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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

Seed and initial dried extract.
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Figure 2: Seed and initial dried extract.

Mentions: From 150 kg of seed, we obtained 8.3 kg of dried extract. This extraction resulted in an 80% recovery of ergovaline and an 18-fold increase in concentration. Initial seed and the resulting freeze-dried extract are depicted in Figure 2. Because we had to use the longer times of extraction for our large batch extractions, we measured the epimerization that may occur between ergovaline and ergovalinine in the solvents to be used. Chemically pure ergovaline dissolved in 80% methanol had no measureable epimerization immediately after being dissolved. However, by 22 and 47 h significant epimerization occurred, 16 and 26 %, respectively (data not shown). Similarly, seed extract solubilized in 80% ethanol (Table 2) or acetonitrile (data not shown) also had significant epimerization, about 40% conversion and was very consistent across treatments during extraction and measurement.


Tall fescue seed extraction and partial purification of ergot alkaloids.

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

Seed and initial dried extract.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Seed and initial dried extract.
Mentions: From 150 kg of seed, we obtained 8.3 kg of dried extract. This extraction resulted in an 80% recovery of ergovaline and an 18-fold increase in concentration. Initial seed and the resulting freeze-dried extract are depicted in Figure 2. Because we had to use the longer times of extraction for our large batch extractions, we measured the epimerization that may occur between ergovaline and ergovalinine in the solvents to be used. Chemically pure ergovaline dissolved in 80% methanol had no measureable epimerization immediately after being dissolved. However, by 22 and 47 h significant epimerization occurred, 16 and 26 %, respectively (data not shown). Similarly, seed extract solubilized in 80% ethanol (Table 2) or acetonitrile (data not shown) also had significant epimerization, about 40% conversion and was very consistent across treatments during extraction and measurement.

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