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Rapid purification of gram quantities of β-sitosterol from a commercial phytosterol mixture.

Srividya N, Heidorn DB, Lange BM - BMC Res Notes (2014)

Bottom Line: An improved method for the rapid purification of β-sitosterol from a commercial phytosterol extract is presented.Fractional crystallization of soybean oil yielded a soluble and an insoluble fraction. β-Sitosterol was purified by silica gel and Na-Y zeolite chromatography.The rapid and cost effective three-step purification described here afforded β-sitosterol in gram quantities with high purity (>92%) and yield (>22%).

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biological Chemistry and M,J, Murdock Metabolomics Laboratory, Washington State University, Pullman, WA 99164, USA. lange-m@wsu.edu.

ABSTRACT

Background: β-Sitosterol, a plant sterol or phytosterol, has commercial uses in the nutraceutical and pharmaceutical industries, but is also employed frequently in biological research. Phytosterols always accumulate as mixtures, and obtaining highly pure β-sitosterol in larger quantities for biological assays has been a challenge.

Findings: An improved method for the rapid purification of β-sitosterol from a commercial phytosterol extract is presented. Fractional crystallization of soybean oil yielded a soluble and an insoluble fraction. β-Sitosterol was purified by silica gel and Na-Y zeolite chromatography.

Conclusion: The rapid and cost effective three-step purification described here afforded β-sitosterol in gram quantities with high purity (>92%) and yield (>22%).

Show MeSH
Flow chart of β-sitosterol purification from a crude soybean oil extract. Representative GC-MS chromatograms are shown for each step. Yields are provided in parentheses. Numbering of metabolites as in Figure 1.
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Figure 2: Flow chart of β-sitosterol purification from a crude soybean oil extract. Representative GC-MS chromatograms are shown for each step. Yields are provided in parentheses. Numbering of metabolites as in Figure 1.

Mentions: Fractional crystallization has been used by various groups to obtain a solid (S) and a liquid (L) phytosterol fraction, of which only the L fraction (fairly low phytosterol content but with few other contaminants) was further processed. The phytosterol-rich S fraction contained high amounts of 1, which was very difficult to remove when 2 was the primary target, and was thus discarded[6,9-11]. To avoid substantial losses of 2, we first tested several solvents for obtaining an L fraction with a significant reduction in 1, while maintaining options to also process the S fraction for purifying 2. The solvent selection was based on previously published reports and included (1) acetone, (2) hexane/toluene/ethanol (4:2:1; v:v:v) and (3) diethyl ether (Table 1)[6,9-11]. Crude soybean oil extract was dissolved in 500 ml of each solvent and placed at −80°C overnight. After removal from the freezer, the contents were immediately filtered using a Büchner funnel with sintered glass insert to yield an L and an S fractions (Figure 2). The solvent of the L fraction was evaporated, and the S and dried L fractions separately dissolved in 10 ml chloroform. At this stage, an aliquot from each fraction was analyzed by GC-MS[16]. The use of diethyl ether resulted in the most significant reduction of 1 in the L fraction (Table 1). Campesterol (3) was enriched but can be more easily removed using column chromatography[12-14]. None of the solvents were effective in differentially reducing the amounts of 1 in the S fraction, while diethyl ether was the most appropriate solvent for maintaining high quantities of 2 (Table 1). All further experiments were thus performed using diethyl ether as a solvent. Phytosterols were distributed among the S and L fractions at 85% and 15%, respectively (Figure 2).


Rapid purification of gram quantities of β-sitosterol from a commercial phytosterol mixture.

Srividya N, Heidorn DB, Lange BM - BMC Res Notes (2014)

Flow chart of β-sitosterol purification from a crude soybean oil extract. Representative GC-MS chromatograms are shown for each step. Yields are provided in parentheses. Numbering of metabolites as in Figure 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3974218&req=5

Figure 2: Flow chart of β-sitosterol purification from a crude soybean oil extract. Representative GC-MS chromatograms are shown for each step. Yields are provided in parentheses. Numbering of metabolites as in Figure 1.
Mentions: Fractional crystallization has been used by various groups to obtain a solid (S) and a liquid (L) phytosterol fraction, of which only the L fraction (fairly low phytosterol content but with few other contaminants) was further processed. The phytosterol-rich S fraction contained high amounts of 1, which was very difficult to remove when 2 was the primary target, and was thus discarded[6,9-11]. To avoid substantial losses of 2, we first tested several solvents for obtaining an L fraction with a significant reduction in 1, while maintaining options to also process the S fraction for purifying 2. The solvent selection was based on previously published reports and included (1) acetone, (2) hexane/toluene/ethanol (4:2:1; v:v:v) and (3) diethyl ether (Table 1)[6,9-11]. Crude soybean oil extract was dissolved in 500 ml of each solvent and placed at −80°C overnight. After removal from the freezer, the contents were immediately filtered using a Büchner funnel with sintered glass insert to yield an L and an S fractions (Figure 2). The solvent of the L fraction was evaporated, and the S and dried L fractions separately dissolved in 10 ml chloroform. At this stage, an aliquot from each fraction was analyzed by GC-MS[16]. The use of diethyl ether resulted in the most significant reduction of 1 in the L fraction (Table 1). Campesterol (3) was enriched but can be more easily removed using column chromatography[12-14]. None of the solvents were effective in differentially reducing the amounts of 1 in the S fraction, while diethyl ether was the most appropriate solvent for maintaining high quantities of 2 (Table 1). All further experiments were thus performed using diethyl ether as a solvent. Phytosterols were distributed among the S and L fractions at 85% and 15%, respectively (Figure 2).

Bottom Line: An improved method for the rapid purification of β-sitosterol from a commercial phytosterol extract is presented.Fractional crystallization of soybean oil yielded a soluble and an insoluble fraction. β-Sitosterol was purified by silica gel and Na-Y zeolite chromatography.The rapid and cost effective three-step purification described here afforded β-sitosterol in gram quantities with high purity (>92%) and yield (>22%).

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biological Chemistry and M,J, Murdock Metabolomics Laboratory, Washington State University, Pullman, WA 99164, USA. lange-m@wsu.edu.

ABSTRACT

Background: β-Sitosterol, a plant sterol or phytosterol, has commercial uses in the nutraceutical and pharmaceutical industries, but is also employed frequently in biological research. Phytosterols always accumulate as mixtures, and obtaining highly pure β-sitosterol in larger quantities for biological assays has been a challenge.

Findings: An improved method for the rapid purification of β-sitosterol from a commercial phytosterol extract is presented. Fractional crystallization of soybean oil yielded a soluble and an insoluble fraction. β-Sitosterol was purified by silica gel and Na-Y zeolite chromatography.

Conclusion: The rapid and cost effective three-step purification described here afforded β-sitosterol in gram quantities with high purity (>92%) and yield (>22%).

Show MeSH