JPS58180426A - Antitumor substance - Google Patents

Abstract

PURPOSE:An antitumor substance, obtained by removing a solid substance from a suspension of crushed bacterial cells belonging to acetic acid bacteria, and consisting of a water-soluble intracellular fraction without side effects. CONSTITUTION:An antitumor substance consisting of a water-soluble intracellular fraction obtained by removing a solid substance from a suspension of crushed bacterial cells of bacteria, e.g. Acetobacter MK-17 strain (FERM-P No. 5747), belonging to acetic acid bacteria. The aimed water-soluble intracellular fraction is obtained by cultivating the various bacteria belonging to the genus Acetobacter aerobically in an ordinary culture medium for the acetic acid bacteria at 20-45 deg.C, preferably 28-35 deg.C, usually by the shaking or spinner culture method with aeration, collecting the bacterial cells from the resultant culture by the centrifugation, etc., destroying the bacterial cells, centrifugally precipitating the cells, dialyzing the resultant supernatant liquid, and freeze-drying the dialyzate. The resultant fraction is prepared from the nonpathogenic bacteria, and will not cause side effects, e.g. reduction in leukocytes, body weight and appetite, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an antitumor substance obtained from a disrupted bacterial cell solution of bacteria belonging to the Acetobacter group.

In recent years, there have been many reports that certain plants, mushrooms, microorganisms, etc. exhibit antitumor activity through their hosts, and they have come to be used clinically as anticancer agents.

However, the conventionally reported antitumor agents containing bacterial cells as active ingredients are Corynebacterium (Oryne-ba,
cterium) and hemolytic streptococcus (S
trθptococcus hemol, yticw
) etc., which are pathogenic to the human body, and therefore, when preparing an actual antitumor agent, a complicated purification process is required to remove the pyrogens contained.

On the other hand, Bifidobacterium (B
In the case of strains belonging to the genus Ifidobacterium and Lactobacillus, they are anaerobic and have a slow growth rate, and require complicated operations such as avoiding contact with air during culturing. There were drawbacks.

The present inventors have long been involved in the study of acetic acid bacteria (Acetic acid bacterium).
I am currently engaged in research on aerobic fermentation using bacteria belonging to the genus A. The present invention was completed by discovering that the intracellular water-soluble fraction of bacterial cells has excellent antitumor activity. That is, the present invention relates to an antitumor substance comprising an intracellular water-soluble fraction obtained by removing a water-insoluble fraction from a suspension of disrupted bacterial cells belonging to the Acetobacter group.

The present invention will be explained in more detail below.

The acetic acid bacteria used in the present invention include bacteria belonging to the genus Acetobacter and Gluconobacter, and specific examples of these bacteria include "Purges of Determinative Bacteriology, 18th Edition, pp. 251-253, 276. 278 (1974), and examples of bacteria belonging to Gluconobacter include Gluconobacter oxidans (Gluconobacter oxidans).
conobacter oxydans)■Fo-s
2s7 strain, Gluconobacter suboxidans (
G, 5ubdoxydans) IFO-1252
8 strains, Gluconobacter industrius (G,
ind, ustrius) ■AM-1816 strain, Gluconobacter melanogenus (G, melan
, ogenus) IFO-3294 strain, and as a bacterium belonging to the genus Acetobacter, Acθtobaatθr-aceti (Acθtobaatθr-aceti)
s28s strain, Acetobacter lancens jA, ra
ncens) NRRL-B2S3 strain, Acetobacter busturianus (A. pasteurianus)
)■FO-5225 stock.

Acetobacter aceti, subsvicii xylinum (
A, aceti 5ubsp, glinum) FO-5288 strain, etc. can be suitably used.

In addition, according to the above classification method, among the strains classified as Acetobacter in that they have a strong ability to produce acetic acid from ethanol and at the same time peroxidize acetic acid and lactic acid to decompose them into carbon difluoride, terminal electron transfer Ubiquinone-1 as a coenzyme in the system
It exhibits similar properties to Acetobacter aceti sapsubicyi xylinum in that it contains 0 as the main y component, but on the other hand, it does not form a thick film made of cellulose when cultured statically. Strains of the genus Acetobacter, which are essentially different from Subs. xylinum and whose taxonomic position is still unclear, can also be suitably used in the present invention.

Acetobacter MK- is an example of a strain belonging to the genus Acetobacter whose taxonomic position cannot be determined.
There are 17 strains (?ERM P-57'$'7). The mycological properties of Acetobacter MK-17 strain are disclosed in a patent application filed in 1982.
It is disclosed in the specification of No. 145724.

The medium used for culturing these strains may be a normal medium for acetic acid bacteria, and carbon sources include, for example, ethanol, glycerol, glucose, and gluconic acid.

Manlnitol, sucrose, dextrin.

A saccharified solution of cereals containing phlegm is used as a nitrogen source, and inorganic and organic nitrogen sources such as ammonium hyalate, yeast extract, and peptone are used alone or in combination. Also potassium and sodium.

Salts such as calcium and magnesium, and microcapsules such as pantothenic acid and nicotinic acid are used as necessary.

Cultivation is carried out aerobically. Usually, shaking or aerated agitation culture is preferred, but surface culture can also be used. The culture temperature can be changed as appropriate within the range in which the bacteria grow, but is usually 2°C to 45°C, preferably 28°C to 55°C. The culture time varies depending on various conditions, but in the case of shaking or aerated agitation culture, the culture is usually 20 to 120 hours, and in the case of surface culture, it is usually 120 to 480 hours.

To obtain the intracellular water-soluble fraction of bacterial cells from cultures of bacteria belonging to the genus Acetobacter or bacteria belonging to the genus Gluconobacter cultivated under the conditions described above, various known treatment methods may be arbitrarily employed. can do. For example, bacteria are collected by centrifugation, washed with physiological saline, etc., to remove medium components and extracellular products, and then collected, for example, by Dyn0-M1llKDL type (Switzerland Wiely B).
achOf6u Nhxgilleering Wor
KS) to destroy the bacterial cells, and then a low speed (7,000
10 minutes at rpm.

The undestroyed bacteria that settle by centrifugation is removed at 0°C), and the supernatant liquid is then centrifuged at high speed (28,000 rpm for 60 minutes at 0°C).Then, the precipitated insoluble substances are removed, and the The precipitated supernatant liquid is placed in a dialysis tube and dialyzed overnight against 20 to 50 times more distilled water, and then the dialyzed liquid is freeze-dried to obtain a powdered intracellular water-soluble fraction of the bacterial cells.

The intracellular water-soluble fraction of the bacterial cells obtained in this way is extracted from non-pathogenic bacteria and is not toxic to cells. No cytotoxicity was observed, and there was a decrease in white blood cells9.
Weight loss 9 No unfavorable or fatal symptoms such as loss of appetite were observed.

The actual dose of the antitumor substance according to the present invention is appropriately selected depending on the animal species, route of administration, and administration schedule, but for example, in the case of intraperitoneal administration to mice,
Next, the intracellular water-soluble fraction of the bacterial cells belonging to the acetic acid bacterium according to the present invention was tested for its growth-inhibiting effect on Sarcoma type 180 solid cancer by the following method. Examined. That is, ascites was collected from Sarcoma 180 ascites-bearing mice on the 7th day after transplantation, and 5 x 10791 m was collected using sterile physiological saline.
/Create a cell suspension of 0.1d (5
A group of 10 aay (Zan) mice weighing 19±11 were subcutaneously transplanted into the left tremor region of each group. From 24:00 on the S implantation, 20 Q of powders obtained by the methods shown in Examples 1 to 5 below were applied.

A sample of 1011 g and 2 mg dispersed in 1 g of physiological saline was administered once per animal at a rate of 0.1 - 10 g every other day.
The drug was intraperitoneally administered twice, and the antitumor activity was determined by calculating the dispersion ratio of tumors in the treated group to those in the untreated group. The antitumor activity was calculated using the following formula.

T: Weight of tumor in mice administered with the product of the present invention.
The antitumor activities of strains belonging to the genus Acetobacter or Gluco7bacter prepared according to the methods described in Examples 1 to 3 below were as shown in Table 1 below.

Table 1 Example 1 A liquid medium consisting of 20% gulfur sodium, 20% ethanol, 0.5% glucose, 0.3% glycerol, 0.5% polypeptone, 0.2% yeast extract, pH 4.5 The mixture was placed in a jar fermenter, inoculated with Acetobacter acechee FO-3283 strain, and cultured with aeration at 50°C for 4 days. Immediately after completion of the culture, the culture solution was centrifuged to collect the bacterial cells, which were washed twice with physiological saline.

The bacterial cells obtained as described above were transferred to Dyno-Mill K.
Cells were disrupted using the DL type. Centrifuge the disrupted cell suspension at 7000 rprn for 10 min.
The supernatant with undestructed bacterial cells removed is further heated to 2800 Orpm.
1. Centrifuged at 0°C for 0 minutes, put the centrifuged supernatant into a dialysis tube and dialyzed against 15 tons of distilled water at 5°C overnight.The resulting dialyzed fluid was freeze-dried to form 581 powder. I got it. The component analysis values of this fraction were as follows.

The analysis method was based on the following methods.

1) Carbohydrate phenol sulfuric acid method (M, Dubois, K,
A.

G1.11es, J, K, Ham'l1ton,
P, A, Reber III.

ava F, Sm1th, Analytical
Chem1str728, 550.1956) 2) Protein Lowry method (0, D, 5 tauffer
, Anal, ytical Biochemistr.
y 69.646.1975) 5) Lipid Bligh-Dia method (E, G, Bligh and W, J,
Dyer, Oa.n., J., Biochem,
Physiol, H1911, 1959) 4) ff acid Schmidt-Tannhauser, Schneider method (W, O, 5chneider, J,
Eiol-, Ohemo, Black, 747.1946),
fractionation using the diphenylamine method (K, Burto
n, Bj, ochem.

Jo, 62.315.1956) and the orcinol method (W
, Mejbau7Il, Z, Physiol, Oh
em, 258.117.1959).

Example 2 Yeast extract 5%, sake lees 71. Glucose 107. Ethyl alcohol 24P, vinegar! 569-containing medium was placed in an 801-volume jar fermenter, and this was mixed with 5 y-1 of yeast extract and 1 oz of sake lees per 11 ml of tap water.
1. gu/l/course 201i'.

2 t of a seed culture of Gluconobacter suboxidansae strain FO-12528, which had been shake-cultured for 30 DEG C. Cl2O hours in a medium containing 5 ay of ethyl alcohol and 2 z of acetic acid, was inoculated, and culture with aeration and agitation was started. Culture was terminated 25 hours after the acetic acid concentration reached 5.6%.

The cultured solution was centrifuged to collect bacterial cells, and diluted with physiological saline for 2 hours.
After washing twice, the bacterial cells are suspended in physiological saline and crushed using an Aminco French press. The obtained cell suspension was centrifuged at 7,000 rpm for 10 minutes at 0°C, and the supernatant was further centrifuged at 28,000 rpm for 60 minutes at 0°C to obtain a supernatant fraction. Both supernatants were put into a dialysis tube and dialyzed against distilled water overnight, and the dialyzed solution was freeze-dried to obtain powder specimen 6.1i.
I got it.

Example 5 Sake lees yeast was inoculated into the saccharified liquid of rice malt, alcoholic fermentation was carried out, and the sugar content (Brix) obtained by pressing was 5% and alcohol was 6.
Pour 150 tons of alcoholic fermented liquor into a 200% surface fermenter, and add sugar content (Br1x) containing 2.0% alcohol.
) 50'C in 5% rice malt saccharified liquid medium.

Acetobacter MK-j7 strain (I
Add 20t of seed culture solution of PBRM P-5747),
Surface fermentation was carried out while keeping the temperature at 0°C, and the culture was terminated 240 hours after the residual alcohol concentration reached 0.2%. The cultured solution was centrifuged to collect the bacterial cells, washed with physiological saline, the obtained wet bacterial cells were suspended in physiological saline, and 5ONI
FIERMOD”fGL 550 (output 117W,
The material was crushed using a device with a frequency of 20 KH2).

The disrupted suspension was centrifuged at 7,000 rpm for 10 minutes, and the resulting supernatant was further centrifuged at 28,000 rpm in an ultracentrifuge.
.

Centrifuge for 60 minutes to remove insoluble fraction. Both supernatants are dialyzed against distilled water for 24 hours. The dialyzed fluid was freeze-dried to obtain standard sample 4.91.

Patent applicant: Nakano Suten Co., Ltd.

Claims (1)

[Scope of Claims] 1) An antitumor substance consisting of an intracellular water-soluble fraction obtained by removing solid content from a suspension of crushed bacterial cells belonging to the Acetobacter group. 2) The antitumor substance according to claim 1, wherein the bacteria belonging to Acetobacter belong to the genus Acetobacter. 5) The antitumor substance according to claim 2, wherein the bacterium belonging to Acetobacter is Acetobacter MK-17 strain (IPERM P-5747). 4) The antitumor substance according to claim 1, wherein the bacteria belonging to Acetobacter belong to the genus Gluconobacter.