JPS5934896A - Preparation of antitumor polysaccharide - Google Patents

Abstract

PURPOSE:To prepare a polysaccharide exhibiting antitumor activity, by culturing a polysaccharide-producing microorganism belonging to Acremonium genus. CONSTITUTION:A microbial strain belonging to Acremonium genus and capable of producing a polysaccharide, e.g. Acremonium sp. SBT7016 (FERM-P No.6601), is cultured on an agar plate medium. The obtained mycelia or conidospores are inoculated in a liquid nutrient medium and cultured at 20-30 deg.C for 3-5 days without agitation or under aeration and then at 25-27 deg.C for 4-6 days under shaking or under aeration and agitation. The objective antitumor polysaccharide is separated from the cultured product.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to Acremonium 4@ (Acremonium
The present invention relates to a method for producing polysaccharides exhibiting antitumor activity by culturing polysaccharide-producing m belonging to ).

In recent years, many methods for producing antitumor substances using polysaccharides as active ingredients by culturing basidiomycetes have been reported, and some of these methods are being implemented industrially.

On the other hand, it has been reported that some fungi Deuteromycetes produce polysaccharides outside the pepidermal cells when cultured in a liquid medium, but no fungi have yet shown effective antitumor activity. unknown.

As a result of examining fungi Deuteromycetes in soil that have the ability to produce polysaccharides that exhibit effective anti-cancer activity, the present inventor discovered that Acremonium, a rare species belonging to the genus Acremonium, and Subicii ( Ac
They discovered that polysaccharides exhibiting effective anti-tumor activity can be produced by culturing the Q strain, ``remonium sp,'')'', leading to their invention.

Incidentally, the term "hexapolysaccharide" used in the present invention means a heteropolysaccharide in which different types of sugars and substances other than sugars are bonded.

The present invention will be explained in detail below. The present invention involves culturing mycelia or conidia obtained by culturing polysaccharide-producing bacteria belonging to the genus Acremonium in an agar plate medium in a liquid medium, and obtaining antitumor activity from the culture. It is characterized by collecting polysaccharides exhibiting

The polysaccharide-producing bacterium belonging to the genus Lemonium used in the present invention was isolated from soil (mountain soil, Chichibu City, Saitama Prefecture), and was isolated from the subphylum Deuteromycota.
tina), Hypho-myce
tθB)
belonging to the Acremonium Society (A
cremonium sp,) Japanese BT 7016 display No. 6601 (FBRMP-6601) 1
7) The number has been entrusted to the Institute of Microbial Technology, Agency of Industrial Science and Technology.

In addition, Acremonium sp, SBT 701
6 was identified by W, Game, [Oephal
osporium-artige Shim-1
8 (1980) and Seiji Tokumasu: “Anti-bacterial and anti-mildew Jvol.

8, No. 2.18 (1980).

AcremoHlum sp, EIBT 70 below
16 mycological properties.

(1) Growth Growth on malt agar medium is slow, colonies are fluffy and yellow-brown, dark brown due to conidia formation, and the underside and agar become dark brown. Produces brown water droplets on the surface. Growth on potato dextrose agar medium is rapid;
It is fluff-like and white, and it remains white due to conidia formation, and the back side remains white.

(2) Microscopic findings on slide culture on potato dextrose agar medium show that the hyphae are colorless, and the conidiophores are colorless and do not have septa, growing upright from the side of the aerial hyphae. Although it is a branch, it contains only one branch.

The phialides are elongated and tapered. The conidia are phialoid, colorless, ovoid, and unicellular, forming slime balls and clustering at the tip of the conidiophore.

Utilizes ucrose, maltose, lactose and starch, but does not utilize xylose.

In the present invention, Acremonium e2-, SBT
7016 f'! The mycelia or conidia, or a mixture thereof, grown on the surface of the medium are obtained by culturing on an agar plate medium.

This culture is usually carried out at 20-30°C, preferably at 25-27°C.
Do this for 10 to 14 days. In addition, the agar plate medium used here is a potato dextrose agar medium normally used for culturing filamentous fungi, yeast, etc., with the addition of 0.1 to 0.3 weight percent yeast extract and the pH adjusted to 4 to 7. This is what I did.

Next, in the present invention, the white mycelium or conidia (spores) obtained by culturing as described above, or a mixture thereof, is used as a whole seed mother, and this is cultured in a liquid medium. When culturing in this liquid medium, the initial stage of culture (generally 3 to 6 days)
It is preferable to perform static culture or aerated culture, followed by shaking culture or aerated agitation culture for about 4 to 6 days. The reason for adopting this culture method is that when the mycelium as a seed mother is inoculated into a liquid culture and immediately subjected to shaking culture or aerated agitation culture without static culture, the mycelium does not grow. On the other hand, poly1i, which is the target substance,
! This is due to a decrease in the production of anti-cancer active substances whose main component is @. The culture temperature in the liquid medium is 20 to 30°C, preferably 25 to 27°C throughout the two stages.

The medium used for the above-mentioned liquid culture may be a known liquid medium that is generally applied to the culture of microorganisms, for example, it may be a medium that uses glucose as a sugar source and contains peptone and #mother extract.

Furthermore, it is also possible to use a medium to which inorganic substances, amino acids, vitamins, milk components, etc. are added.

Further, the pH of the liquid medium is suitably in the range of 4 to 7, and it is particularly preferable to use a liquid medium with a pH of 5.5 before sterilization.

Although culture in a liquid medium can be carried out successively, the productivity of polysaccharides gradually decreases as the number of passages increases, so it is recommended to inoculate bacteria only in a liquid medium for 3 to 3 days.
However, from the viewpoint of polysaccharide productivity, it is advantageous to perform a new liquid culture using whole mycelia grown on an agar plate medium.

However, when the culture scale becomes large (for example, when carrying out liquid culture of 10 cells or more), it becomes operationally difficult to use the entire mycelium as a seed mother for the agar plate culture, so static culture in a liquid medium is used as fungal culture. It is preferable to use the resulting culture as a seed mother. Alternatively, shake or aerate the first bound culture from the agar plate culture for 3 to 5 minutes with sterile physiological saline.
Double diluted and homogenized? , can also be used with seeds.

Aaremonium ep, 5B used in the present invention
When T7016 mycelia (or mineral conidia) are cultured in liquid as described above, a highly viscous substance is produced and the culture itself becomes highly viscous. Extraction techniques can be applied to collect the desired polysaccharide with antitumor activity from this highly viscous culture, but the extraction efficiency is not good if the culture is used as it is because of its high viscosity. Dilute with 3 times as much distilled water, mix thoroughly with a mixer, and apply techniques such as centrifugation and filtration to this mixture to obtain an aqueous solution fraction. In addition, the residue (mycelium) obtained by the above fractionation is washed with distilled water two to three times, and after adding water again, the mycelium is crushed with, for example, a Waring blender, and the aqueous solution fraction obtained by centrifugation is used. Furthermore, both fractions may be combined with the aqueous solution fraction. Furthermore, it is also possible to use the mycelial suspension as it is.

Next, free proteins, free nucleic acids, and
Reduction a! In order to remove impurities such as, both components are treated with activated carbon, a weakly basic ion exchange resin, or chromatography. These treatments also decolorize the fraction and remove low-molecular substances.

Furthermore, treatments such as precipitation with ammonium sulfate, precipitation using lower alcohols or acetone, molecular sieves, and ultrafiltration may be applied alone or in combination, and other treatments such as sepag, which is used for extracting polysaccharides from cultures, may be applied. method or enzyme method can also be applied.

The polysaccharide collected from the culture as described above is subjected to dialysis against distilled water, concentrated if necessary, and then freeze-dried to obtain a product.

Next, the physical properties of the polysaccharide thus obtained (hereinafter referred to as the present substance) will be explained.

Appearance: White in color and decomposes and carbonizes when heated.

Melting point: This substance does not show a clear melting point.

Infrared absorption spectrum: The results measured by the KBr tablet method are as shown in the attached figure.

Solubility: Soluble in water and dimethyl sulfoxide, but insoluble in general organic solvents and organic acids.

Note that an aqueous solution of this substance does not pass through a semipermeable membrane, a 2-10% aqueous solution has a pH of 6.2-6.4, and a 0.1% aqueous solution exhibits no ultraviolet absorption.

Color reaction: Molisch reaction, Anthrone reaction, and ninhydrin reaction are positive, and Elson-Morgan reaction, Bahaal reaction, and iodostarch reaction are negative.

The origin of this substance does not shift even when thin layer chromatography is performed under various conditions. This substance was hydrolyzed with 2-4N sulfuric acid, neutralized, and then trimethylsilylated and analyzed by gas chromatography. As a result, glucose and small amounts of mannose and galactose were detected. In addition, as a result of analyzing a sample obtained by hydrolyzing this substance with 4N hydrochloric acid for 14 hours using an automatic amino acid analysis method, galactosamine and a small amount of glucosamine were all detected.

Furthermore, this substance was made into a suspension of 50#v150rnl in water, treated with ultrasonic waves to sufficiently solubilize it, centrifuged, and the resulting supernatant was subjected to gel filtration or ion exchange system. Column chromatography or solution pH
As a result of analysis using a fractionation method that takes advantage of the difference in solubility due to adjustment, it was found that it is composed of polymers of neutral sugars whose main constituent is glucose, amino sugars whose main constituent is galactosamine, and proteins. .

The content ratio of these components varies depending on the culture conditions for obtaining the authentic Wk and the extraction conditions of the culture, but is generally as follows.

Neutral sugars: 50 to 60 subunits (as total hexose by phenol-sulfuric acid method) Amino sugars: 25 to 35 subunits (as hexosamines by indole-hydrochloric acid method) Proteins: 5 to 15 quintillion units % (as protein by the Lowry-Folin method) Next, the pharmacological properties of the polysaccharide obtained by the present invention will be explained.

(1) Acutely toxic mice are OR-JOL strain, 4-5 weeks old, body weight 20-2
5f rats were all Wistar strain, 4 to 5 weeks old, and weighed 110 to 140 y, and were administered orally or intraperitoneally to 25 rats in each test group for all acute toxicity tests. This was done as follows.

After administration of this substance, each test animal was observed for one week for general symptoms, changes in body weight, and deaths, followed by necropsy. The results are shown in the table below, and it can be seen that the LD5Q value is extremely high.

(2) Anti-1111 tumor activity The anti-tumor activity of this substance was assayed in vivo in mice subcutaneously inoculated with Sarcoma 180 solid saliva and in mice intraperitoneally implanted with Fihrlish tumor cells.
It was conducted using the o test method.

Antitumor activity against Sarcoma 180 solid tumor: Test animals were 6-week-old female mice (body weight 2
Sarcoma 180 tumor cells were subcultured intraperitoneally in mice in the ascites form every week. For the test, cells from the ascites in the mouth were removed one week after inoculation and subcutaneously transplanted into the lower right flank of saline test mice containing approximately 4 million cells. 24 hours after transplantation, add this substance to physiological saline for 50~
The solution was dissolved to a concentration of 0.25ml/ml and sterilized at 120°C for 15 minutes, and then administered intraperitoneally to 0.25rnl mice, followed by continuous administration for 10 days.

Control mice were similarly administered 0.25 rnt of sterile saline water.

30 days after transplantation of one tumor, the mouse was dissected and solid llj! A comparison with a control group was made by removing the tumor and measuring its weight. Each group consisted of 10 mice.

As a result, this substance has a tumor suppression rate of 87. The trowel showed strong antitumor activity, and the l11 tumors in 6 of the io animals in the treatment group had completely disappeared.

Here, the field lagoon suppression rate for Sarcoma 180 solid/i@tumor is a value calculated using the following formula.

0B CsE Average tumor weight TB of control group: The effective dose of this substance for Sarcoma 180 solid tumor in the test group is 5 to 15 cm/cm (body weight) per day,
It can be applied by converting it into an iR agent by a conventional method.

(Anti-tumor activity against ascites saliva: Test animals were 8-week-old female mice (
Four groups each consisting of 11 mice (body weight 252±3F) were administered with K I x 106 Ehrlis mice each.
hl111 tumor cells were transplanted, and 24 hours later, this substance was administered to each of three groups other than the control group at 25η/ume (body weight) for 7 days and 10 ri/k.
y (body weight) 7 days and 2.5η/mm (body weight)/8 for 10 days, respectively, to observe the survival effect and healing effect in each group. In addition, only physiological saline was administered to the control group.

The results were as follows.

25 >200 11 sea urchins 0 sea urchins 10
>200 11 sea urchins 0 sea urchins 2.5
>200 9 out of 11 animals (Note) 1) The survival rate was calculated as follows.

2) Number of cured tumors The number of tumor cells in each group that survived 60 days after transplantation and showed no accumulation of ascites is shown.

The substance obtained according to the present invention is Sarcoma18.
It also showed the same antitumor activity as above against 0.0 ascites ulcer.

EXAMPLES The present invention will be explained in more detail with reference to Examples below.

Example 1 Medium composition: Polypeptone 1. Of yeast extract 5.02 f glucose 30 f water 1 t pH = 5.5 A liquid medium consisting of the following ratio was dispensed into 50 Erlenmeyer flasks with a capacity of 500 d each, and after attaching cotton stoppers, the liquid medium was heated at 120°C.
A.
cremonium sp, SBT 7016 f was inoculated into the above liquid medium, and statically cultured at 23 to 27°C for 3 days. This was continued for a total of 5 days at 23-27℃.
Rotary shaking culture was performed at 180 rpm to obtain 10 tons of highly viscous culture. Add 10 tons of hot water to this,
After stirring and mixing with a mixer, the mixture was centrifuged for 20 minutes using TO, OOO Y to remove mycelium and obtain a viscous transparent liquid. Into this solution was activated carbon-7lite (7L), which had been treated with concentrated hydrochloric acid, thoroughly washed with water, then washed with ethanol and acetone, and dried.
1:1) mixture was added in an amount of 4% by weight, and the mixture was stirred at room temperature for 16 hours.

The activated carbon-celite mixture was removed by filtration, and the resulting solution was dialyzed against distilled water at 5° C. for 48 hours, and then freeze-dried to obtain an off-white powder of 27.5 F. The substance obtained in this way is a polysaccharide consisting of polymers of neutral sugars whose main component is glucose and amine sugars whose main component is galactosamine, and proteins.As a result of testing for its antitumor effect, it was found that JOL, Sarcoma 1 at ft101'f/kf intraperitoneally administered in 6 week old mice
The inhibition rate against 80 solid tumors was 87.3.

Example 2 Medium composition: Polypeptone 5y Yeast extract 32 Glucose 302 - Potassium phosphate 0.52 Dipotassium phosphate 0.5f Magnesium chloride 0.32 Manganese chloride 0.05f Water 1t A liquid consisting of the following ratios: pH=5.5 Dispense 100 d of culture medium into 500 m Erlenmeyer flasks, attach cotton plugs and sterilize at 120°C for 15 minutes, and add Acremonium sp, 8BT 7016 f, which had been separately cultured on an agar slant.
The cells were inoculated in a conventional manner and cultured stationary at 25°C for 4 days.

On the other hand, put 10 tf of a liquid medium of the above composition into a jar fermenter, sterilize it at 120°C for 30 minutes, cool it, homogenize the above culture with a Waring blender, transplant it, and raise the temperature to 25°C. After static culture for 2 days, aeration agitation culture was performed for 5 days under conditions of aeration II of 0.4 vvm and agitation number of 25 rpm.

The obtained culture was diluted twice, and the mycelium was separated and removed using F cloth.

To the thus obtained transparent viscous liquid was added 10 g of Amberly XAD-2 (container/cap ratio), and the mixture was mixed and stirred at room temperature for 2 hours. After separating the Amberlite resin, ethanol and the like were added to the supernatant liquid, and the resulting white precipitate was vacuum-dried to obtain a dark gray powder 24.52. This powder is a polysaccharide composed of polymers of neutral sugars whose main component is glucose and amine sugars whose main component is galactosamine, and as a result of testing its antitumor activity, the IOR-JO
L. The inhibition rate against Sarecoma 180 solid Ill tumor was 8315 cm after intraperitoneal administration of 10q/mK in 6-week-old mice.

[Brief explanation of the drawing]

The attached diagram illustrates an infrared absorption spectrum measured by the KBr tablet method of an antitumor substance composed of a polysaccharide obtained according to the present invention.

Claims (2)

[Claims] (1) Mycelia or conidia or a mixture thereof obtained by culturing polysaccharide-producing bacteria belonging to the genus Acremonium in an agar plate medium are cultured in a liquid medium, and the antitumor activity is determined from the culture. 1. A method for producing an antitumor polysaccharide, which comprises collecting a polysaccharide exhibiting the following. (2) For culture in a liquid medium, culture at 20 to 30°C for 3 to 5 days, followed by 4 days at 25 to 27°C.
The manufacturing method according to claim 1, which is carried out by shaking culture or aerated stirring culture for 6 days.