Isolation of Ergot Alkaloids from Claviceps purpurea Culture Suspensions Using Clay
Source: LSD. Otto Snow. Thoth Press (2003). 124–126. DOWNLOAD
It has been found surprisingly, that adsorbent clays such as bentonite, nontronite, bleaching earth, and Fuller’s earth have an unusually high adsorption capacity at the natural pH value of the culture suspension not only for the ergometrine, but for all the investigated ergot alkaloids of the most diverse structures.
It has further been discovered that the mycella-adsorbent mixture can be separated from the liquid component in a relatively facile manner through filtration or other physical separation means.
A still further surprising discovery was that after physical separation, the mycella-adsorbent mixture containing the alkaloids can be subjected to fluidized bed drying according to the known physical parameters at the normally unfavorably high temperature for the alkaloids of 80°–90° C. without isomerization or decomposition of the compounds. After a washing with a weak aqueous alkaline solution, the alkaloids can then be extracted easily and virtually completely from the dry mycella-adsorbent mixture with an organic aprotic solvent.
In further investigations it was determined that this drying process was also applicable to those cultures in which the alkaloids are contained in the cell mass; thus, the addition of the adsorbent in these systems was not necessary. In this case, after mechanical separation and drying in the fluidized bed, dry mycella containing the alkaloid are obtained, the further work-up of which is not dependent upon the time of the termination of the drying process, unlike in the prior art.
This inventive process can be used to isolate a whole spectrum of ergot alkaloids, such as ergocryptine, ergotamine, ergocristine, ergocornine, ergometrine, lysergic acid, agroclavine, ergosine and others. This is achieved through the addition of 4–5% by weight, based on the water soluble recovered material, of an adsorbent clay and stirring for about 30 minutes. After mechanical separation of the solid material, for example through filtration, the wet mycella-adsorbent mixture is placed in a fluidized drying bed at 80°–90° C. for about 20 to 40 minutes until an exhaust temperature of between 50° and 70° C. and a residual moisture content of no more than about 15% is achieved. The mycella-adsorbent mixture is then mixed with an aqueous, weakly alkaline solution, for example, 15% ammonia water, and then extracted with an appropriate organic solvent, for example, a lower carboxylic acid ester, acetone or a halogenated hydrocarbon. This extract is then further treated by liquid-liquid extraction with an aqueous acidic phase-in a ratio of 2:1 to 1:1 by volume, in order to eliminate the non-basic residue. The aqueous solution containing the total alkaloid content is then made basic to a pH in the range of 8 to 9.5 and the alkaloids are extracted with an organic solvent not miscible with water, preferably ethyl acetate, in a ratio of 1:1 to 3:1 by volume.
The further treatment of the extract follows the known procedure with recognition of the chemical and physical properties of the alkaloids.
Bentonite is a montmorillonite-containing clay, named after its source, Fort Benton, Mont. It is among the group of clays commonly referred to as bleaching earths which require activation by an acidic treatment process. Fuller’s earth, another montmorillonite-containing clay, is not treated by any activation process before use; hence its description as a “raw bleaching earth”. These and other adsorbent clays may be employed in the inventive process.
Example 1
200 L. of culture suspension of Claviceps purpurea (Fr.), Tul. IMET PA 130 (obtained from the Jena Central Institute for Microbiology and Experimental Therapy, GDR) is stirred for 30 minutes with 8 kg bentonite. It is then filtered through a 10 mm layer of calcium sulfate dihydrate over a rotary vacuum cell filter; the practically alkaloid-free filtrate is discarded. The recovered mycella-bentonite mixture is dried in a fluidized bed at an influx temperature of 90° C. until an exhaust temperature of 60° C. is achieved. This takes approximately 30 minutes. There is recovered 20–24 kg of mycella-bentonite mixture with a residual moisture content of from 3–5%; this mixture contains 95–100% of the ergotoxine and 92–96% of the ergometrine contained in the culture suspension.
Example 2
100 L. of the culture suspension of Example 1 is stirred for 1 hour with 5 kg of bleaching earth, filtered, and the mycella-adsorbate mixture is dried in the fluidized bed at 80°–90° C. for about 15 minutes until an exhaust temperature of 50° C. is reached. There is recovered 12 kg of the dried mycella-adsorbent mixture with a residual moisture content of 10–12% and an alkaloid content of 92–100% based on the culture suspension.
Example 4
60 Kg. of the dry mycella-adsorbent mixture of Example 1 or 2 is mixed with 7.5 L. of 1:1 diluted ammonia, extracted in a mechanical extractor with 150 L. chloroform or methylene chloride over 45 minutes, filtered under pressure and the process repeated with the same volume of solvent for 30 minutes. The total extract is added to an equal volume of 5% aqueous acetic acid and processed in a separator, the aqueous phase constantly adjusted to pH 9; the process is repeated a second time with the same solvent or with ethylene acetate at a double volume. After evaporation and isolation of the ergometrine and ergotoxine, the adducts and yields of an alkaloid content of 96 to 103% calculated as the bimaleinate. The yield is (approx.) 351.6 g ergotoxine-toluol adduct, 72% calculated from the culture suspension and 243 g ergometrin-chloroform adduct, 82% from the suspension.
Example 5
30 L. culture solution of Claviceps purpurea IMET PA 134 at its natural pH value (5–6) is stirred for 30 minutes with 1.2 kg bentonite. It is then filtered through a layer of calcium sulfate dihydrate and the filtrate discarded. Drying of the mycella-adsorbent mixture according to Example 1 yields 2.8 kg of the dry mixture, which contains practically all of the alkaloid content of the culture suspension (ergosine, ergosinine, traces of chanoclavine). Rotary extraction of the alkaloids with a 10 to 12 fold volume of ethyl acetate yields up to 90% of the alkaloid content of the dry mycella-bentonite mixture.
Source: Grawert 1980
The previous methods can be used in the extraction of ergot alkaloids from Claviceps paspali cultures.
Grawert, W; Schiedt, L.; Neumann, B.; Heidenbluth, K; Dauth, C; Schirutschke, R.; Müller, M.; Process for the Isolation of Ergot Alkaloids from Culture Suspensions; 1980; US 4,237,291
This article can be viewed here: https://www.google.com/patents/US4237291?dq=4,237,291
NOTE: On pg. 178, Otto Snow follows up to the above information by describing ergot mixtures filtered using bentonite and other clays as consisting of predominantly ergine and isoergine. A list of all the ergoline alkaloids found in morning glory seeds can be found on page 227 in this PDF: DOWNLOAD.
Source: LSD. Otto Snow. Thoth Press (2003). 124–126. DOWNLOAD
It has been found surprisingly, that adsorbent clays such as bentonite, nontronite, bleaching earth, and Fuller’s earth have an unusually high adsorption capacity at the natural pH value of the culture suspension not only for the ergometrine, but for all the investigated ergot alkaloids of the most diverse structures.
It has further been discovered that the mycella-adsorbent mixture can be separated from the liquid component in a relatively facile manner through filtration or other physical separation means.
A still further surprising discovery was that after physical separation, the mycella-adsorbent mixture containing the alkaloids can be subjected to fluidized bed drying according to the known physical parameters at the normally unfavorably high temperature for the alkaloids of 80°–90° C. without isomerization or decomposition of the compounds. After a washing with a weak aqueous alkaline solution, the alkaloids can then be extracted easily and virtually completely from the dry mycella-adsorbent mixture with an organic aprotic solvent.
In further investigations it was determined that this drying process was also applicable to those cultures in which the alkaloids are contained in the cell mass; thus, the addition of the adsorbent in these systems was not necessary. In this case, after mechanical separation and drying in the fluidized bed, dry mycella containing the alkaloid are obtained, the further work-up of which is not dependent upon the time of the termination of the drying process, unlike in the prior art.
This inventive process can be used to isolate a whole spectrum of ergot alkaloids, such as ergocryptine, ergotamine, ergocristine, ergocornine, ergometrine, lysergic acid, agroclavine, ergosine and others. This is achieved through the addition of 4–5% by weight, based on the water soluble recovered material, of an adsorbent clay and stirring for about 30 minutes. After mechanical separation of the solid material, for example through filtration, the wet mycella-adsorbent mixture is placed in a fluidized drying bed at 80°–90° C. for about 20 to 40 minutes until an exhaust temperature of between 50° and 70° C. and a residual moisture content of no more than about 15% is achieved. The mycella-adsorbent mixture is then mixed with an aqueous, weakly alkaline solution, for example, 15% ammonia water, and then extracted with an appropriate organic solvent, for example, a lower carboxylic acid ester, acetone or a halogenated hydrocarbon. This extract is then further treated by liquid-liquid extraction with an aqueous acidic phase-in a ratio of 2:1 to 1:1 by volume, in order to eliminate the non-basic residue. The aqueous solution containing the total alkaloid content is then made basic to a pH in the range of 8 to 9.5 and the alkaloids are extracted with an organic solvent not miscible with water, preferably ethyl acetate, in a ratio of 1:1 to 3:1 by volume.
The further treatment of the extract follows the known procedure with recognition of the chemical and physical properties of the alkaloids.
Bentonite is a montmorillonite-containing clay, named after its source, Fort Benton, Mont. It is among the group of clays commonly referred to as bleaching earths which require activation by an acidic treatment process. Fuller’s earth, another montmorillonite-containing clay, is not treated by any activation process before use; hence its description as a “raw bleaching earth”. These and other adsorbent clays may be employed in the inventive process.
Example 1
200 L. of culture suspension of Claviceps purpurea (Fr.), Tul. IMET PA 130 (obtained from the Jena Central Institute for Microbiology and Experimental Therapy, GDR) is stirred for 30 minutes with 8 kg bentonite. It is then filtered through a 10 mm layer of calcium sulfate dihydrate over a rotary vacuum cell filter; the practically alkaloid-free filtrate is discarded. The recovered mycella-bentonite mixture is dried in a fluidized bed at an influx temperature of 90° C. until an exhaust temperature of 60° C. is achieved. This takes approximately 30 minutes. There is recovered 20–24 kg of mycella-bentonite mixture with a residual moisture content of from 3–5%; this mixture contains 95–100% of the ergotoxine and 92–96% of the ergometrine contained in the culture suspension.
Example 2
100 L. of the culture suspension of Example 1 is stirred for 1 hour with 5 kg of bleaching earth, filtered, and the mycella-adsorbate mixture is dried in the fluidized bed at 80°–90° C. for about 15 minutes until an exhaust temperature of 50° C. is reached. There is recovered 12 kg of the dried mycella-adsorbent mixture with a residual moisture content of 10–12% and an alkaloid content of 92–100% based on the culture suspension.
Example 4
60 Kg. of the dry mycella-adsorbent mixture of Example 1 or 2 is mixed with 7.5 L. of 1:1 diluted ammonia, extracted in a mechanical extractor with 150 L. chloroform or methylene chloride over 45 minutes, filtered under pressure and the process repeated with the same volume of solvent for 30 minutes. The total extract is added to an equal volume of 5% aqueous acetic acid and processed in a separator, the aqueous phase constantly adjusted to pH 9; the process is repeated a second time with the same solvent or with ethylene acetate at a double volume. After evaporation and isolation of the ergometrine and ergotoxine, the adducts and yields of an alkaloid content of 96 to 103% calculated as the bimaleinate. The yield is (approx.) 351.6 g ergotoxine-toluol adduct, 72% calculated from the culture suspension and 243 g ergometrin-chloroform adduct, 82% from the suspension.
Example 5
30 L. culture solution of Claviceps purpurea IMET PA 134 at its natural pH value (5–6) is stirred for 30 minutes with 1.2 kg bentonite. It is then filtered through a layer of calcium sulfate dihydrate and the filtrate discarded. Drying of the mycella-adsorbent mixture according to Example 1 yields 2.8 kg of the dry mixture, which contains practically all of the alkaloid content of the culture suspension (ergosine, ergosinine, traces of chanoclavine). Rotary extraction of the alkaloids with a 10 to 12 fold volume of ethyl acetate yields up to 90% of the alkaloid content of the dry mycella-bentonite mixture.
Source: Grawert 1980
The previous methods can be used in the extraction of ergot alkaloids from Claviceps paspali cultures.
Grawert, W; Schiedt, L.; Neumann, B.; Heidenbluth, K; Dauth, C; Schirutschke, R.; Müller, M.; Process for the Isolation of Ergot Alkaloids from Culture Suspensions; 1980; US 4,237,291
This article can be viewed here: https://www.google.com/patents/US4237291?dq=4,237,291
NOTE: On pg. 178, Otto Snow follows up to the above information by describing ergot mixtures filtered using bentonite and other clays as consisting of predominantly ergine and isoergine. A list of all the ergoline alkaloids found in morning glory seeds can be found on page 227 in this PDF: DOWNLOAD.