Isolation and characterization of two sterols from the green alga, Selenastrum capricornutum

The green alga, Selenastrum capricornutum, was cultured in artificial nutrient medium utilizing five-gallon carboys, each of which contained 16 1. of culture. The algal cells were separated from the nutrient medium by continuous-flow centrifugation at 7500 RPM, and were then lyophilized. The lyophilized cells were extracted by refluxing with ether, acetone, and chloroform:methanol (2:1). Free sterols and sterol esters were separated from the crude extract using preparative thin-layer chromatography. Sterol esters were saponified and both the sterols and the fatty acids were recovered. Individual sterols were separated from the free sterol fraction using argentation thin-layer chromatography. Gas chromatograms, mass spectra, and ultraviolet spectra were obtained for these sterols. The free sterol fraction was found to contain approximately 40% 24-methylcholesta-5,7-dien-3B-ol and 60% 24-ethylcholesta-5,7-dien-3B-ol. The sterol ester fraction also contained these two sterols; however, the composition and amount of esterified sterols varied as a function of culture age. Sterol ester content was higher for older cultures, and in older cultures the composition of the esterified sterols more closely resembled that of the free sterols. The fatty acids obtained from the saponification of the sterol esters were methylated and were analyzed using gas chromatography. Tentative identifications, based upon comparative retention times, were made for several of these acids. Sterols were extracted from the nutrient medium after harvest of the algal cells. Extraction was accomplished by mixing large quantities of nutrient medium with ether for several days, or by shaking small aliquots with ether. 24-methylcholesta-5,7-dien-3B-ol and 24 ethylcholesta-5,7-dien-3B-ol were isolated from the nutrient medium in approximately the same relative amounts as from the algal cells. The concentration of sterols in the nutrient medium was approximately equal to the water-solubility of cholesterol (25-29)t g./l.). Extraction procedures which release sterols from water soluble complexes were carried out on extracted cells and on extracted nutrient medium. These procedures failed to yield measurable quantities of sterols. Treatment of extracted cells with strong base and subsequent extraction showed that all sterols had been extracted without prior cell lysis or pretreatment. An extraction of algal cells was carried out using DMSO:ether as the extraction solvent. This extraction resulted in complete removal of sterols from the cells, and the sterols were accompanied by only small amounts of other lipidsoluble material.