JPS6191127A - Preparation of antitumor polysaccharide - Google Patents

Abstract

PURPOSE:To prepare an antitumor polysaccharide free from side effects, by culturing an antitumor polysaccharide-producing strain belonging to Flavobacterium genus, collecting the antitumor polysaccharide from the culture liquid by alcohol precipitation process, and heating the polysaccharide in an acidic solution thereby removing the factor positive to luminous test from the solution. CONSTITUTION:A bacterial strain belonging to Flavobacterium genus and capable of producing an antitumor polysaccharide (marinactan) [e.g. Flavobacterium uliginosum MP-55 strain (FERM P-5793)] is cultured in a nutrient medium. The polysaccharide is collected from the culture liquid by alcohol precipitation process, and heated in a 0.05-1.0N acidic solution to remove the factor positive to luminous test. The acid treatment is carried out preferably at 70-100 deg.C for 5-30min. An antitumor polysaccharide essentially devoid of the factor positive to luminous test and free from side effect can be produced by the acid treatment.

Description

[Detailed Description of the Invention] The present invention provides anti-II! The present invention relates to a method for producing a cough polysaccharide, and specifically relates to a method for producing a purified cough polysaccharide.

The antitumor polysaccharide (hereinafter referred to as marinactan) according to the present invention is a heteroglycan having the following properties and produced by a microorganism belonging to the genus Flavobacterium.

b) Contains (a) fucose, (b) mannose, and (c) glucose as constituent components, whose composition ratio is expressed as a molar ratio la)
: (bl : (cl =0.15±0.05:0.
31±O, OS: l) It has an infrared absorption spectrum shown in FIG. 1. Details of the physicochemical properties of multi-υ3 marinac are described in JP-A-57-96001.

This polysaccharide marinade has strong resistance to Sarcoma S-180 solid cancer type, Sarcoma S5-1AO water cancer type, etc.
It is already known that it has T damage activity and also has an immunostimulatory effect (The Journal of
Antibiotics, vol', 36+
No, 5. 4 7 1 to 4 7 7 (1983)
, Japanese Unexamined Patent Publication No. 57-96001).

For example, as described in some of the above-mentioned documents, the method for purifying the polysaccharide Marinacucum is to add alcohol such as ethanol to the culture supernatant to precipitate it, and then extract marinactan from the precipitate with hot water. After the solution is dialyzed to remove low-molecular substances, a known protein removal method such as the Seberg method is applied to remove contaminating proteins.

However, Gram-negative I such as Flavobacterium spp.
Bacteria II are generally known to have bacterial toxins, such as lipopolysaccharide (LPS), which is an endotoxin of Escherichia coli, and are positive factors in the luminance test. This kind of LPS-like active substance (luminus test positive factor)
The existence of is considered to be a problem. That is, although this LPS or luminous test positive factor exhibits an immunostimulatory effect, it is accompanied by pyrogenic and toxic effects as side effects, and therefore it is necessary to reduce the amount of this factor as much as possible.

However, it is very difficult to remove the luminous test positive factors contained in Marichikukun, and it has not been possible to remove them even by purification methods such as gel filtration.

Therefore, the present inventors have conducted intensive research on a purification method to obtain a preparation of marinaactan that does not exhibit LPS-like activity, and have found that a preparation of marinaactan that exhibits LPS-like activity does not cause hydrolysis of marinaactan in an acidic solution. We have discovered that a purified marinaactan preparation can be obtained by removing LPS-like activity by heat treatment under these conditions, and based on this knowledge, we have completed the present invention.

That is, the present invention cultivates an antitumor polysaccharide marinactan-producing bacterium belonging to the genus Flavobacterium in a nutrient medium, produces and accumulates marinactan in the culture solution, collects marinactan from the culture solution by alcohol precipitation, and then Concentration 0.05-1. The present invention relates to a method for producing purified antitumor polysaccharide marinade, which is characterized by heat treatment in an acidic solution of ON to remove luminance test positive factors.

A method for cultivating a microorganism of the genus Flavobacterium, which is a producing bacterium of the polysaccharide Marinacucum, is described in the above-mentioned Japanese Patent Application Laid-Open No. 5-7-9.
The method described in Japanese Patent No. 6001 may be applied. In addition, Flavobacterium uriginosaum (FlavoI) acLerium is an example of marinactan-producing bacteria.
uliginosum) MP-5
There are 5 strains (FIF, RM I)-5793), and the actual mycological substance is published in JP-A-5-1-96001. This is shown below.

In the present invention, marinactan is collected from a culture solution of marinactan-producing bacteria by alcohol precipitation, or generally obtained from the culture solution by a solid-liquid separation operation such as centrifugation.
[After adding alcohol such as ethanol to the liquid, leave it in an ice chamber to generate precipitated dG.

After collecting marinade by alcohol precipitation, acid treatment is performed, but preferably, before the acid treatment, low-molecular substances are removed using the dialysis method as described above, and protein deproteinization is performed.

There are various acids used in the method of the present invention,
Examples include organic acids such as acetic acid, citric acid, and malic acid, and inorganic acids such as hydrochloric acid and sulfuric acid. The concentration of acid is 0.05-1. ON, preferably in the range of 0.1 to 0.5N is suitable. Acid treatment is performed at 50°C or higher, preferably at 70°C.
~100°C for 5-60 minutes, preferably 5-30 minutes
This is done by heating for a minute. If the acid treatment conditions are too severe, the antitumor activity of Marinaccum will decrease, so it is necessary to carry out the acid treatment within the above range. This acid treatment can remove the luminance test positive factors, and usually the acid treatment is immediately cooled, and the insoluble substances produced by the acid treatment are removed by centrifugation, filtration, etc., and then an alkali such as caustic soda is removed. Use pl+6.0~
Neutralize to 8.5. After neutralization, alcohol precipitation is performed again and a marinactan specimen is collected. A luminance test was performed on this marinactan sample to measure LPS-like activity, and it was found that LPS (E, col) per 1.0 g of sample.
i 0111 ・1134), it is about 2 to 30 cm, and the acid treatment of the present invention reduces the active substance by 1/10 to 1/10.
The amount can be reduced to 1/100, and a specimen containing substantially no luminance test positive factors can be obtained.

Therefore, the marinade obtained by the method of the present invention is very useful as an antitumor substance without side effects.

Next, the present invention will be explained in detail with reference to Examples.

Example 1 Glucose 0.6% in 30g of commercially available artificial seawater (Jermaline S, Jamaline Laboratory-W).

0.5% polypeptone and 001% yeast extract were added to adjust the pH to 7.3, and this medium was poured into a 5001 volume tank and heated at 120° C. for 15 minutes to sterilize it. Seed culture solution 30e obtained by culturing in the same medium was inoculated into this, and 2
Culture with aeration at 7°C for 24 hours (1/2V, V, M, 3
000r, p, m,). The culture solution was centrifuged to remove bacterial cells, and the resulting culture supernatant was concentrated under reduced pressure to obtain a 501 concentrate. An equal amount of ethanol was added to this, and the resulting precipitate was collected by centrifugation. Extract this precipitate twice with 2ON hot water at 90°C for 2 hours).
, after removing insoluble residue (centrifugation method),
Dialysis was performed for days.

1/3 volume of chloroform and 1730 for 1 volume of dialysis fluid
of n-butanol and shaken vigorously for 30 minutes, the resulting precipitate was centrifuged (30.0 Or, p, m, .
15 minutes) to obtain a supernatant. Furthermore, this operation was repeated twice to remove protein.

A 1.5-fold volume of ethanol was added to the supernatant thus obtained, and the resulting precipitate was collected, washed with pure ethanol, and dried under reduced pressure to obtain 30 g of a crude marinaactan sample.

The sugar content (phenol-sulfuric acid method) of this crude marinactan sample was 70%, the protein content measured by the Lowry method was 1.5%, and the activity measured by the luminance test method was 70%.
It was 120 mg/g in terms of PS. 1.2g of this specimen
was dissolved in 100mff1 of water, 20ml (l) of 2.4N acetic acid aqueous solution was added thereto, and the mixture was heated under reflux for 15 minutes. After heating, centrifugation was performed to remove insoluble substances, and ION
Neutralized to ρ117.5 with caustic soda.

Add 1.5 times the volume of ethanol to the neutralized solution, filter and collect the resulting precipitate, repeat the ethanol precipitation, wash the resulting precipitate with ethanol, and dry under reduced pressure.
A specimen of 70 rvt was obtained. This specimen was acid-treated in the same manner as described above, except that the heating time was changed. For each of the obtained preparations, ps-like activity and antitumor activity against Sarcoma 180 solid cancer in mice were measured, and the relationship with the heating time of acid treatment was investigated.

The results are shown in Table 1.

'Knee' Table 1 The tumor growth inhibition rate in Table 1 was determined by subcutaneously implanting 3x106 Sarcoma 180 cells into ICR mice (5 weeks old, female), and administering the specified amount of the sample intraperitoneally for 10 days starting the next day. After 5 weeks, the tumor weight was measured and the tumor growth inhibition rate of the sample was determined.

On the other hand, the survival rate is ICR mice (5 weeks old, 1ltff)
Sarcoma 18011 in 7 animals! 1 x 106 water cancers/
The sample was administered intraperitoneally to 25 mice for 10 days starting from the next day.
It was administered intraperitoneally at a rate of ■/kg, and the survival rate was determined.

As shown in Table 1, by heat treatment for 5 to 30 minutes, most of the luminance test positive factors could be removed with almost no loss of antitumor activity.

Next, 1.2 g of the above-mentioned crude marinaactan sample was dissolved in 100 mβ of 0.1 N hydrochloric acid and heat-treated at 80° C. for 20 minutes. After heating, the mixture was filtered to remove insoluble substances, neutralized to pH 7.5 with ION caustic soda, and 1.5 times the volume of ethanol was added to collect the resulting precipitate. Next, after washing with ethanol, the product was dried under reduced pressure to obtain a purified sample of 800#.

The tumor growth inhibition rate, survival rate, and luminance test (LPs conversion) of this preparation were 78%, 165%, and 8%, respectively.
．． It was 5 mg/g.

[Brief explanation of drawings]

FIG. 1 is an infrared absorption spectrum of the heteroglycan according to the present invention. Patent Applicant Microbial Chemistry Research Foundation Continued from page 1 9 Inventor Katsumi Suzuki 3'9 Ikebukuro, Toshima-ku, Tokyo Inventor Tsuyoshi Shiio 1631 2-32-chome Terabun, Kamakura City

Claims (2)

[Claims] (1) An antitumor polysaccharide that belongs to the Flavobacterium genus and has the following properties.It consists of (a) fucose, (b) mannose, and (c) glucose, and the composition ratio is (a) in molar ratio.
:(b):(c)=0.15±0.05:0.31±0
．． 05:1 b) Cultivate the producing bacteria having the infrared absorption spectrum shown in Figure 1 in a nutrient medium, produce and accumulate the polysaccharide in the culture solution, and extract the polysaccharide from the culture solution by alcohol precipitation. 1. A method for producing an antitumor polysaccharide, which comprises collecting and then heat-treating in an acidic solution with a concentration of 0.05 to 1.0 N to remove luminance test positive factors. (2) The antitumor polysaccharide-producing bacterium belonging to the genus Flavobacterium is Flavobacterium uriginozamu strain MP-55 (
FERM P-5793).