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Tall fescue seed extraction and partial purification of ergot alkaloids.

Ji H, Fannin F, Klotz J, Bush L - Front Chem (2014)

Bottom Line: Following elution, ethanol was removed from the eluate by evaporation at room temperature and the resulting syrup was freeze-dried.The aqueous fraction was extracted with chloroform, the aqueous layer discarded, after which the chloroform was removed with a resulting 20-fold increase of ergovaline.The resultant partially purified ergovaline had biological activities in in vivo and in vitro bovine bioassays that approximate that of synthetic ergovaline.

View Article: PubMed Central - PubMed

Affiliation: Kentucky Tobacco Research and Development Center, University of Kentucky Lexington, KY, USA.

ABSTRACT
Many substances in the tall fescue/endophyte association (Schedonorus arundinaceus/Epichloë coenophiala) have biological activity. Of these compounds only the ergot alkaloids are known to have significant mammalian toxicity and the predominant ergot alkaloids are ergovaline and ergovalinine. Because synthetically produced ergovaline is difficult to obtain, we developed a seed extraction and partial purification protocol for ergovaline/ergovalinine that provided a biologically active product. Tall fescue seed was ground and packed into several different sized columns for liquid extraction. Smaller particle size and increased extraction time increased efficiency of extraction. Our largest column was a 114 × 52 × 61 cm (W × L × D) stainless steel tub. Approximately 150 kg of seed could be extracted in this tub. The extraction was done with 80% ethanol. When the solvent front migrated to bottom of the column, flow was stopped and seed was allowed to steep for at least 48 h. Light was excluded from the solvent from the beginning of this step to the end of the purification process. Following elution, ethanol was removed from the eluate by evaporation at room temperature and the resulting syrup was freeze-dried. About 80% recovery of alkaloids was achieved with 18-fold increase in concentration of ergovaline. Initial purification of the dried product was accomplished by extracting with hexane/water (6:1, v/v). The aqueous fraction was extracted with chloroform, the aqueous layer discarded, after which the chloroform was removed with a resulting 20-fold increase of ergovaline. About 65% of the ergovaline was recovered from the chloroform residue for an overall recovery of 50%. The resultant partially purified ergovaline had biological activities in in vivo and in vitro bovine bioassays that approximate that of synthetic ergovaline.

No MeSH data available.


Related in: MedlinePlus

Front and rear view of tub extractor that holds 150 kg powdered seed.
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Figure 1: Front and rear view of tub extractor that holds 150 kg powdered seed.

Mentions: For our large scale extractions tall fescue seed that tested high in ergovaline content were ground to pass a 1 mm sieve. Ground seed were packed carefully into a 114 × 52 × 61 cm tub column (Figure 1). There is a gasket around the rim of the tub that seals the top lid to the tub for the introduction of N2 to aid in the removal of the extraction solvent. The screw jacks on the top are visible to hold the lid in place while the system is under pressure from the N2. The jacks may be used to compress the seed to remove the solvent but we found that using N2 was more efficient. On the left end is the exit for the solvent into a tube covered with foil that drains into a large surface area container for the removal of the ethanol from the extract. Packing was done to provide a uniform column substrate as possible. This was done by adding about 25 kg at a time, leveling and lightly packing. Care in packing the column was done to insure that the solvent would migrate uniformly to the bottom of the column. This column holds 150 kg dry powder seed. The bottom of the column was filled with glass marbles above the outlet for solvent. The glass marbles were covered with a metal screen and non-dyed denim filter to keep seed residue from entering the eluate. The seedbed was compressed slightly by 19 mm thick polyethylene plate. Extraction was done with 80% ethanol and to fill the void volume approximately 160 L were added over 18–20 h period. At this time the solvent had reached the bottom of the column and the flow was stopped and seed steeped for 48 h. The tub column was then sealed and compressed N2 was introduced onto the top of the tub column to aid in removal of the extracting solvent. The eluate was drained into a large surface area tub and the ethanol removed by a fan blowing on the surface. From the point of emergence from the column and throughout the remaining processing the material was kept in the dark. After elution was complete, approximately 80 L of 80% ethanol was added over an 8 h period. Then the flow was stopped and seed steeped again for 48 h. Elution continued as previously described. After most of the ethanol was removed from the extractor and the drying tub, the resulting extract was freeze-dried to remove remaining ethanol and water and then stored at −20°C.


Tall fescue seed extraction and partial purification of ergot alkaloids.

Ji H, Fannin F, Klotz J, Bush L - Front Chem (2014)

Front and rear view of tub extractor that holds 150 kg powdered seed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263182&req=5

Figure 1: Front and rear view of tub extractor that holds 150 kg powdered seed.
Mentions: For our large scale extractions tall fescue seed that tested high in ergovaline content were ground to pass a 1 mm sieve. Ground seed were packed carefully into a 114 × 52 × 61 cm tub column (Figure 1). There is a gasket around the rim of the tub that seals the top lid to the tub for the introduction of N2 to aid in the removal of the extraction solvent. The screw jacks on the top are visible to hold the lid in place while the system is under pressure from the N2. The jacks may be used to compress the seed to remove the solvent but we found that using N2 was more efficient. On the left end is the exit for the solvent into a tube covered with foil that drains into a large surface area container for the removal of the ethanol from the extract. Packing was done to provide a uniform column substrate as possible. This was done by adding about 25 kg at a time, leveling and lightly packing. Care in packing the column was done to insure that the solvent would migrate uniformly to the bottom of the column. This column holds 150 kg dry powder seed. The bottom of the column was filled with glass marbles above the outlet for solvent. The glass marbles were covered with a metal screen and non-dyed denim filter to keep seed residue from entering the eluate. The seedbed was compressed slightly by 19 mm thick polyethylene plate. Extraction was done with 80% ethanol and to fill the void volume approximately 160 L were added over 18–20 h period. At this time the solvent had reached the bottom of the column and the flow was stopped and seed steeped for 48 h. The tub column was then sealed and compressed N2 was introduced onto the top of the tub column to aid in removal of the extracting solvent. The eluate was drained into a large surface area tub and the ethanol removed by a fan blowing on the surface. From the point of emergence from the column and throughout the remaining processing the material was kept in the dark. After elution was complete, approximately 80 L of 80% ethanol was added over an 8 h period. Then the flow was stopped and seed steeped again for 48 h. Elution continued as previously described. After most of the ethanol was removed from the extractor and the drying tub, the resulting extract was freeze-dried to remove remaining ethanol and water and then stored at −20°C.

Bottom Line: Following elution, ethanol was removed from the eluate by evaporation at room temperature and the resulting syrup was freeze-dried.The aqueous fraction was extracted with chloroform, the aqueous layer discarded, after which the chloroform was removed with a resulting 20-fold increase of ergovaline.The resultant partially purified ergovaline had biological activities in in vivo and in vitro bovine bioassays that approximate that of synthetic ergovaline.

View Article: PubMed Central - PubMed

Affiliation: Kentucky Tobacco Research and Development Center, University of Kentucky Lexington, KY, USA.

ABSTRACT
Many substances in the tall fescue/endophyte association (Schedonorus arundinaceus/Epichloë coenophiala) have biological activity. Of these compounds only the ergot alkaloids are known to have significant mammalian toxicity and the predominant ergot alkaloids are ergovaline and ergovalinine. Because synthetically produced ergovaline is difficult to obtain, we developed a seed extraction and partial purification protocol for ergovaline/ergovalinine that provided a biologically active product. Tall fescue seed was ground and packed into several different sized columns for liquid extraction. Smaller particle size and increased extraction time increased efficiency of extraction. Our largest column was a 114 × 52 × 61 cm (W × L × D) stainless steel tub. Approximately 150 kg of seed could be extracted in this tub. The extraction was done with 80% ethanol. When the solvent front migrated to bottom of the column, flow was stopped and seed was allowed to steep for at least 48 h. Light was excluded from the solvent from the beginning of this step to the end of the purification process. Following elution, ethanol was removed from the eluate by evaporation at room temperature and the resulting syrup was freeze-dried. About 80% recovery of alkaloids was achieved with 18-fold increase in concentration of ergovaline. Initial purification of the dried product was accomplished by extracting with hexane/water (6:1, v/v). The aqueous fraction was extracted with chloroform, the aqueous layer discarded, after which the chloroform was removed with a resulting 20-fold increase of ergovaline. About 65% of the ergovaline was recovered from the chloroform residue for an overall recovery of 50%. The resultant partially purified ergovaline had biological activities in in vivo and in vitro bovine bioassays that approximate that of synthetic ergovaline.

No MeSH data available.


Related in: MedlinePlus