CS245159B1 - A method for preparing a mixture of secondary anthraquinone metabolites - Google Patents

Abstract

The essence of the method lies in the fact that the microorganism Trichoderma viride CCM F-742 is cultivated on a liquid medium containing sources of carbon, nitrogen and mineral substances, then the mycelium and filtrate of the culture medium are subjected to extraction! with an organic solvent of the type of chlorinated hydrocarbons, acetic acid esters or aliphatic alcohols that are limitedly miscible with water, the obtained extract is dissolved in acetone after the solvent has evaporated, adsorbed on silica gel, eluted with a benzene-acetone mixture and the solvent is evaporated.

Description

The invention relates to a process for preparing a mixture of atrachinone-type secondary metabolites.

Many secondary metabolites of microorganisms are characterized by biological effects and can be used to combat pathogenic microorganisms, parasites such as cancerostatics and otherwise acting pharmacologically active substances.

Some of them are used as starting compounds for the preparation of semisynthetic derivatives with more advantageous or novel biological effects. The field of use of secondary metabolites or their semisynthetic derivatives with biological activity includes human and veterinary medicine, plant and animal production, food processing and other industrial production.

Among the most important secondary metabolites of microorganisms are anitbiotics and alkaloids, which have the most varied application. Searching for secular metabolites of microorganisms and exploring the possibilities of their practical use continue worldwide.

Different strains of Trichoderma viride microscopic fungus produce secondary metabolites of diverse chemical structures, e.g. peptide suzuucillins and alameticine, steroid viridine, epiditiodioxopiperazine gliotoxin and trichotecene trichodermin / R. J. Cole and R. H. Coxi Handbook of Toxic Fungal Metabolites, Academic Press, New York 1981, V. Betinai The Chemistry and Biology of Antibiotics, Elsevier, Amsterdam 1983 /.

Anthraquinone derivatives - pachybazine (3-methyl-1-hydroxyantraquinone) and emodine (3-methyl-1,6,8-trihydroxyantraquinone) / (C.-K) were also isolated from T. viride, which were not characterized further. Wati Microbial Quinones, in A.I. Laskin and H.A. Lechevalier, editors of the CRC Handbook of Microbiology, vol. III, Microbial products, CSC Press Inc., Cleveland, 1973, pp. 183-260].

According to PV 08189-83 by V. Betina and A. Fargash microorganism Trichoderma viride CCM P-742 produces anthraquinone derivatives. Anthraquinone secondary metabolites have a variety of biological effects. E.g. anthracycline antibiotics are cancerostatic and some anthraquinone derivatives from fungi have antimicrobial, insecticidal / e.g. ruglozin, J. Dobias, V. Betina and P. Nemec, Biology 35/1980 / 431-434, and other biological effects.

The process for preparing anthraquinone-type secondary metabolite mixtures according to the invention is characterized in that at a temperature of 20 to 25 ° C on a liquid medium again containing 6.0 to 6.5 containing sources of carbon, nitrogen such as yeast or corn extract and minerals, C cultures Trichoderma viride CCM F-742 for 15-25 days, then the mycelium and the culture medium filtrate are subjected to extraction with organic solvents such as chlorinated hydrocarbons, acetic acid esters or aliphatic alcohols limited to water miscibility, the extract obtained is dissolved in acetic acid It is adsorbed onto silica gel, eluted with a benzene-acetone mixture and the solvents are evaporated.

The crude mixture of anthraquinone metabolites from mycelium and culture medium is dissolved in acetone and adsorbed onto a 5-fold amount of silica gel by evaporation of the solvent under reduced pressure. The anthraquinone metabolites adsorbed onto silica gel are loaded onto a silica gel column prepared by suspending the silica gel in a benzene-acetone mixture. Elution with benzene-acetone (III or 2: 3) gave an anthraquinone metabolite mixture.

The mixture does not dissolve in water, dissolves in methanol, acetone, ethyl acetate and chloroform, weakly dissolves in benzene. The mixture can be qualitatively separated by thin-layer chromatography into several components.

Said mixture serves as a starting material for the preparation of pure anthraquinone-type substances.

Example 1 pieces 1000 ml Erlenmyer flasks are filled with 300 ml of a culture solution of this composition: sucrose 40 g, sodium nitrate 3 g, potassium hydrogen phosphate 1 g, magnesium sulfate 0.5 g, potassium chloride 0.5 g, ferrous sulfate 0.01 g, yeast atuolysate 5 g and 1000 ml drinking water, pH before sterilization 6.0 to 6.5. Sterilize at 120 ° C.

In the culture flasks, the solution is inoculated with 4 ml of a spore suspension of Trichoderma viride CCM F-742, which is prepared by pouring the spore culture in 1,000 ml Erlenmeyer flasks with 125 ml sterile 0.05% aqueous Tween 80 solution and releasing the spores with a sterile glass rod. . The culture flasks are incubated at room temperature statically under constant illumination or alternating day and night for 21 days when maximum production of anthraquinone metabolites is reached.

Example 2

The culture medium after cultivation of the Trichoderma viride strain of Example 1 is carefully decanted and filtered. The mycelium in Erlenmeyer flasks are coated with 200 ml of ethyl acetate, which is left to work for 16 to 24 hours at room temperature. The mycelium is then separated from the extract by vacuum filtration and the combined extracts are dewatered by filtration over anhydrous sodium sulfate and the filtrate obtained is evaporated under reduced pressure. 6 g of crude preparation are obtained.

Example 3

To 12 l of the culture medium filtrate obtained according to Example 2, 6 liters of ethyl acetate are added, mixed thoroughly with a stirrer for 20 minutes and the two phases are separated. 3 L of ethyl acetate is added to the aqueous phase and the procedure is as for the first extraction. The combined acetic phases are dehydrated by filtration through a pad of anhydrous sodium sulfate and the filtrate is evaporated to dryness under reduced pressure. 1 g of crude preparation is obtained.

EXAMPLE 4 g of the crude preparation obtained from the mycelium of Example 2 and the culture medium filtrate of Example 3 are extracted with 200 ml of n-hexane, the extract is filtered off and the solvent is evaporated off under reduced pressure. 4.45 g of an oily component are obtained, which is not further processed. The residue of the crude preparation is extracted with 1000 ml of ethyl acetate, the extract is filtered and the solvent is evaporated under reduced pressure. 1 g of a crude mixture of anthraquinone metabolites is obtained.

The crude mixture was dissolved in acetone, 5 g of silica gel was added and the solvent was evaporated under reduced pressure. The silica gel with adsorbed metabolites is loaded onto a 3 x 30 cm silica gel slurry suspended in benzene-acetone (95> 5) and eluted with benzene-acetone (1> 1-23). A yellow to orange fraction containing 6.5 g of anthraquinone metabolite mixture was collected.

Claims (1)

Process for the preparation of a mixture of secondary anthraquinone-type metabolites, in particular hydroxyanthraquinones, characterized in that at a temperature of 20 to 25 ° C a liquid medium having a pH of 6.0 to 6.5 containing sources of carbon, nitrogen such as yeast or corn extract and minerals is c cultures the Trichodirus viride CCM F-742 for 15 to 25 days, then the myoelum and the culture medium filtrate are subjected to extraction. The organic extract obtained is dissolved in acetone after evaporation of the solvent, adsorbed onto silica gel, eluted with a benzene-acetone mixture and the solvents are evaporated from the eluate.