. The dose of the given culture was the same as in (1).

The test group / control group (lifetime equipment) was higher than 112%.

(3) Inhibitory effect of Ehrlich cancer cells:

Two groups of 10 mice were inoculated in one group and inoculated with Errich's ascites tumor cells. The culture solution was administered to the abdomen of one group of mice continuously for 5 days. Thus, the inhibitory effect was confirmed by the tumor cell number on day 10 as follows. The dosage of the culture solution was the same as in (1).

(4) Inhibitory effect of Sarcoma 180 cells:

As described above, two groups of ICR mice, in which one group consists of 10 mice, were inoculated with Sarcoma 180 cells in the abdomen. The culture solution was administered to one group for 5 days. The number of cells at day 10 was examined and the following results were obtained. The dosage of the culture solution was the same as in (1).

For comparison, the effect of the culture solution of other known microorganisms obtained by the same culture method was investigated. Cultures of the following unknown microorganisms were administered to mice inoculated with Friend Leukemia virus. In each case no effect was observed:

Pseudomonas

Klebsiella

Staphylococcus aureus

(4) Isolation and Purification of Active Ingredients:

The active ingredient contained in the culture of Staphylococcus epidermidis can be isolated and purified, for example, by the following method:

(1) The culture was centrifuged at 3,000 to 12,000 rpm for 30 minutes to 10 minutes to separate the solution and precipitate. The precipitate was then washed with distilled water and centrifuged again. Wash solution was combined with the solution. This method was repeated 1 to 5 times (usually 3 times).

(2) The solution thus collected was concentrated by evaporation at a temperature of 10-30 ° C. under a reduced pressure of 5-30 mmHg for 1-6 hours. Thus, the precipitated solid material was washed with distilled water and dissolved. This solution was concentrated again by evaporation. This process was repeated once to five times (typically three times) to obtain a white, yellow or brownish, white crystalline powder soluble in water.

(3) This crystalline powder is classified by water reclamation operation such as, for example, Sphadex (Sweden, adsorption filtration gel manufactured by Biochemical Co., Ltd.) G-25.

The active fractions were selected repeatedly by investigating the activity of each fraction. This process yielded a material with a single active peak.

This active material was again dissolved in water and purified by purification method such as ion exchange chromatography or dialysis.

(4) The active ingredient thus purified can be separated by adding various organic solvents to the aqueous solution, changing the pH from the acidic zone to the alkaline zone, followed by reprecipitation after salting and concentration by filtration or evaporation.

This material has the following properties.

Thus, the antitumor activity of the purified and purified active ingredient was confirmed as follows by adding its diluted aqueous solution to the culture solution of L-1210 cells and culturing it at 35 ° C. for 48 hours to determine the cell proliferation inhibition rate.

Thus, the material produced by Staphylococcus epidermidis has the effect of controlling various malignancies such as liquid and solid cancers. The substance is not acutely toxic.

(5) Administration of the active ingredient:

Dosage depends on the route of administration but is about 3-300 mg / kg. The culture medium from which the solid material has been removed can be used directly. However, it is of course better to use the isolated and purified active ingredient in order to precisely control the dosage. Various dosage methods are possible, such as intravenous, subcutaneous, intramuscular, oral, suppositories, tablets and emulsions. Therefore, the form of the formulation may be appropriately selected, and examples thereof include powders, tablets (monolayer tablets, multilayer tablets, enteric agents, etc.), capsules, ampoules, or vials, injections, sublingual tablets, troches, suppositories, or fat emulsions.

The following examples illustrate the invention. The following examples in no way limit the invention.

Example 1

Staphylococcus epidermidis STF was incubated at 37 ° C. for 48 hours in the media below:

The culture solution was centrifuged at 10,000 rpm for 30 minutes to divide the supernatant and the precipitate.

The precipitate was redispersed with 1 liter of distilled water and the dispersion was left at 18 ° C for 18 hours and centrifuged again at 10,000 rpm to separate the supernatant and precipitate. This process was repeated five times. The obtained supernatant was collected and concentrated by evaporation under reduced pressure of 10 mmHg at room temperature for about 2 hours. After about 1/30 enrichment was achieved, the liquid passed through the Sephadex D-25 layer and an indicator of OD (optical density) 265 was used to determine U.V. Spectroscopic analysis was performed.

The separation of fractions uses polysaccharide detection methods such as the Molisch reaction. Chromatogram of L-1210 cell proliferation inhibitory activity was investigated to isolate the active fraction. This process was repeated three times to obtain the highest active fraction. The aqueous solution of this active fraction was concentrated by evaporation to obtain a white powder.

This powder was washed with water, dissolved again and concentrated by evaporation. This process was repeated five times to obtain a white crystalline powder. The yield from 1 liter of culture was 0.3 g.

Example 2

A 25 liter liquid medium constructed as in Example 1 was made. The culture was carried out at 37 ° C. for 2 days. The culture was centrifuged (6,000 rpm for 20 minutes) to remove bacteria. The obtained supernatant (about 23 meters) was prepared at about 500c.c. Concentrated to volume.

The precipitate thus formed was washed three times with 100 c.c. of distilled water. This wash was combined with the concentrate, all of which was concentrated to a volume of 500 c.c. at 40 ° C. under reduced pressure.

The concentrate is an Amicon Far East Diaflon # 10,000 moleclave membrane, with two fractions (one fraction having a molecular weight of 10,000 or more and the other having a molecular weight of 10,000 or less). Divided into

A solution with a molecular weight of 10,000 or less was passed through a dimethyl aminoethyl cellulose tower (OH-type) to divide into acidic, neutral and basic substances.

The neutrals and bases were then divided into two separate fractions, diaphragm # 5,000, one with a molecular weight of 5,000 to 10,000 (original solution) and another with a molecular weight of 5,000 or less. The molecular weight less than 5,000 was again divided into two fractions (with molecular weight 1,000-5,000 and those with molecular weight 1,000 or less). Thus, the concentrate could be divided into individual fractions.

U.V. of each fraction. Spectrum and IR spectrum were determined (see FIGS. 1-4).

The individual fractions were then administered to animals infected with various induced diseases to obtain the following results.

(A) Anti-malignancy effect of fractions with 5,000 <molecular weight <10,000 and 1,000 <molecular weight <5,000 on in vitro cultured leukemia cells L-1210:

(B) Anti-malignancy effect of fractions with 5,000 <molecular weight <10,000 and 1,000 <molecular weight <5,000 on tumor cells cultured in vitro:

(C) Anti-malignant tumor effect of styeen fraction with 5,000 <molecular weight <10,000 and 10,000 <molecular weight <5,000 on in vitro cultured hepatic hepatocellular tumor AH41C:

(D) Anti-malignant tumor effect of styrene fractions containing 5,000 <molecular weight <10,000 and 10,000 <molecular weight <5,000 in mice inoculated with Aerrich ascites tumor cells (influence on total tumor cell number on day 11 after cell inoculation) ).

Claims (1)

It is produced by incubating the Staphylococcus epidermidis STF (non-performance deposited NO.3706, ATCC 31310) in a liquid culture medium containing a carbon source, a nitrogen source, and an inorganic salt, removing solid matter from the culture medium, and concentrating the residue. The method for producing an anti-malignant tumor, characterized in that the precipitate is washed with water, the precipitate is redissolved, separated into several fractions, and the anti-tumor active fraction is concentrated and purified.

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