JPS5857153B2 - Manufacturing method for anti-cancer substances - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION CI) BACKGROUND OF THE INVENTION Technical Field The present invention relates to a method for producing water-soluble anticancer substances from yeast cell walls, and more particularly from the insoluble fraction of yeast autolysis products.

PRIOR ART Many methods for producing substances with anticancer activity from yeast cells are already known.

Most of these methods involve treating yeast cells with hot water, alkaline treatment, or self-extinguishing treatment, and obtaining a water-soluble anticancer substance from the resulting solution.

However, with this method, the utilization rate of yeast cells is low, and problems such as disposal of waste liquid in the case cannot be ignored.

In terms of obtaining anticancer substances from the yeast cell wall itself, for example, there is the invention described in Japanese Patent Publication No. 47-15712, No. P, and No. 53-44614.

The former involves subjecting a specific yeast to a process consisting of self-extinguishing, enzyme treatment, and dilute alkali heating treatment, and then recovering the cell wall fraction, which is then subjected to a strong alkaline heat treatment.The latter involves brewer's yeast. The water-insoluble fraction obtained by removing the water-soluble extract from the autolysed product is used as an antitumor agent.

Although the latter anticancer substance was not water-insoluble in the past, the former method is also water-soluble and is described as an anticancer polysaccharide consisting only of glucose. The substance becomes a gel in neutral or acidic water;
This gel-like material is said to be soluble in alkaline water (column 3, lines 33-36 of the same publication).

The anticancer activity of any of these yeast cell-derived anticancer substances has been described only when administered intraperitoneally to Sarcoma 180 solid tumor in mice; Details regarding other routes of administration are unknown.

[Summary of the Invention The present invention aims to provide a novel water-soluble anticancer substance derived from yeast cell walls, and a method consisting of enzymatic treatment of an insoluble fraction of yeast autolysis products. The aim is to achieve this goal by

Therefore, the method for producing an anticarcinogenic substance according to the present invention involves allowing a yeast cell wall lytic enzyme to act on the autolyzed insoluble matter of yeast of the genus Satucharomyces to obtain a water-soluble anticarcinogenic substance in the resulting solution fraction; It is characterized by:

Effect: A water-soluble anticancer substance is produced from the cell wall of yeast of the genus Satucharomyces that has undergone a specific history of autolysis and insoluble matter.

This substance is mainly composed of mannose.

This substance has been found to have anticancer effects not only on Sarcoma 180 solid tumor in mice but also on some other tumors when administered intraperitoneally.

This homogeneous σ is thought to act on the immune system of tumor-bearing animals to exhibit anticancer effects, but the fact that the effect is observed even after oral administration is a major advantage in actual use.

In addition, this substance was found to have an anticancer effect on rat ascites liver cancer AH130, but considering that it is said that anticancer drugs that promote the immune system are not effective against this tumor, It can be said that there is a large difference in the mechanism of action between the substance according to the present invention and the anticancer polysaccharides that have been previously reported.

In the present invention, the yeast extract extraction residue obtained by the autolysis method can be used as a raw material, which leads to effective use of yeast extract production by-products, and the production process itself is simple.

Detailed description of the invention
Except for S), the autolysis step p and the insoluble matter and its recovery method are essentially the same as those conventionally known.

Examples of the yeast of the genus Satucharomyces include beer yeast, sake yeast, and baker's yeast.

In the present invention, brewer's yeast and baker's yeast are representative.

Specific bacterial strains include Satucharomyces cerevisiae (S, cerevisiae), Satucharomyces
cursis bergensis (S.

carlsbergensis), and others.

Yeast autolysis can be carried out by a method commonly used for yeast extract production.

Specifically, for example, yeast is suspended in water or a suitable aqueous solvent, a small amount of an organic solvent such as toluene is added, and then self-digestion is carried out at 30 to 50C for 30 to 60 hours.

The autolyzed insoluble matter can be obtained from the autolyzed reaction product by means such as centrifugation.

One suitable autolytic insoluble for use in the present invention is
This is yeast extract extraction residue that is produced as a by-product during the yeast extract manufacturing process using the autolysis method.

2. Enzyme treatment ■) Yeast cell wall lytic enzyme The typical yeast cell wall lytic enzyme used in the method of the present invention is Arthrobacter (7) Bacterial production enzyme (Japanese Patent Publication No. 47-32674 Tokuko Showa 4
No. 8-2790) p and Oelscobia genus (
OerSkcvia) enzymes produced by bacteria (J, B
acteriol, vol. 111, p. 821 (1972
year)).

It goes without saying that yeast cell wall lytic enzymes similar to these enzymes can also be used if desired.

Preferably, the enzyme is at least partially purified.

Specifically, a commercially available product is "Zymolyase" (registered trademark) (eg, Zymolyes 60000), which is an Arthropacter enzyme.

2) Enzyme reaction Add an appropriate amount of enzyme to a suspension of autolyzed insoluble material at an appropriate concentration, e.g. For units, see Japanese Patent Publication No. 47-32674),
to a suitable pH 1, for example pH 6.0 to 9.01.
The reaction is preferably carried out at a pH of 7.0 to 8.0 and at an appropriate temperature, such as 20°C to 50°C, preferably 35°C to 45°C, for an appropriate period of time, such as 0.5 to 6 hours, with stirring. By doing so, a reactant can be obtained.

The yield of the anticancer homogeneous product of the present invention can be increased by stirring the autolyzed insoluble material of the raw material in an aqueous alkali solution having a pH of about 12 or more at room temperature for about 5 to 60 minutes.

During this treatment, known sulfites (eg, sodium sulfite, potassium sulfite) or SH group-containing compounds (eg, mercaptoethanol) can be present as agents for promoting the action of yeast cell wall lytic enzymes.

Additionally, these valuable compounds can also be present in the reaction mixture during the above enzyme treatment.

After reacting for a predetermined period of time, the pH of the reactant is adjusted to pH 3.0 to 6.0.
After adjusting the pH to about 3.5 to 4.5, the insoluble matter is removed by centrifugation or other methods.
A clear solution is obtained.

Before recovering the anticarcinogenic substance of the present invention from this solution, it is usually subjected to protein removal treatment according to a conventional method.

For protein removal treatment, for example, trichloroacetic acid, which is an appropriate protein precipitant, is added and dissolved to a concentration of about 2 to 4%, left to stand at a low temperature, for example, 5°C or less, for about 2 hours or more, and then centrifuged. The process consists of removing the formed precipitate by means such as , filtration, etc.

When a water-miscible non-solvent such as methanol or ethanol is added to the solution thus obtained, the anticancer substance of the present invention is precipitated.

A treatment consisting of collecting this precipitate, dissolving it in water, adding the above-mentioned water-miscible non-solvent thereto and precipitating it again is carried out in step 1.
Repeat this process one or more times (at least once in this process).
The anticancer substance of the present invention can be obtained in the form of an aqueous solution by further performing a dialysis treatment on water to remove the low molecular weight fraction. It will be done.

If this solution is freeze-dried, the anticancer substance of the present invention as a solid standard can be obtained.

This substance can also be further purified by conventional methods such as gel filtration and ion exchange, if necessary.

3. Anticancer substance The anticancer substance of the present invention obtained in this manner is a protein polysaccharide mainly composed of mannose, containing 7 to 90% carbohydrate as mannose and (15 to 3.0 g) of nitrogen. It is presumed that this is the case.

It is soluble in water, insoluble in organic solvents such as methanol, ethanol, and acetone, and shows a positive color reaction in any of the following reactions: Anthrone reaction, Molisch reaction, ninhydrin reaction, and biuret reaction.

It is easy to infer that the detailed physicochemical properties of the substance obtained by the present invention vary depending on the type of yeast, the type of yeast cell lysing enzyme, the type of substance purification method, and the degree of purification. - Shows remarkable anticancer effects in all cases.

As a specific example, the physicochemical properties and physiological effects of the preparations obtained in the Examples described later are as follows.

l) Physicochemical properties (1) Elemental analysis C40.9% ± 1.2 Rice cake H6.0% ± 0.2 φN 1.9% ± 0.1 Poly0 50.7% ± 1.5 Ash content 0.5% ±0.05 average molecular weight by ultrafiltration method 300,000-340,
It is 000.

(3) Melting point (decomposition point) Polysaccharides generally do not have a melting point, but they turn brown at around 265°C and turn black at around 270°C.

(4) Specific optical rotation [α] 25D = +57.4 to 63.2 (C21.0 ratio)
(5) Ultraviolet absorption spectrum as shown in FIG.

No specific absorption is observed.

(6) Infrared absorption spectrum As shown in FIG.

(7) Solubility in solvents Soluble in water, methanol, ethanol, - Insoluble in Teru and Ascent.

(8) Positive for color reactions Anthrone reaction, Molisch reaction, ninhydrin reaction, biuret reaction, Jakuyo and xanthoprotein reaction.

(9) Basic, acidic, and neutral The pH of the 1% aqueous solution is 5.5-6.5.

-color of substance White (1]) Types and composition of constituent sugars Mannose 79-35φ, glucose 15-21φ, a small amount of glucosamine is detected.

(12) Sugar binding mode The release of sugar by α-annanase Therefore, mannose is bonded through α-bonds.

0 Types and composition of constituent amino acids Serine 20-25%, Threonine 17-20%, Alanine 15-18%, Glutamic acid 7-9%
, aspartic acid 7-9%, valine 7-8%, proline 6-8%, glycine 3-4%, inleucine 2-3%,
2-3% lysine, 1-2% leucine, 1-2% tyrosine
, phenylalanine 0.3-0.7%.

2) Anticancer effect (1) Effect on mouse Sarcoma 18o Five million cells of Sarcoma 180 in the ascites were subcutaneously inoculated into 5-week-old ddY male mice, and from the next day the substances according to the present invention are shown in Table 1. Administration route and dose were administered once daily for 10 times, animals were sacrificed 5 weeks after tumor cell inoculation, tumors were excised, weighed, and tumor growth compared with that of the control group. The inhibition rate was calculated.

The results are shown in Table 1.

As a reference example, a commercially available anticancer drug derived from Coriolus versicolor was also tested in the same manner.

The substance according to the invention can be administered intraperitoneally or orally. Together, they have anticancer effects that are superior to commercially available products. Indicated.

(2) Effect on rat ascites liver cancer AH130, AH66 and AH44 were used as tumor cells, and female Donryu rats (approximately 150 g) were used as animals.

10 million tumor cells were implanted into the tail vein of a rat, and from 3 days after the tumor implantation, the substance of the present invention dissolved in physiological saline was administered orally at a dose of 9 in 100 η/dose. It was administered once a day for 10 days.

The survival rate was observed for 60 days after tumor transplantation, and Fig. 3 (
The results shown in Part 1 (3) were obtained.

In the group treated with the substance of the present invention shown by the solid line, the tumors were AH l 30 (1), AH66 (2), and AH44 (3), respectively.
The control group indicated by the dotted line indicates the case in which only physiological saline was administered.

Although all of the control group died from any tumor before 20 days, the group administered with the substance of the present invention had an AHI of 30% even after 60 days.
In XAH44, 50% survived, and in AH66, 83 survived.

A similar test was carried out on a commercially available anticancer drug derived from Coriolus versicolor, but no difference in survival rate was observed between the drug-administered group and the control group.

3) Toxicity Acute toxicity in mice is as follows.

The mice used were DD male, 5 weeks old, and weighing 20 to 27 g.

There were two routes of administration: oral and intraperitoneal administration.

After administration of this substance, the presence or absence of death and general symptoms were observed for 7 days.

As a result, no deaths were observed even at the maximum technically administrable dose, and the LD was estimated to be 5.00 oWIV kg or more for both oral and intraperitoneal administration.

5. Experimental Examples In the following examples, Zymolyase 60000 (trade name) was used as a yeast cell wall lytic enzyme.

In addition, as a raw material, yeast extract extraction residue produced as a by-product during the yeast extract manufacturing process by the autolysis method manufactured by Beer Yeast Kirin Beer (2) was used.

Example 1 200 g of raw material was warmed in water to make 2 liters, 25.6 g of sodium sulfite was added and dissolved, and the pH was adjusted to 8.0.

200 η of enzyme was added and reacted at 38°C for 3 hours with stirring.

After the reaction was completed, the pH of the reaction product was adjusted to pH 4.0 using hydrochloric acid, and centrifugation was performed to obtain a supernatant.

Trichloroacetic acid was added to the supernatant to give a concentration of 2.5%, and the mixture was left at 2°C overnight.

The precipitate deposited by centrifugation was removed, twice the amount of ethanol was added, the precipitate was collected and redissolved in water, and twice the amount of ethanol was added again to form a precipitate.

The precipitate was collected and dissolved in water, and the pH of the solution was adjusted to pH 12 with a caustic soda solution, and then twice the amount of ethanol was added to form a precipitate.

The precipitate was collected and dissolved in water to make the pH neutral, and then dialyzed against running water for 2 days.

Subsequently, after dialysis against pure water for 1 day, insoluble matter in the dialyzed solution was removed by centrifugation and freeze-dried.

Yield was 21.6g.

Example 2 200 g of the raw material was suspended in water to make 2 liters, and after adjusting the pH to 13 using a caustic soda solution, the suspension was stirred at room temperature for 30 minutes.

The pI was adjusted to 8.0 using hydrochloric acid, 500 μl of enzyme was added, and the mixture was reacted at 38° C. for 3 hours with stirring.

After the reaction was completed, 17.3 g of an anticancer substance was obtained from the reaction product in the same manner as in Example 1.

Example 3 200g of raw material was suspended in water, and 25.0g of sodium sulfite was added.
After adding 6 g, adjust the pH to 1 using caustic soda solution.
It was set as 2.8.

After stirring at room temperature for 10 minutes, adjust the enzyme to pH 8.0 with hydrochloric acid.
00■ was added thereto, and the reaction was allowed to proceed at 38°C for 3 hours with stirring.

After the reaction was completed, 5 g of anticancer substance 3o was obtained from the reaction product in the same manner as in Example 1.

Example 4: Suspend 40g of raw materials in water to make 400ml, adjust the pH to 8.0 using caustic soda solution, and then add 27w1 of enzyme.
! was added and reacted at 35°C for 2 hours with stirring.

After the reaction was completed, 4.3 g of an anticarcinogenic substance was obtained from the reaction product in the same manner as in Example 1.

[Brief explanation of drawings]

1 and 2 are reproductions of the ultraviolet absorption spectrum and infrared absorption spectrum of the anticancer substance according to the present invention, respectively. 3 are graphs showing the effect of the anticancer substance according to the present invention on rat ascites liver cancer.

Claims (1)

[Scope of Claims] 1. An anticancer substance characterized by causing a yeast cell wall lytic enzyme to act on the self-extinguishing insoluble matter of yeast of the genus Satucharomyces to obtain a water-soluble anticancer substance in the resulting solution fraction. Method of manufacturing a substance. 2. A patent claim in which the yeast is selected from the group consisting of brewer's yeast and Kung yeast, and the yeast cell wall degrading enzyme is an enzyme produced by a bacterium selected from the group consisting of bacteria of the genus Arthronophyta p and bacteria of the genus Oerscobia. The method described in item 1.