DD265636A1 - METHOD FOR OBTAINING INDOINDIVES FROM MUSCLES OF FUNGI AND MEDIUM MEDIUM HERE - Google Patents

Abstract

The invention relates to the microbial recovery of indole derivatives. It is applicable for obtaining model substances for neurobiological and psychiatric examinations. The invention consists in the use of nutrient media consisting of complex natural or synthetic substrates to which tryptamine or its salts are added. The surface cultivation is carried out at p H 3-7 and 16-32C, the extraction and isolation of the active ingredients in a known manner. The solution according to the invention leads to a high and largely reproducible yield of indole derivatives.

Description

Field of application of the invention

This invention relates to a process for the recovery of indole derivatives from the mycelia of Basidiomycetes and to a suitable nutrient media composition. It can be used in the pharmaceutical industry for the synthesis of drugs for neurobiology and psychiatry.

Characteristic of the known state of the art It is known that several higher fungi of the genera Psilocybe, Inocybe, Panaeolub, Pluteus and Stropharia in Fruits and mycelia Indole derivatives wib psilocybin and psilocin, which have neurotropic activity. A method (DE 1087321) describes the recovery of this Wirkstoff «from substrate mycelium and sclerotia, dio in Surface cultures were grown using malt extract solutions with ZusaC * various mineral salts. The disadvantage of the method is that using these substrates in various concentrations, only a content of mycelia

of 0.28% psilocybin, calculated on the dry masses can be achieved.

Also, it must be fermented at a constant temperature to the same substrate and a relatively constant content of To achieve mycolys en psilocybin and psilocin. Furthermore, they vary by batch to batch Composition of this complex substrate also large variations in the content of Indolderivaten determine, so often

only Alkaloidspurfsn be detected in the mycelia.

It has already been proposed that indole derivatives can be obtained by cultivating the fungi species in synthetic. Nutrients to be used .. As a result, an increase in the yield of alkali metal can be achieved. The disadvantage of the method is iJaß only with some

Non-European mushroom species a significant increase in yield of psilocybin can be achieved.

Moreover, it is not possible by this method, minor alkaloids such as djS Monorr -thy! Homologues of psilocybin, Baeocystin, present in some mushroom species as an ingredient of naturally occurring fruiting bodies such. B. in the

native Psilocybe semilanceata (Fr.) Kumm. occurs and also no-h is not synthetically accessible. These types of fungi also do not form significant amounts of psilocybin in the synthetic nutrient media described.

Object of the invention

The aim of the Erfir Jung is to obtain indole derivatives such as psilocybin and its secondary alkaloids with increased Ausboute, where all such iletsarten, which form in their fruit bodies, the alkaloids in question. Should be used.

Explanation of the essence of the invention The invention is based on the object, by suitable additives to NShrmedium dio biosynthesis of indole derivatives and

to influence their accumulation in the mycelia. The cultivation of the fungi in question on known liquid synthetic or natural Nährmedinn done according to the invention with the addition of Trypi imine in the form of the free

Base or water-soluble in salts. The di - tryptamine concentration is in the range of 0.001-0.1%. As culturing conditions, the following parameters are advantageous:

- pH 3-7

- temperature 16-32 * C

- Cultivation time 20 - & \ days.

The extraction, chromatographic isolation and purification of the indole derivatives is carried out in a known manner. As Grundnahrrmdium all suitable for the cultivation of higher fungi can be used. The addition of 0.2% Ajjar allows t ^; n rapid spreading of fungal hyphae on the surface. By the addition of the Tryptamine takes place essentially, the accumulation of inulin derivatives in the mycelia by biotransformation

this connection.

This abbreviation of the biosynthetic pathway of the indole derivatives also leads to the formation of neban alkaloids such as Baeocystin and Psilocin, which are otherwise not or only slightly formed. The yield of psilocybin is increased depending on the type of fungus on the 2- to 5-fold or only possible in mycelium culture such. Psilocybin semilanceata (Fr.) Kumm.

The procedure is such that a fine suspension of mycelium flakes to the sterilized nutrient solutions under aseptic conditions in suitable vessels such. B. Penicillin- or Fernbachkolben cyht>> nd the solutions incubate at temperatures between 16 and 32 ⁰ C 20 bit 50 T ige. After filtration, washing the Mycel'.en with water, drying at 20 to 5O ⁰ C, the mycelium is after known, extracted and the active substances are separated by means of chromatographic methods.

By this procedure, high yields of biomass can be obtained with a large percentage of Indoldorivaten, a constant fermentation temperature is not necessary.

By the following examples, the invention will be explained in more detail.

example 1

A spore culture of the subtropical species Psilocybe cubensis (Earle) Sing is grown on 6% Malzacar (agar: 1.5%). After 3 weeks aseptically a piece of mycelium of about 2 χ 2cm is removed and shaken for 10 minutes in a 100 ml sterile flask containing 20 g of saddle filling body, whereby the filling bodies crush the mycelium to form a fine suspension. Each 5 ml of the suspension is used to inoculate one liter of nutrient solution, which has the following composition and was prepared in 2.41 penicillin flasks6

 Composition of the nutrient solution:

40g maltose, 3g ammonium succinate, 0 0.1g KH ₂ PO ₄ , 20mg yeast extract, 5rng thiamine hydrochloride, 2g agar, 71mg magnesium sulfate, 0.5mg zinc sulfate, 0.8mg manganese (II) sucrose, 0.8mg Copper sulfate, 0.25 mg sodium molybdate and 5 mg Fison (II) sulfate and 300 mg tryptamine hydrochloride. The substances were diluted with water to 11 or dissolved and autoclaved at 120 ° C for 20 min, the thiamine salt was added as a sterile solution only on cooling. With hydrochloric acid was adjusted to pH 3.5, inoculated and cultured at IU to 26 ⁰ C. After 30 Togen was filtered through a Dederonsieb, washed with 500ml of water, 12 hrs. Freeze-dried and then dal dry mycelium extracted and isolated the active ingredients. Yield: 18g dry mycelium Psilocybin content: 1.20% Psilocingehtilt: 0.3%

A comparison with an analog nutrient solution without the addition of tryptamine yielded 16 g of dry mycelium containing 0.73% psilocybin and traces of psilocin.

Example 2

There was a culture of Psilocybe cyanescens Wakefield emend. Kriegisteiner created as described in Example 1.

The nutrient medium used was a 6% malt extract containing 0.2% agar.

With the addition of 100 mg of tryptamine phosphate to 500 ml of this solution in 1.61 Fernbachkolben at 20 to 25 ⁰ C were under analogous cultivation and inoculation methods as in Example 1 22g Trcckenmycel per liter obtained that contained 0.92% psilocybin noben traces of psilocin.

In contrast, in the analogous cultivation without the addition of the tryptamine salt, only 19.5 g of dry mycelium with a psilocybin content of 0.21% were obtained. Psilocin was undetectable here.

Example 3

One spurs culture of the native mushroom Psilocybe semilanceata (Fr.) Kumm. whose plant was made until the preparation of the Impfsuepension analogous to Example 1.

Composition C ¹ Jr nutrient solution:

80g glucose, 2g agar, 1 g ammonium succinate, 9g glycine 10mg thiamine hydrochloride, 20mg yeast extract, 0.1g KH ₁ PO ₄ , 100mg tryptamine hydrochloride and trace salts analogous to Example 1, then was made up to 11H ₂ O and then adjusting the pH Value of 4.0 was carried out with dil. Hydrochloric acid.

Yield after 30 days cultivation: 25 g dry mycelium

Content a ·· Psilocybin: 0.8%

Content of Baeocystin: 0.25%

A reference mixture without tryptamine salt gave 23 g of dry biomass containing only traces of psilocybin and no baeocystin.

Claims (3)

1. A process for the production of indole derivatives from mushroom mycoses from surface cultures Psilor.ybin- and psilocinhaltiger higher fungi, preferably the genera Psilocybe, Panaoolus, Stropharia, Inocybe and P 'Jteus by per se known cultivation on synthetic or natural, liquid nutrient media and known separation and purification operations are obtained, characterized in that the cultivation is carried out with the addition of tryptamine. 2. The method according to claim 1, characterized in that tryptamine is used as the free base or in the form of its water-soluble salts in concentrations of 0.001 to 0.1%. 3. nutrient medium composition for surface cultures of higher fungi which can be used for the production of indole derivatives, characterized in that they are Components known per se, which constitute a natural or synthetic liquid nutrient medium suitable for the growth of higher fungi, and - Tryptamine as the base or water-soluble salt in a concentration of 0.001 to 0.1%.