JPH04104795A - Novel substance sbs and production thereof - Google Patents

Abstract

NEW MATERIAL:SBS having the following physicochemical properties. Elemental analysis (X); C 73.06, H 11.85; mol.wt.: 738 (by FAB-mass spectrum); molecular formula: C45H86O7; coloring reaction: negative to ninhydrin reaction and phenol sulfuric acid reaction, positive to iodine reaction and potassium permanganate decolorization reaction; solubility: soluble in methanol, acetone, chloroform and ethyl acetate, slightly soluble in water and hexane; appearance: white powder; specific rotation: [alpha]<20>=+5.4 ; neutral, etc. USE:The SBS exhibits a platelet aggregation-inhibiting activity and an anticarcinogenic promoter activity and is useful as an anti-thrombogenic agent, anticarcinogenic agent, etc. PREPARATION:A SBS-producing bacterium belonging to the genus Lyophyllum such as Lyophyllum.ulmarium Lul-8 (FERN BP-1416) is cultured at 20-25 deg.C at a pH of 7-8 for 5-15 days by a liquid culture method.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a novel substance SBS having platelet aggregation inhibitory activity and anti-cancer promoter activity, and a method for producing the same.

[Conventional technology]

In recent years, there has been an active search for physiologically active substances from Basidiomycetes.
Various reports have been made. For example, anti-tumor substances have been found in Corsiella versicolor and Swahirotake mushrooms, antihypertensive substances have been found in Cypress mushrooms, and immunostimulating substances, anti-allergic substances, anti-ulcer substances, etc. have also been found in various Basidiomycetes. In addition, pharmacological effects have been found in commonly eaten mushrooms, with antitumor substances found in shiitake and enokitake, and blood pressure lowering effects in oyster mushrooms (Japanese Unexamined Patent Publication No. 2-7).
No. 2121) is known. In addition, functional foods that promote the tertiary functions of foods are currently attracting attention, with the Ministry of Education's specific research "Systematic analysis and development of food functions" as an opportunity, and research on the bioregulatory functions of foods we ingest on a daily basis. Searches for physiologically active substances in foods are also actively conducted.

[Problem to be solved by the invention]

An object of the present invention is to provide a novel physiologically active substance produced by Liophyllum ulmarium, which is commonly eaten as a food mushroom along with shiitake and enokitake mushrooms, and a method for producing the same.

[A means of saving to solve problems]

To summarize the present invention, the first invention of the present invention is a novel substance S
Regarding BS, it is characterized by having the following physical and chemical substances.

Elemental analysis: C73,06% H11,85% Molecular weight:
738 (according to FAB-mass spectrum) Molecular formula: C,5H8,0° ultraviolet absorption spectrum (in methanol): As shown in Figure 1, it exhibits maximum absorption at 208 nm.

Infrared absorption spectrum (KBr tablet): 3400.2990. as shown in Figure 2. 2960.2920.1380. Absorption is shown at 1180.945 C[l+-'.

Color reaction: Negative ninhydrin reaction, negative phenol sulfuric acid reaction, positive iodine reaction, positive potassium permanganate decolorization reaction The second invention of the present invention relates to a method for producing a new substance SBS, which is a new substance SBS belonging to the genus Liophyllum. It is characterized in that the novel substance SBS of the first invention is collected from the culture of the producing bacteria or the fruiting bodies of the producing bacteria.

To give an example of the present invention, as the fruiting body of the new substance SBS-producing bacterium, one that is artificially cultivated and commercially available in large quantities under the trade name "Yamabikohan Shimeji ■" can be used. As an example of a culture of a bacterial strain, a culture of Liophyllum ulmarium Lui8 (FERM BP-1416) can be used, and the microbiological properties of this strain are described in Japanese Patent Application Laid-open No. 63-27.
Although described in Japanese Patent No. 3467, the novel substance SBS-producing bacteria used in the present invention is not limited to the above-mentioned strains, but any strain belonging to the genus Liophyllum can be used.

The method for producing the new substance SBS consists of three steps: obtaining fruiting bodies or cultured bacterial cells, extraction, and purification.

The fruiting bodies are easily available commercially, and the cultured bacterial bodies are
- Obtained by liquid culture method.

For example, assimilated organic carbons such as glucose, sucrose, starch, dextrin, glycerin, and fats and oils are used as carbon sources, and yeast extract, peptone, corn stew liquor as nitrogen sources, defatted soybeans, soybean hulls, meat extracts, amino acids, and ammonium salts are used as nitrogen sources. Organic nitrogen compounds and inorganic nitrogen compounds such as can be used. In addition, inorganic salts such as magnesium salts, phosphates, and potassium salts may be added. The culture temperature is suitably 15-30°C, preferably 20-25°C. The culture pH is suitably 5 to 9, preferably around 7 to 8. Culture may be either shaking culture or static culture, and bacterial cells can be obtained in 5 to 15 days, but static culture produces a higher amount of SBS. Extraction is then performed using an organic solvent such as methanol, ethanol, acetone, ethyl acetate, or chloroform.

Purification was performed using normal phase and reversed phase silica gel, and reversed phase ODS.

Adsorption chromatography of cellulose etc., gel filtration method,
General separation and purification methods such as methods that utilize differences in solubility in various solvents can be used.

The novel substance SBS obtained as described above has the following physical and chemical properties.

1. Appearance: White powder 2. Elemental analysis: C73.06% H11.85%3
, Molecular weight (according to FAB mass spectrum) = 4, Molecular formula: C, 5H, 60° 5, Specific optical rotation: [α] 20 = +5.4° (c = 1.
0, methanol) 6. Ultraviolet absorption spectrum (in methanol): As shown in Figure 1, maximum absorption is shown at 208 nm. That is, Figure 1 7゜8゜9゜ shows the ultraviolet absorption spectrum when this substance is dissolved in methanol at a concentration of 0.25 mg/-, and the absorbance (vertical axis) and wavelength (nm, horizontal axis). FIG.

Infrared absorption spectrum (KBr tablet): As shown in Figure 2, 3400.2990. 2960.2920.1380. It exhibits an absorption at 1180.945 cm-'. That is, FIG. 2 is a diagram showing the infrared absorption spectrum of the KBr tablet of the present substance in terms of the relationship between transmittance (%, vertical axis) and wave number (cm-', horizontal axis).

Color reaction: Ninhydrin reaction was negative, and phenol-sulfuric acid reaction showed no sugar coloration. Positive for iodine reaction and potassium permanganate decolorization reaction.

Solubility: Soluble in methanol, acetone, chloroform, and ethyl acetate, slightly soluble in water and hexane10, Distinction between acidic, neutral, and basic: Neutral No known substances that match the above physical and chemical properties have been reported. ,SBS
was determined to be a new substance.

[For production]

Next, the physiological activity of SBS, a novel substance of the present invention, will be illustrated by experimental examples.

Experimental Example 1 Platelet aggregation inhibitory effect: Final concentration of platelet aggregation stimulant: 5 μg/m
l-collagen was used. Japanese domestic colored rabbit platelets (
4X IO5/μfl) 0.2rnj7, final concentration 1-50μg/mf! 30 seconds later, collagen was added and platelet aggregation was observed. As a result, as shown in Table 1 below, the new substance S
BS had a clear platelet aggregation inhibitory effect.

Table 1 SBS 1.0 10.0 3.0 18.6 Experimental Example 2 Anti-cancer promoter action potent promoter teleocidin B-4
-4) induced by Nibstein-Pearl (Bpstei)
The effect of completely suppressing early antigens of N-Barr virus was investigated in vitro using Raji cells.
tro) system.

Raji cells (5X 10'/rnl) at 20ng/m
The cells were cultured for 48 hours in a medium containing f-teleosidin B-4, 5 μM dimethyl sulfoxide, and 3 mM n-butyric acid, and cells that induced early antigens were detected by indirect immunofluorescence. Similarly, when a new substance, SBS, was added at a final concentration of 2 μg/ml, the number of cells that induced early antigens was suppressed to 40% compared to when no addition was made. As a result, it was revealed that the new substance SBS has an anti-cancer promoter effect.

Experimental Example 3 Acute toxicity test The novel substance of the present invention was orally administered to ICR mice. 10
No toxicity was observed at 00 mg/kg administration.

As shown in the above experimental examples, the substance of the present invention exhibits platelet aggregation inhibitory activity and anti-carcinogenic promoter activity,
It is useful as an antithrombotic agent, an anticarcinogenic agent, etc.

In addition, this substance suppresses the rejuvenation reaction of lymphocytes caused by concanavalin A, and is useful as an antiallergic agent.

〔Example〕

Next, the present invention will be explained in more detail with reference to examples, but the present invention is not limited to the scope of the following examples.

Example 1 1.5 kg of the fruiting body of "Yamabikohan Shimeji ■" was freeze-dried and made into a fine powder using a pulverizer. Ethyl acetate 31 was added and extracted for 3 hours with stirring to obtain 5.4 g of extract.

This extract was dissolved in a small amount of chloroform and applied to a column filled with 75 g of silica gel 60 (manufactured by Merck & Co., Ltd.) and washed with chloroform. After washing with 11 chloroform, elution was performed with a chloroform-methanol (4:1.V/V) solution to obtain 1.1 g of eluate. This eluate was subjected to preparative reverse phase high performance liquid chromatography [Column: Capsule Pack C86, φ10 x 250 mm, manufactured by Shiseido Mobile phase:
Methanol-water (4: 1.V/V):
The chromatographic pattern shown in the figure was obtained. That is, FIG. 3 is a diagram showing a chromatographic pattern of reversed phase high performance liquid chromatography, where the vertical axis shows absorbance (210 nm) and the horizontal axis shows time (minutes).

Separate both portions of Figure 3, A: 18■, B: 3
9■, C: 44■, D: 38■, E: 56■, F:
61 mg, G: 48 mg and SBS: 106 ■ were obtained.

Example 2 Lyophyllum ulmarium ul-8 (FERM
100- seed medium (
Glucose 2%, yeast extract 0.3%, peptone 0.5
%, 0.1% of KH, PO, and 0.05% of Mg5O1.7H20) and cultured with shaking at 25°C for 14 days to obtain a seed culture. 200 d of this seed culture solution was inoculated into a 30 f capacity jar fermenter containing 10 A production medium (same composition as the seed medium), and cultured at 25°C for 6 days with aeration. (Amount of ventilation 101!/
20 min). Bacteria were collected using a filter press to obtain 830 g of wet microbial cells. After freeze-drying this bacterial cell, 5B349■
I got it.

Example 3 The platelet aggregation inhibitory effect and anti-carcinogenic promoter effect of each plate obtained in Example 1 were investigated, and the results shown in Table 2 were obtained. The platelet aggregation inhibitory effect was assayed in an in vitro system using collagen as an aggregating agent, and the anti-carcinogenic promoter activity was assayed by inhibiting Ebstein Le virus activation by an oncogenic promoter.

Bar No. 2 Table B ++
++C++ 10+ D ++++++ E 10
+10F 10 Dried C+ +SBS
＋＋＋＋ ＋＋＋＋ (In the table, 10 represents the strength of activity, and the strength is in the order of ＋＋＋＋＞＋＋＋＞10＞10. In addition, - indicates no activity) [Effects of the invention] The present invention SBS is a new substance produced by the edible fungus Rophyllum ulmarium and has anti-platelet aggregation and anti-carcinogenic promoter effects.
It is useful as a functional food material, etc.

[Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption spectrum of the new substance SBS, Figure 2 shows the optical external absorption spectrum (KBr tablet) of the same substance, and Figure 3 shows the reversed phase high performance liquid chromatography of the same substance. It is a figure showing a result.

Claims (1)

[Claims] 1. A new substance SBS having the following physical and chemical properties. Elemental analysis: C73.06% H11.85% Molecular weight: 738 (by FAB-mass spectrum) Molecular formula: C_4_5H_6_6O_7 Ultraviolet absorption spectrum (in methanol): Maximum absorption at 208 nm Infrared absorption spectrum (KBr tablet): 3400, 299
0, 2960, 2920, 1380, 1180, 945 cm^-^1 Color reaction: Negative ninhydrin reaction, negative phenol sulfuric acid reaction, positive iodine reaction, positive potassium permanganate decolorization reaction 2, new substance SBS-producing bacteria belonging to the genus Liophyllum A method for producing the novel substance SBS, which comprises collecting the novel substance SBS according to claim 1 from a culture of or a fruiting body of the producing bacterium.