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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

Chromatograms of ergovaline from initial seed to a partially purified material. Panel (A) is chromatogram of routine analysis of powdered seed (EV, ergovaline; EVI, ergovalinine). Panel (B) is a chromatogram from dried material following 80% ethanol extraction. Panel (C) is chromatogram of dried material following the water/hexane/CHCl3 purification. Panel (D) is a chromatogram of HPLC purification of ergovaline from (C).
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Figure 3: Chromatograms of ergovaline from initial seed to a partially purified material. Panel (A) is chromatogram of routine analysis of powdered seed (EV, ergovaline; EVI, ergovalinine). Panel (B) is a chromatogram from dried material following 80% ethanol extraction. Panel (C) is chromatogram of dried material following the water/hexane/CHCl3 purification. Panel (D) is a chromatogram of HPLC purification of ergovaline from (C).

Mentions: Based on the above results we selected the following protocol for the second step in purification. Three hundred mL of water was added to 600 g of the dried extract and mixed to a smooth slurry to remove all “crystalline” pieces. Slurry was added to a 4 L brown bottle and 1800 mL of hexane was added. The mixture was vigorously shaken for 10 min and the hexane and aqueous layers were allowed to separate. This could take up to 2 h. Hexane removed many of the lipids and the alkaloids remained in the aqueous fraction. The hexane layer was decanted and the aqueous fraction was extracted with hexane two more times. The aqueous fraction was extracted with 1800 mL chloroform 3-times and the chloroform fractions combined and removed in a rotary evaporator. Residue was stored at −20° C for use. Ergovaline concentration increased from the seed about 350- to 400-fold (Figures 3A–C). Chromatogram in Figure 3A is from initial seed extract with ergovaline (EV) and ergovalinine (EVI). The initial crude 80% ethanol extract alkaloid content is illustrated in Figure 3B. This chromatogram is a 100-fold dilution of the extract for comparison with Figure 3A. Ergovaline present in the CHCl3 of the water/hexane/CHCl3 cleanup had no lysergic acids and the solution for the chromatogram had been diluted 600-fold (Figure 3C, Table 3). Lysergic acid and loline alkaloids were not detected in the final purified extract (not shown on the chromatograms). Fragment ion spectrum of ergovaline and of the partially purified ergovaline m/z 534 fragment are indicative of ergovaline (Figure 4). Further purification may be done with HPLC separation and collection of ergovaline and ergovalinine. The HPLC purification of the water/hexane/CHCl3 extract is illustrated in Figure 3D. The ergovalinine is most likely from epimerization that occurred during the chromatography or processing. A final purification by HPLC was reported earlier by Moubarak et al. (1993).


Tall fescue seed extraction and partial purification of ergot alkaloids.

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

Chromatograms of ergovaline from initial seed to a partially purified material. Panel (A) is chromatogram of routine analysis of powdered seed (EV, ergovaline; EVI, ergovalinine). Panel (B) is a chromatogram from dried material following 80% ethanol extraction. Panel (C) is chromatogram of dried material following the water/hexane/CHCl3 purification. Panel (D) is a chromatogram of HPLC purification of ergovaline from (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Chromatograms of ergovaline from initial seed to a partially purified material. Panel (A) is chromatogram of routine analysis of powdered seed (EV, ergovaline; EVI, ergovalinine). Panel (B) is a chromatogram from dried material following 80% ethanol extraction. Panel (C) is chromatogram of dried material following the water/hexane/CHCl3 purification. Panel (D) is a chromatogram of HPLC purification of ergovaline from (C).
Mentions: Based on the above results we selected the following protocol for the second step in purification. Three hundred mL of water was added to 600 g of the dried extract and mixed to a smooth slurry to remove all “crystalline” pieces. Slurry was added to a 4 L brown bottle and 1800 mL of hexane was added. The mixture was vigorously shaken for 10 min and the hexane and aqueous layers were allowed to separate. This could take up to 2 h. Hexane removed many of the lipids and the alkaloids remained in the aqueous fraction. The hexane layer was decanted and the aqueous fraction was extracted with hexane two more times. The aqueous fraction was extracted with 1800 mL chloroform 3-times and the chloroform fractions combined and removed in a rotary evaporator. Residue was stored at −20° C for use. Ergovaline concentration increased from the seed about 350- to 400-fold (Figures 3A–C). Chromatogram in Figure 3A is from initial seed extract with ergovaline (EV) and ergovalinine (EVI). The initial crude 80% ethanol extract alkaloid content is illustrated in Figure 3B. This chromatogram is a 100-fold dilution of the extract for comparison with Figure 3A. Ergovaline present in the CHCl3 of the water/hexane/CHCl3 cleanup had no lysergic acids and the solution for the chromatogram had been diluted 600-fold (Figure 3C, Table 3). Lysergic acid and loline alkaloids were not detected in the final purified extract (not shown on the chromatograms). Fragment ion spectrum of ergovaline and of the partially purified ergovaline m/z 534 fragment are indicative of ergovaline (Figure 4). Further purification may be done with HPLC separation and collection of ergovaline and ergovalinine. The HPLC purification of the water/hexane/CHCl3 extract is illustrated in Figure 3D. The ergovalinine is most likely from epimerization that occurred during the chromatography or processing. A final purification by HPLC was reported earlier by Moubarak et al. (1993).

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