JPS59155322A - Antitumor substance ks-pm and its preparation - Google Patents

Abstract

NEW MATERIAL:An antitumor substance KS-PM having the following characteristics. Elementary analysis (%), C 40.6+ or -1.2, H 6.3+ or -0.2, N 1.4+ or -0.04, O 49.9+ or - 1.5, ash content 1.8+ or -0.05; molecular weight (ultrafiltration), 30,000-100,000; melting point (decomposition), about 250 deg.C; specific rotation, [alpha]<23>D=+40-+55 (C=1.0, H2O); solubility, soluble in water, insoluble in methanol, ethanol, etc.; color reaction, positive to Molish reaction, etc., and positive to ninhydrin reaction, etc. after hydrolysis; exhibits neutral - slightly acidic nature (5.4-5.8pH) as 1% aqueous solution; color, white - pale yellow. USE:Remedy for tumor. PREPARATION:A KS-PM-producing microbial strain is cultured aerobically in a solid or liquid medium at 10-35 deg.C and 4-9pH for 1-8 days. The microbial cells are removed from the cultured product by filtration, and the filtrate is subjected to the salting-out to remove proteins, and to the dialysis, etc. to remove the substances having low molecular weight. The objective KS-PM is obtained by removing the solvent from the product.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an antitumor substance KS-13M, a method for producing the same, and a tumor therapeutic agent comprising the substance.

Antitumor polysaccharides obtained from microorganisms include those obtained from heel yeast (11) and cell walls (special 13tl If
155-147224 minutes), produced by filamentous fungi belonging to the genus Pestaloteia (Japanese Patent Application Laid-open No. 56-49395), and those produced by autolysed heel yeast belonging to the genus Satulomyces It is also known that immune-promoting 10E substances can be obtained (Japanese Patent Application Laid-open No. 7878/1987), but these are not produced by bacteria belonging to the genus Penicillium (0°) but are produced by bacteria belonging to the genus Penicillium. As a polysaccharide that
No. 634) It is an acidic polysaccharide, not a neutral polysaccharide, and also has antitumor properties.This invention They found that Penicillium Jg+ produced an almost neutral polysaccharide, and succeeded in isolating the polysaccharide and called it K5-1'M. First, after examining its properties, it was found that it had a remarkable 3rjCIt heavy-duty "pL", and the present invention was completed.

Antitumor substance K S , -p M used in this invention
'4':.

Among the +1'(- bacteria, this strain is tentatively referred to as the K1-o-1-32 strain. Its Dutch characteristics are as follows.

Observation records on various differentiation media are as follows.

[Medium] [Growth condition] Wort agar Growth is vigorous. On the 5th day of culture, the mycelium becomes a medium.
The color is yellow to orange, with some parts tinged with red, and the center swollen. Some greenish-gray conidia are observed.

The underside of the hyphae is orange-brown. yellow diffusivity Elution of the dye is observed. Culture 7 As the days pass, the central part of the bacterial flora becomes reddish-orange. Green conidia grow around it.

Potato grapes Medium growth. After 5 days of culture, the outside of the bacterial flora turns yellow on the dextrose agar side and orange on the inside, and green conidia grow in the center of the medium medium.

The back side of the bacterial flora is yellowish brown, and the elution of the pigment is unacceptable. Orange area after 7 days of culture The fungus is covered with conidia. .

・Zapek Growth is vigorous. The bacterial flora becomes do-shaped, yellow-orange, and the surface exhibits folds, and eventually the entire surface becomes covered with deep-green conidia. It exhibits an orange-brown color and elutes a diffusible dye of the same color.

Automy Growth is medium 71V. After 5 days of culture, the outside of the bacterial flora was white, the inside turned yellow, and pale green-gray conidia were observed on it. On medium heat, the growth of some single-shaped aerial mycelia of the fungus H is yellow and there is no diffusible pigment. After 7 days of culture, the entire surface is covered with gray conidia.

YpS s Cold Growth is vigorous. After 5 days of culture, the bacterial flora leaves 2 to 3111 M of white hyphae on the outer large medium and a large number of velvety conidia are attached to the entire surface.

The underside of the bacterial flora is yellowish brown and no diffusible pigments are visible. I can't stand it.

Saburo cold Growth is poor. The mycelium is thin and white. After 5 days on the large culture medium, some green conidia were observed on the knob-shaped strain in the center.

The back side of the mycelium is white. There are no diffusible pigments.

Corn mee Growth is slow and poor. The vegetative hyphae are thin and white. A yellow sticky substance appears in the center.

Adhesion of conidia was observed in culture 78 years ago. I can't. The back side is almost the same as the front side. Same thing. There is no diffusible C3 accumulation.

Yeast YM Cold Growth is good. After 5 days of culture, the bacterial flora took on the shape of a large folded medium, the outer edge was 1 to 3 cm wide and the inside was pale orange, and the center had greenish-gray conidia. Exudate is seen on the back side of Bacteria M. The underside of the bacterial flora becomes orange-brown, with pigment diffusion of the same color.

In addition, other physiological and ecological properties include growth pH.
Range: 3.5-8.5 (optimal 4.5-6.0) Growth temperature range: 20-35°C (optimal 25-30''C) Growth on nitrite medium: impossible.

Benicillus: Unicycle.

Conidiophore: IVt erect from J locus, tip is enormous.

Iyo: 8-14° finely woven.

Chain of conidia: circle (live shape).

Conidia bispheroid, 2-3 B. Smooth surface.

The strain listed in ``2'' is 1 in Penicillium multicolor.
~do. Penicillium multicolor (Penici-
Hiunm+IticoJor) is The Japanese Huetation Off Culture Collection Off Microorganisms b, Su(-i'he J
apaneseF'ederation of C
u1cure Co11ection of M
icroorganis+ns) 1957 edition No. 81
The KU-0-132 strain described in page 2 is
It has been deposited with the Research Institute of Kokuri Gi 1, 1q Institute of Biotechnology Industrial Technology I + I as accession number 4375.

In producing the antitumor substance K5-PM of this invention, K5-PM producing bacteria are cultured in a medium.

In principle, the culture method is similar to that of common organisms, but aerobic culture using solid or liquid media is usually used.
There is 1 to C. The 4:1 medium contains bran, rice bran, defatted fat 8, natural products such as cottonseed flour, starch, sugars such as glucose, r-ravinose, xylose, ammonium sulfate, sodium nitrate, peptone, malt extract, nitrogen such as yeast extract, etc. sources, inorganic phosphates, magnesium salts, calcium salts, etc.;I
IE j salts can be added. Culture is from 10 to
Temperature of 35°C, preferably 25-30°C, 4-9
It is suitable to carry out aerobically, preferably at a pH of 4 to 7, for 1 to 8 days.

The antitumor substance K S -1), M accumulates in the culture;
It generally reaches its peak after 2 to 6 days of culture.

Antitumor substances from the culture medium! j K S -I'
In order to collect one pot of M, conventional methods for separating and collecting natural products may be applied. That is, culture l proboscis r
Bacterial cells are removed using a method such as salting out, proteins are removed from the ρ solution by salting out, low-molecular substances are removed by means such as dialysis, and acidic substances, basic substances, etc. are removed using conventional methods as necessary. Remove by. When the solvent is removed from the K S -1)M-containing solution thus obtained by concentration, freeze-drying, etc., the antitumor substance K S -J,) M is obtained. Ice crystals can be purified by appropriately combining commonly used purification methods.

4 yuan 1 stamped 1 yen 1 yen 1
(The characteristics of S -1'M are as follows.

■ I and basic content are good C2 40.6-112 %Jl
= 6.3 f 0.2 %N' = 1.4 l
-0.04$ 0 = 49.9-ql, 5 type ash content = 1.8-0.05% Q) Molecular clearing The average molecular -fN according to the ultra-rho method is 300
00 not L], 00000 (:'There is.

■ A' + lI 1 point (minute y 9 points) In general, polysaccharides) are 1 g;
0 ”C appended IJi and minutes jl, l〆.

■ Hinozu “Likelihood” 3 [α capital 240~+55 (C=], 0.1120) [F]) Ultraviolet gland prefectural spectrum As shown in Figure 1, no specific absorption was observed.

(0 infrared absorption spectrum, as shown in Figure 2, approximately 3400.295o, 1660o,
1530, 1360~1460゜1 () 10~11
40, 970, 91 (), 810.500~
670C No. 1 has 11 pores. (1) Solubility in solvents Soluble in water. Insoluble in methanol, ethanol, ether and acetone.

■ Color reactions such as Molisch reaction, phenol-sulfuric acid reaction, and Anthrone reaction were positive. After hydrolysis, ninhydrin reaction and Elson-Molcan reaction were positive.

■ Distinction between basic, acidic, and neutral 1% aqueous solution is neutral or slightly acidic (PI-15, 4-5,
8).

■ Color of substance: white or pale yellow.

From the above characteristics, the antitumor substance KS-PM is considered to be a new substance belonging to polysaccharides.

The antitumor activity of KS-PM was assayed using Ehrlich T/L.
/ shi/ -7 (Ehrl ich carcinom
Line 1C using the in vivo tumor support test method in mice inoculated subcutaneously with a).

(Experiment) As a test material, I) Injected a 51-week-old S strain 1m mouse, and inserted a Nielrich-6 lucinoma into the cavity of the mouse 11 with an 11-dose water burr. On the 7th day after irrigation, the ascites was subcutaneously transplanted into the dorsal region at a number of 06 cells (7 days, 3'x to another mouse).

24 hours after the outpost, K S-P M was injected into the abdominal cavity of the mouse as a physiological saline solution for the first time, and thereafter in the same manner.
111 times, this was repeated 5 times for 10 days, the tumor was removed on 8th day of the 15th, and a control group in which only physiological saline was administered and l'
The amount of li tumor was compared. In addition, KS-PM throwing school! ■ is 6
One group consisted of 10I71. was set to 1 m. Tumor 1t1. -+1-, the rate was found by the following formula.

-T Tumor 1 ratio ---= The anti-tumor properties of the 14I tumor substance KS-PM and Krestin (commercially available) were tested and the results are shown below. It is clear that it has a sexual nature.

The acute toxicity of KS-1) M obtained in Example 1 was determined by 11 trials using 1) S strain mice (10 animals weighing 20-23 g), and the following results (I- 1) So'l direct) was obtained.

Orally [-1 dose-'g, more than 20 F/' worms Intraperitoneal injection 1. From the results described above, it is clear that the toxicity is extremely low.

Next, examples will be shown.

Example 1 Liquid medium I containing 10% Arabicum and 1.0% dry yeast
Partial pressure of 100 ytrt of base (pl(6,0)) was placed in a Sakalo flask and sterilized by steam at 121°C for 20 minutes. The collar was inoculated with 1 vtl of the cuttings of the U-0-1,32 strain, and 1.4 Q r, p, +n
0) 71 at 28°C while vibrating with a rotating vibrating reed
Incubate for ”. Combine 20 bottles of culture solution and mix with Nutsche J5.
The bacterial cells were removed by filtration to obtain 1 volume of culture solution F 1.5e.

This culture solution was heated at 120"C for 30 minutes, and after removing insoluble matter, it was concentrated under reduced pressure to a volume of 150 ml. This was saturated with 90% ammonium sulfate to precipitate proteins, and the supernatant was collected. After drying, the 1'11 antitumor substance K S -P M 2.2 y was obtained by freeze-drying.

Example 2 IT, described in Example 1.
S-13M20g 1) 118.0:'A #J
'a D5r j & l OOrut, add proteolytic enzyme pronase P to digest and decompose the protein as tumor, remove the decomposed product by dialysis, and freeze-dry the solution to prepare anti-tumor storage MS-PM1. Obtain 4g.

Example 3 Uniformly mix 1.51 g of defatted rice and 1.51 g of cottonseed powder (1 3 e of water), put it in a vat, and incubate it in an autoclave. Benithulium multicolor I comb 1-〇-1,3 cultivated for 3 days
Inoculate 150 g of seed koji of 2 strains and leave to stand at 28°C for 5 days. 5.4 kg of the culture was crushed and packed in a Norarum, and extracted by pouring water through -1-. Quickly extract the extract 18e.
b minutes to remove a small amount of solid matter, concentrate by ultrafiltration to 4 parts, add acetone 2,7e, collect the resulting precipitate, and dry under reduced pressure to obtain powder 165y. ' Dissolve 100g of this powder in 1β of water and heat at 120'C for 20
Immediately heat for 500 ryt and pass through a cotton bag.
get l. This was saturated with ammonium sulfate to 90% to remove protein, and the supernatant was dialyzed and lyophilized to produce crude antitumor substance K.
Obtain S-PM 15.4'l. 10g of ice crystals
i8. The product was dissolved in a mild phosphoric acid solution of Q, digested with the proteolytic enzyme pronase I', dialyzed and lyophilized to obtain a 4-b lung, I-labile substance K 5-11 M 7.29.

[Brief explanation of drawings]

X'! Figure 3J1 shows the anti-11 (I tumor rate K S-13
The spectrum of the ultraviolet stage 11'y of M in FIG. 2 is also an infrared absorption spectrum. Patent applicant Shionogi & Co., Ltd. and one representative
Person: Patent attorney (Mae), 1 other person

Claims (1)

[Claims] (]) Following characteristics 1 (1) Elemental analysis C=4o, 6±1.2%N=6.
3±0.2% N=1.4±0.04% 0=49.9±1.5% Ash content -18±0.05% ■Molecular cell The average molecular weight by the ultra-dialysis method is 30,000
It is from 1oooooo. ■ Sub-1 point (decomposition point) Generally, for polysaccharides, 1%1
Although one point was not observed, it decomposes at around 250°C. ■ Specific optical rotation [α] 23-+40 to +55 (to = 1.0, H2O) 1
] ■ Ultraviolet absorption spectrum As shown in Figure 1, no specific absorption was observed. ■ Infrared absorption spectrum As shown in Figure 2, approximately 3400.2950°16601
1530.1360-1460.1010-1140.
970. 910°810.500~670c? 〃'
It has one stage L1 orifice. ■Soluble J111 Soluble in water. Insoluble in methanol, ethanol, ether and acetone. ■ Presentation 1: Molisch reaction, phenol-sulfuric acid reaction, and Anthrone reaction were positive. Hydrolysis 'l&, ninhydrin reaction and Ernne-Morgan reaction positive. ■ ``Distinction between acidic, acidic, and neutral 1% aqueous solution is neutral to slightly acidic (Pt15.4-5.8
). [Stock] Color of substance White [Kabuto or Danken [Ha. Antitumor substance KS-1) Mo (2) Belongs to the genus Penicillium and has the following properties: ■ Elemental analysis C = 40.6 ± 1.2 %) l = 6.3 ± 0.
2〆ぢN=1. , 4-j, 0.04%〇249.9
i-1,5 type ash = 1.8 ± 005 + type (■ Molecular weight The average molecular weight by the ultrapolar filtration method is 3000
It is 0 to 1oooooo. ■ h) 11 points (decomposition point) In general, 1 for multi-layer
．． Go! l! Although the point is recognized as I'λ, it is 19If at around 250°C. (4) Torsion) C degree 3 [α], ) = -1-40 to +55 (C = 1.0.112
0)1'9 Ultraviolet absorption spectrum As shown in FIG. 1, no specific fl absorption was observed. (■ Infrared absorption +15j spectrum As shown in Figure 2, shadow J: 3400.2950.166
0.1530.1360-1460.1010-114
0.970.910.810.500~670Cn! =
It has 11 prongs. ■ Solubility in solvents Soluble in water. Insoluble in methanol, ethanol, ether and acetone. ■ Color reactions such as Molisch reaction, phenol-sulfuric acid reaction, and Anthrone reaction were positive. After hydrolysis, ninhydrin reaction and Elson-Morgan reaction were positive. ■ Distinction between basic, acidic, and neutral 1% aqueous solution is neutral or slightly acidic (pH 5.4 to 5.8)
. ω1 color: white to pale yellow. A method for producing an anti-tumor substance KS-PM, which comprises separating and collecting the anti-tumor substance KS-PM from a hip culture cultured in a culture medium using a producing bacterium of M. . (3) The following characteristics■ Elemental analysis C=40.6±1.2 %H=6
．． 300.2% N=1,4''20.04% 0=49.9±1.5% Ash content=1.8±0.05% ■ Molecular weight The average molecular weight by the 1-view Tato r-filtration method is 300
00 to 100000. (3') Melting point (decomposition point) Polysaccharides generally do not have a melting point, but they decompose at around 250°C. ■ Toji no 亦) 1'; degree 3 [α] D2 +40 ~ +5.5 (C = 1..0゜It
20) (9 As shown in the ultraviolet absorption spectrum in Figure 1, no specific absorption is observed.) seconds The infrared absorption spectrum in Figure 2 shows a stop of 1, approximately 3400.2950.1660
．． 1530.1360~1460°1010~1140
．． 970.910.810.500~670cm'
has a suction of +1J. ■ Solubility in solvents Soluble in water. Insoluble in methanol, ethanol, needles and acetone. ■ Color reactions such as Molisch reaction, phenol-sulfuric acid reaction, and Anthrone reaction were positive. After hydrolysis, ninhydrin reaction and Elson-Morgan reaction were positive. ■ Distinction between basic, acidic, and neutral 1% aqueous solution is neutral to slightly acidic (pal 5.4-5.
8). ■ Color of substance: white or pale yellow. A tumor therapeutic agent characterized by containing an antitumor substance K5-I) M as an active ingredient.