JPS6216424A - Antitumor active composition - Google Patents

Abstract

PURPOSE:To obtain the titled composition, obtained by extracting microbial cells of the genus Candida with a two-phase solvent consisting of water and a solvent partially miscible therewith, having improved antitumor activity without participation of T-cells and consisting of a low-molecular weight nonsugar substance. CONSTITUTION:An antitumor active composition, obtained by extracting microbial cells of the genus Candida, e.g. Candida albicand or Candida utilis, with a two-phase solvent consisting of water and a solvent partially miscible therewith, e.g. butanol or phenol, dialyzing the resultant aqueous layer against distilled water, ultrafiltering the dialyzate to remove components having about >=30,000 molecular weight, passing the resultant filtrate through a sugar adsorption column. e.g. ConA sepharose, to adsorb and remove sugar, dialyzing the resultant eluate and freeze-drying the dialyzed eluate. The above-mentioned composition contains about 5-8% sugar, about 60-70% protein and about 25-30% nucleic acid and is capable of exhibiting antitumor activity different from that of an immunological activator (participation of T-cells).

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel composition with antitumor activity obtained from cells of the genus Candida and a method for producing the same.

BACKGROUND OF THE INVENTION It is already known that antitumor active substances can be obtained from cells of the genus Candida. This known anti-RtL tumor active substance is composed of a mixture of water-soluble polymeric polysaccharides, such as mannan, glucan, and glucomannan, obtained by hot water or alkali extraction of bacterial cells, and is a substance that is associated with T-cells. It is an immune-enhancing substance.

The present inventors have demonstrated that T-cells are not involved in the low molecular weight non-sugar substance obtained by extracting Candida cells with a two-phase solvent consisting of water and a solvent partially miscible with water. They discovered that it has excellent antitumor properties and completed the present invention.

Purpose of the invention That is, the present invention provides the following methods: (1) extracting Candida microbial cells with a two-phase solvent consisting of water and a solvent partially miscible with water; (2) separating an aqueous layer from the extract; (3) Dialyze the aqueous phase against distilled water, (4) ultrafiltrate the dialyzed aqueous layer to remove components with a molecular weight of approximately 30,000 or more, and (5) use the filtrate obtained in (4). Sugars are adsorbed and removed through a sugar adsorption column (6
) Next, the present invention provides a method for producing an antitumor active composition, which comprises dialyzing the eluate obtained in (5) and then freeze-drying it.

The present invention also provides an antitumor active composition produced by the above method.

A preferred bacterial species of the genus Candida that can be used in the production method of the present invention is Candida albicans (Candida albicans).
da albicans), Candida utilis (
Candida utilis), Candida guillermon
dii) etc.

In the present invention, these bacterial cells in the logarithmic growth phase are
Extract with a two-phase solvent of 5 to 40 times the weight of the bacteria. The two-phase solvent consists of approximately equal volumes of water and a solvent partially miscible with water, and the latter solvent is preferably butanol, phenol, or the like. Extraction takes 30 minutes at a temperature of 4 to 100℃
This is carried out under stirring for 2 hours.

The extract is centrifuged to remove bacterial cell residue, then the aqueous layer is separated and dialyzed against distilled water for 3 to 5 days at approximately 2 to 10°C, and the precipitate generated during dialysis is removed by centrifugation. . Further, the dialysate is filtered using a membrane filter (pore size: 022 to 150 μm, preferably 10 to 50 μm), and then substances having a molecular weight of about 30,000 or more are removed using an ultrafiltration membrane. In addition to the above-mentioned ultrafiltration method, gel filtration method, etc. can be used for this molecular weight fractionation.
Although other methods may be used, ultrafiltration is preferred due to its short operating time. The obtained filtrate containing low molecular weight substances with a molecular weight of about 30,000 or less is passed through a column packed with a suitable sugar adsorbent to remove sugars. Examples of the sugar adsorbent include soybean lectin and wheat germ lectin, but in the method of the present invention, adsorbents containing concanavalin A as a ligand (e.g., Con A Sepharose) are particularly preferred. . Next, the sugar-free eluate that passed through this column was dialyzed against distilled water at 2 to 10°C for 2 to 5 days, and then dried, resulting in the antitumor active composition of the present invention as a yellowish-white to reddish brown powder. You can get things.

The antitumor active composition according to the present invention obtained in this manner is a powder that is slightly soluble in water. Its composition is approximately 5 to 5 sugars
8%, proteinaceous 60-70% and nucleic acid 25-3
It is 0%.

The antitumor active composition according to the present invention has tumor suppressive activity even in nude mice (leg line-deficient mice) and exhibits high tumor suppression after one administration. It is thought that the antitumor activity is expressed by a mechanism of action (not involving T cells) that is different from that of conventional immunostimulants (involving T cells).

The acute toxicity value of the antitumor active composition of the present invention is at least 1 g/kg or more when administered intraperitoneally to mice. Accordingly, the compositions of the present invention are useful in the treatment of malignant tumors in various animals, including humans, and can be formulated with conventional carriers or excipients and administered by various routes. The preferred route of administration is intravenous injection, intramuscular injection or subcutaneous injection, and in this case, the dose for an adult human is
It can be about 100-500 Q/day.

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 A medium consisting of 4.5% yeast extract, 9% polypeptone, and 18% glucose was added to 8 5Q flasks, 3Q each, and after sterilization and cooling, Candida albicans IT? '01385
300 ml (l) of the preculture solution was inoculated in each case, cultured at 37°C for 3 hours with shaking, and the culture solution was centrifuged to obtain 166 g of microbial cells in a wet state.

Add 800 m (l) of water to this, suspend, and add 800 m (l) of water.
Add rttQ n-butanol, stir at 4°C for 1 hour, centrifuge, and separate the aqueous layer. After dialysis at 4°C for 3 days, the precipitate was removed by centrifugation. After filtration with an Omm membrane filter, two fractions having a molecular weight of about 30,000 or less are obtained using an ultrafiltration membrane (Diaflow Hollow Fiber Cartridge tlP30-20 manufactured by Amicon). This solution was concentrated under d pressure and passed through a ConA Sepharose column (ConA 5epharosX, a polysaccharide with Concananophosphorus A as a ligand, an adsorbent for glycoproteins, etc. manufactured by Pharmacopoeia) column, and the fraction passing through the column was heated at 4°C for 2 hours. The antitumor active composition of the present invention can be obtained by lyophilization after dialysis for several days. Yield: 113 mg.

The infrared absorption spectrum of the antitumor active composition thus obtained is shown in FIG.

Example 2 Candida utilis IFOO was added to the same medium as in Example 1.
396 was inoculated to obtain 1,359 wet cells. to this,
700 mQ of water was added to suspend the mixture, 700 mQ of n-butanol was added, and the mixture was stirred at 4°C for 1 hour. The same operation as in Example 1 was carried out to obtain the target substance 138H.

Example 3 Candida albicans FOI3 as in Example 1
85 bacterial cells were obtained in a wet weight of 1589. Add water at 800 degrees Celsius to this, suspend it, add 800 mQ of phenol and bring it to 50 degrees Celsius.
After stirring for 1 hour, the same operation as in Example 1 was performed to obtain 75 mg of the target substance.

Pharmacology experiment 1 The antitumor activity of the composition of the present invention was tested in animal experiments.

A group of 6 BALB/C mice was implanted with 26 colon adenocarcinoma tumors, each of which was a syngeneic tumor.
Animals in the treatment group received test substance 25 shoulder 9-4 on day 5 after implantation.
00 m9/kg was administered once intraperitoneally. Control group animals received physiological saline in the same manner. On the 9th day after tumor implantation, the tumor was excised and weighed, and the tumor suppression rate based on comparison with the control was calculated according to the following formula.

Tumor suppression rate (%) As a result, the tumor suppression rate when the composition 100B of the present invention was administered was 72%.

Furthermore, as a result of testing BΔLB/C nude mice in the same manner as above, the tumor suppression rate at a dose of 200 m9/9 was 53.2%.

[Brief explanation of drawings]

FIG. 1 is a graph showing the Irt spectrum of the composition of the present invention.

Claims (1)

[Scope of Claims] 1. A composition obtained by extracting Candida microbial cells with a two-phase solvent consisting of water and a solvent partially miscible with water, comprising: (a) a molecular weight of 30,000; (b) Its composition is approximately 5-8% sugar and approximately 60-7% protein.
0% and about 25 to 30% of nucleic acids, and (c) exhibits antitumor activity not involving T cells. 2. (1) Extract Candida bacteria with a two-phase solvent consisting of water and a solvent partially miscible with water, (2) separate the aqueous layer from the extract, and (3) separate the aqueous phase. Dialysis is performed against distilled water, (4) the dialyzed water layer is ultrafiltered to remove components with a molecular weight of approximately 30,000 or more, and (5) the filtrate obtained in (4) is passed through a sugar adsorption column. Sugars are adsorbed and removed, (6) then (
5) A method for producing an antitumor active composition, which comprises dialyzing the eluate obtained in step 5) and then freeze-drying it. 3. The method according to item 2, wherein the bacterial cells are in a logarithmic growth phase. 4. The method according to item 2, wherein the bacterial cells are selected from the group consisting of Candida albicans, Candida utilis, and Candida guillermondi.