JPS62274000A - Substance h-13-2 - Google Patents

Abstract

NEW MATERIAL:A substance H-13-2 having the following physico-chemical properties. Melting point; 157-159 deg.C. Elementary analysis (%); C, 57.56; H, 9.07; N, 11.89. Contained amino acids; Alanine, proline and isoleucine. Specific rotatory power [alpha]<20>D=-31.3 (C=0.03, chloroform). Soluble in methanol, ethanol, chloroform, ethyl acetate and dimethyl sulfoxide, sparingly soluble in water and diethyl ether and insoluble in hexane. Molecular weight; 1205 (fast atom bombardment mass spectroscopy method). Positive to the sulfuric acid and iodine vapor and negative to hydrazine reagent, cerium sulfate reagent, Ehrlich regent and ninhydrin reagent. Neutral substance. White power. USE:An antimicrobial agent and carcinostatic agent PREPARATION:A microorganism, belonging to the genus Trichoderma and capable of producing the substance H-13-2 (FERM PBP-1009) is cultivated in culture medium at 5-6 pH and 20-40 deg.C for 4-8 days by a submerged culture method with aeration.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a novel H-13-2 substance. The H-13-2 substance of the present invention has antibacterial, antifungal, and anticancer effects and is useful as a medicine.Conventional technology The H-13-2 substance of the present invention is a novel substance that has not been described in any literature. It is.

Problems to be Solved by the Invention An object of the present invention is to provide a new substance having antibacterial, antifungal, and anticancer effects.

Means to solve problems The above object is achieved by the substance of the present invention described below.

The H-13-2 substance of the present invention has the following physical and chemical properties.

(a) Melting point: 157-159°C0 (b) Elemental analysis: 0 57.56%, H9.07%, N 11.89%.

(C) Containing amino acids: alanine, proline, isoleucine.

(d) Specific rotation: [α]20=-31,3 (C=0.0
3. In chloroform>.

(e) Ultraviolet absorption spectrum, λ (nm) in methanol:
No characteristic absorption is shown between 220 and 360. The chart is shown in Figure 1.

Br (f > infrared absorption spectrum, ν (cm-1): ma
× 3330.2940.1675.1539.1465.
A 1396° chart is shown in Figure 2.

(Cl) Solubility: Well soluble in methanol, ethanol, chloroform, acetic acid ethyl ester, and dimethyl sulfoxide. Slightly soluble in water and diethyl ether, and insoluble in hexane.

(h) Molecule ffl: 1205 (by fast atomic bombardment mass spectrometry).

(i>¥ Color reaction: positive for fiM acid, iodine vapor, negative for hydrazine reagent, cerium sulfate reagent, Ehrlich reagent, ninhydrin reagent.

(j) Distinction between basic, acidic, and neutral: neutral substances.

(k) Color of substance: white powder.

The H-13-2 substance of the present invention is produced by culturing a microorganism capable of producing an H-13-2 substance (hereinafter referred to as an H-13-2 substance-producing bacterium) in a medium, and then producing H-13-2 from the resulting culture. -13
- Can be manufactured by separating and collecting two substances.

An example of the H-13-2 substance-producing bacteria is the H-13 strain belonging to the genus Trichoderma, which the present inventors newly isolated from the soil of Heilong Province, People's Republic of China.

This H-13-2 substance-producing bacterium was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, with the accession number "Feikoken Joki No. 100".
No. 9 (FERM BP-1009>j).

Next, a case will be described in which the H-13-2 substance of the present invention is produced by culturing, for example, the above-mentioned H-13-2 substance-producing bacteria susceptible to Trichoderma in a medium.

The culture method is basically based on the culture method of general microorganisms, but a stirring aeration culture method using a liquid medium is usually advantageous.

The medium used for culture may be any medium containing a nutrient source that can be utilized by the H-13-2 substance producing bacteria.

That is, a synthetic medium, a semi-synthetic medium, or a natural medium can be used. Regarding the composition of the medium, carbon sources such as glucose, mannose, glycerin, sucrose, molasses, starch, liquefied starch, and organic acids are used singly or as a mixture. Examples of nitrogen sources include soybean flour, corn steep liquor, meat extract, yeast extract, cottonseed flour, peptone, wheat germ, and more! ammonium acid,
Ammonium nitrate and the like are used alone or in combination. Examples of inorganic salts include calcium carbonate, sodium chloride, potassium chloride, sodium nitrate, magnesium sulfate, cobalt chloride, and various phosphates, which are added to the medium as necessary.

Further, when foaming is significant during culturing, antifoaming agents such as vegetable oils such as soybean oil and linseed oil, higher alcohols such as octadecanol, and various silicon compounds may be added as appropriate.

During culture, the DH of the medium is about 5 to 6, and the culture temperature is about 2.
It is best to adjust the temperature to about 0 to 40'C. Culture time is 4-8
Approximately one day is appropriate. In addition, when performing mass production, it is preferable to use deep aeration culture.

After the cultivation is completed, the method for collecting the H-13-2 substance from the culture is similar to the method used when separating and collecting ordinary fermentation products from nutrients. For example, the To1-13-2 substance can be obtained by performing a suitable combination of filtration, centrifugation, adsorption/desorption using various active adsorbents, talomadography, extraction using various organic solvents, recrystallization, and the like.

The H-13-2 substance of the present invention thus obtained has a broad antibacterial spectrum against Durum-positive bacteria, Durum-negative bacteria, and fungi such as yeast and mold. In addition, the present invention H-13-2
The substance also has anticancer properties.

The antibacterial activity of the H-13-2 substance of the present invention was determined by the minimum inhibitory concentration (M ,I.

C,). The results are shown in Table 1.

Condolence table 1 Example Next, a more detailed explanation will be given with reference to examples.

Example liquid medium (potato extract (boiled Botel Slice 1009 in deionized water 500IrlQ for 15 minutes and removed solids with Gavi), glucose 2%, DH 5.5) was added to five 500m 12 Erlenmeyer flasks. After dispensing 100 mQ each into
H-13-2 substance producing bacteria grown on Podeto agar medium (
Inoculated with spores and mycelium of ``Feikokenjoki No. 1009'',
Rotary shaking culture at 27°C for 2 days (180 revolutions per minute, 1
0 cm) to prepare a preculture solution. Next, glucose 4
．． 0%, soyton 0.5%, sodium nitrate 0.3%,
Potassium dihydrogen phosphate 1 cum 0.2%, potassium chloride 0.0
5%, magnesium sulfate 0.05%, ferric sulfate 090
Liquid medium with a composition of 0.3% (1))-15,5>1
After dispensing 00 mQ each in the same manner, 5 mQ of the above preculture solution was inoculated as a seed into 80 500 mQ Erlenmeyer flasks that had been sterilized by autoclaving for 120' Cl2O, and rotated at 27°C for 5 to 6 days. Shaking culture (180 revolutions per minute, 10 ra) was performed.

The culture solution was filtered, and the remaining bacterial cells were extracted with 1.5 methanol. The methanol extract was concentrated, and the residue was extracted twice with 0.2 L each with ethyl acetate, and then the ethyl acetate extract was washed with water. The culture solution was extracted twice with 5 L of ethyl acetate each time, and the extract was washed with water, combined with the ethyl acetate extract from the bacterial cell side, dried over anhydrous sodium chloride, and concentrated to obtain the extract. The oily substance obtained was subjected to silica gel column chromatography (manufactured by Merli Co., Ltd., Kieselgel, 5.9 x 19 cm> (chloroform:methanol = 5:1) to separate the active components.The separated active components were again subjected to silica gel column chromatography ( The active ingredient was separated by applying Kieselgel, 5.9 x 19 cm (manufactured by Merck & Co., Ltd.) (acetic acid ethyl ester: methanol = 5:1).The separated active ingredient was recrystallized from acetonitrile, and a white powder of H-13-2 was added. The substance 98m!J was obtained.As an indicator of the active ingredient, Staphylococcus aureus 209P (5taphylococcus aureus
ccus aureus 209P) was used.

[Brief explanation of drawings]

FIG. 1 shows the ultraviolet absorption spectrum of substance H-13-2 in methanol, and FIG. 2 shows the infrared absorption spectrum of substance 1-1-13-2 measured with a potassium bromide tablet. (that's all)

Claims (1)

[Claims] (1) A novel H-13-2 substance having the following physical and chemical properties. (a) Melting point: 157-159°C. (b) Elemental analysis: C57.56%, H9.07%, N11.89%. (c) Containing amino acids: alanine, proline, isoleucine. (d) Specific rotation: [α]^2^0_D=-31.3(C
= 0.03 in chloroform). (e) Ultraviolet absorption spectrum, λ (nm) in methanol:
No characteristic absorption is shown between 220 and 360. The chart is shown in Figure 1. (f) Infrared absorption spectrum, ν^K^B^r_m_a_
x (cm^-^1): 3330, 2940, 1675,
1539, 1465, 1396. The chart is shown in Figure 2. (g) Solubility: Well soluble in methanol, ethanol, chloroform, acetic acid ethyl ester, and dimethyl sulfoxide. Slightly soluble in water and diethyl ether, and insoluble in hexane. (h) Molecular weight: 1205 (according to fast atomic bombardment mass spectrometry). (i) Color reaction: positive for sulfuric acid and iodine vapor, negative for hydrazine reagent, cerium sulfate reagent, Ehrlich's reagent, and ninhydrin reagent. (j) Distinction between basic, acidic, and neutral: neutral substances. (k) Color of substance: white powder.