JPS5893702A - Beta-1,3-glucan and its preparation - Google Patents

Abstract

PURPOSE:The titled neutral polysaccharide having an antitumoric property, which is separated and purified from the mycelia of a specified fungus belonging to Polyporaceae. CONSTITUTION:A glucan, estimated MW of 10,000-1,000,000, which is prepared by subjecting a beta-glucan fraction obtained, as usual, by extraction of the mycelia of oxyporus belonging to Polyporaceae with hot water to separation by affinity chromatography and which has, as a repeating unit, a structure (formulaI) in which beta-1,3-glucoside bonds constitute the main chain and one beta-1,6-glycoside bond of glucose is present per 12 glucose units.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel neutral polysaccharide having antitumor properties, a method for producing the same, and its use, and more particularly, the present invention relates to a novel neutral polysaccharide having antitumor properties, and more particularly, to a novel neutral polysaccharide having antitumor properties, and more particularly, to a novel neutral polysaccharide having antitumor properties, and more particularly, to a novel neutral polysaccharide having antitumor properties, and more particularly, to a novel neutral polysaccharide having antitumor properties, and more particularly, to a novel neutral polysaccharide having antitumor properties, and more specifically to a novel neutral polysaccharide having antitumor properties,
Ganod@rma applanmtum IFO6
Isolated and purified from the mycelium of 499AKU), with a molecular weight of 1
The present invention relates to a glucan having the following repeating structure, which is thought to have 1 million to 1 million repeats.

In recent years, many sl.
It has been reported that ＠substances have been obtained, and the structures of some of them have been clarified. If you ask me, the schizophylran that exists as a component in Suehirotake is 1β-1,3-
The structure has one 1,6-glycosidic bond for every three glycoside units as a repeating self-structure [Noka No. 45
Volume i, No. 4, pp. 1-62' (1971)], the antitumor polysaccharide lentinan contained in shiitake mushrooms contains both 1,3- and 1,6-bonds in the glycosidic bonds in the side chain [Fermentation and Industrial Science]. Volume 31, WJ12, page 912 (1976)]
. In addition, the glucan contained in Kykura-r has a main chain of 1,3-glycosidic bonds, and the ratio of 2 to 3 1,6-glycosidic bonds is 1,6.
- It has a repeating structure with a glycosidic bond side chain (Japanese Patent Application Laid-Open No. 54-63012), and the existence of glucan having a repeating unit similar to Schizophylrane is also known to exist in C. versicolor (Japanese Patent Application Laid-open No. 54-61112). ).

The present inventors previously collected an anti-tumor polysaccharide from the fruiting body of the Coffus chinensis fungus and clarified its structure [Agricultural and Bio-Logical Chemistry, Vol. 45, p. 328 (1981)] However, when the mycelium was further cultured and its polysaccharide components were examined in detail,
The present invention was based on the discovery that one component separated from the glucan fraction by affinity chromatography is C, which is a glucan with a very low degree of branching, which was not known at all in the past and was not found in fruiting body components. completed.

The β-1,3-glucan according to the present invention can be obtained by hot water extraction of the mycelium of the Coffus spp.
Affinity chromatography using
Igaku no Ayumi, Vol. 89, No. 1 (1974)], and its structure is as shown in formula (11), with β-D-1,3-glycosidic bond as the main chain, It is a glucan that has a structure in which glucose is bonded with β-1,6-glycosidic bonds as a repeating unit at a ratio of 1 to 12 glucose units.

The glucan according to the present invention is a simple glucan with a molecular weight of approximately 10,000 to 1,000,000, and its physicochemical properties are as follows.

1. Properties: White powder 2. Solubility: Insoluble in organic solvents such as alcohol and acetone; Soluble in water, saline, dilute aqueous alkaline solutions, etc.

3. Light intensity, [α), +9 (11, H2O) (
UniconGik@n Automatic Dig
ital Polarim@ter PM-101) 4, Infrared absorption spectrum: Figure 1 (KBr) 5, Nuclear magnetic resonance spectrum (C13): Figure 2 (5 regrets, 0.2
5 MNaOD/'DzO) (JEOL JEOL J
NM FX-100 type 25MHz) 6. Constituent sugar composition Nigelcose The structure of β-1,3-glucan according to the present invention can be prepared by digestion with β-glucanase, periodic oxidation, and β-glucanase of limited Smith degradation products. It was confirmed to be the formula CI) by digestion and nuclear magnetic resonance spectroscopy.

Next, the antitumor properties of β-1,3-glucan according to the present invention will be described.Test method and results I Glucan dissolved in sterile physiological saline was intraperitoneally administered once on day -3. The tumor diameter was measured on the 25th day and compared with the control group to calculate the tumor inhibition rate. In addition, the tumor complete disappearance rate on day 45 was examined.As described above, the compound of the present invention exhibits high antitumor activity even at low doses, and is useful as an anticancer agent.

Next, the method for collecting β-1,3-glucan of the present invention will be specifically explained with reference to Examples.

Example Coffus monkey sawfish (Gat+od@rma app
D-
3 glucose, 0.3 g yeast extract,
Potassium dihydrogen phosphate 6. xs, ammonium monohydrogen phosphate 0.15%, crystalline magnesium sulfate o, os*
Culture solution (pH 4, 8) contained in 1 (10dXS bottles)
t”301: for 7 days with shaking, then D-glucose 5, yeast extract 0.51G, potassium dihydrogen phosphate 0.11, ammonium monohydrogen phosphate 0.151!, crystalline sulfuric acid 1 Transplanted into a culture medium (PH4,8) containing 0.05 gnesium (I J ),
Culture with shaking at 0°C for 7 days. The culture solution is separated in a furnace, and the mycelium is washed with cold water and then with acetone, and then dried in a chamber at 40° C. or below with ventilation and powdered. Bacterial yield 7 is 1! The hit is 81.

Mycelium powder 100? Suspended in 100 times the volume of water, 95~
Heat to 1.00°C and stir and extract for 3 hours.

This operation is repeated twice, and the extracts are combined and concentrated under reduced pressure.

Add 7 times the volume of ethanol and collect the precipitate. The obtained solid content (11) is dissolved in water and subjected to column chromatography using a DEAE-cellulose (Ct-type) column (φ2.6×90cIL), and the fraction eluted with water is collected. Adding the same amount of ethanol will precipitate the glucan fraction. When the precipitate was collected and dried, 6.91 was obtained, and this fraction was 5.0? Dissolve 250 ml of water, add 250 d of borate buffer (pH 8,0), 0.15 M and add methyl-trimethyl-ammonium (C@t).
avlon) 250-. While keeping this solution at 20°C, add 3N-hydroxide (Q) rum aqueous solution to adjust the pH.
8,6. The resulting precipitate was centrifuged (85oot).

15 minutes), collected and diluted with 0.1M borate buffer (PH8,
6), dissolve in 100% of 3M acetic acid, and add 2x to this
volume of methanol is added and the resulting precipitate is collected by centrifugation. Wash the precipitate thoroughly with methanol, dissolve it in water, and add V
After dialyzing with tap water in a laking tube, the solid content is 2.7 when the internal solution is freeze-dried. A solid content of 100 q'' Ik0.2 M sodium chloride solution is obtained, and a Sepharose CL4B column (φ2
．． 6 x 97 cm). The eluate was separated into 8.3 d portions and a portion was subjected to the phenol sulfur method [Analytical Chemistry Vol. 28, p. 350 (
(1956)] Develop a color, measure the absorbance at 485 nm, and record the four turns of elution. The first eluted peak portion (tubes 421 to 25) is collected, dialyzed, desalted and lyophilized to obtain 16 solid fractions containing β-glucan.

50 da of solid matter from this fraction was dissolved in 0.1 M IJ sodium phosphate buffer pi-17.0, IM sodium chloride solution) 1.5 s IgKI, and concanavalin A-Sepharose cL4B column (φa, ox27 crIL). Affinity chromatography using
Elute with the same solvent. Aliquot the eluate in 10-hour portions, take a portion, develop color using the phenol-sulfuric acid method, and add 485r
Measure the absorbance at +m and record the elution arm turn. The fraction thus obtained (tube 7f & 5-15) was desalted by dialysis and then lyophilized to obtain 3.5 μl of β-ri-1,3-glucan having the repeating structure of formula [I]. .

[Brief explanation of the drawing]

Figure 1 shows the infrared absorption spectrum of the β-1,3-glucan of the present invention, and Figure 2 shows the cl5 nuclear magnetic resonance spectrum of the same compound. Patent Applicant Sankyo Co., Ltd. Agent Patent Attorney Osamu Kashi De Sho Figure 2 TPS ζ no m 90 80 70 60Pprn Supplemental Procedures/Overwriting (Voluntary) February 15, 1980 Commissioner of the Patent Office Haruki Shimada 1, of the case Indication 1981 Patent Application No. 1.9: (459 No. 2, Name of the invention β-1,:3-glucan and its manufacturing method 3, Relationship with the case of the person making the amendment Patent applicant address: 103 Chuo-ku, Tokyo 6 Name of 3-1 Nihonbashi Honmachi (185) Sankyo Co., Ltd. Representative Director and President Yoshinori Kawamura 4, Agent Residence 1-2-58 Hiromachi, Honbunagawa-ku, Tokyo 140 Sankyo Co., Ltd. 6. Subject of amendment Column 7 for detailed description of the invention in the specification;
Contents of the amendment As shown in Attachment 1, Formula CI) on page 2 of the specification is corrected as follows. "that's all

Claims (1)

[Scope of Claims] 1) β-1,3-glucosidic bond is the main chain, and the ratio of 1 to 12 glucose units is β-glucose.
A glucan having a 1,6-glycosidic bond structure as a repeating unit. 2) Extract the mycelium of Kopkisarnokoscus fungus with hot water and separate it against β-gluca/fractionation affinity chromatography 2 fee obtained by a conventional method to obtain a % image - β-
The main chain is 1,3-glucosidic bond, and one glucose unit for every 12 glucose units is β-1,6-
A method for producing glucan that has a glycosidic bond structure as a return unit.