Acylated steryl glycoside analysis

ACYLATED STERYL GLYCOSIDE ANALYSIS

Acylated steryl glycosides are present in extracts of all plant tissues and in concentrations similar or higher than those of the non acylated forms.
Because of the structure of these molecules, extracts cannot be saponified during their purification.
The extraction process is the same as that described above for steryl glycosides.

Acylated steryl glycosides are generally analyzed after a separation by TLC, the spots being identified, as for steryl glycosides, specific reagents for sugar and sterols. A previous saponification treatment assures also the identity of the compound which must disappear and found at the migration position of steryl glycosides. The quantification may be done by colorimetry of one of the constituents or by HPLC.

Pure standard compounds may be obtained from Matreya

TLC separation:

Lipids dissolved in  hexane are submitted to TLC on silica gel G60 plates with one of the following mixtures as developing solvent:

– chloroform/methanol/conc. ammonia (40/10/2, v/v), the steryl glycoside spot has a Rf of about 0.85, ahead of monogalactosyl diglycerides and near the triglyceride spot (close to the solvent front)
– chloroform/acetone/water (30/60/2, v/v), the steryl glycoside spot has a Rf of about 0.65 and well separated from triglycerides and sterols.
– chloroform/acetone/methanol/acetic acid (73/25/1.5/0.5, v/v), the steryl glycoside spot has a Rf of about 0.4 and well separated from sterols, glycolipids and fatty acids.

The lipid spots are visualized under UV after spraying with primuline. If steryl glycosides are present, a fluorescent spot is observed and the presence of a glucide moiety is confirmed after spraying another lane with the orcinol reagent (pink spot) and the presence of a sterol moiety after spraying another lane with a sterol reagent (violet spot).    

Column chromatography:

Acylated steryl glycosides may be isolated from a complex lipid extract by a chromatographic procedure involving a chloroform elution from a silicic acid column as already described (to see: click here). 
If triglycerides are abundant in the vegetal extract, the silicic acid column is made in hexane and first eluted with 5 ml hexane, 10 ml of hexane/diethyl ether (1/1, v/v) to remove triglycerides, sterols and steryl esters. The acylated steryl glycosides are then eluted with 5 ml of hexane/diethyl ether (3/7, v/v). This fraction is evaporated and readily submitted to TLC or other separation procedures (Lepage  M, J Lipid Res 1964, 5, 587). After a short wash with chloroform, the other glycolipids may be eluted with acetone/methanol (90/10, v/v).

Separation and quantification by HPLC:

It is recommended to isolate previously a glycolipid fraction by TLC or with a  small column of silicic acid (see above). 

Procedure

HPLC conditions: 

column: LiChrospher Si 60, 120 x 4 mm i.d. (Merck).
Mobile phase: solution A: chloroform, solution B: methanol/water (95/5, v/v). 
Flow rate: 1 ml/min
Gradient: linear and continuous gradient from time 0 (99% A and 1% B) to time 15 min (75% A and 25% B) followed by a reequilibration time of about 10 min with the conditions of time 0 prior the next analysis.
If complex glycolipids are removed by a previous silicic acid column chromatography, an isocratic elution may be used (chloroform/methanol/water, 90/9.5/0.5, v/v).
Detection: acylated steryl glycosides can be detected between 210 and 230 nm but a better sensitivity with a fine baseline is obtained with a light-scattering detector. 

Acylated steryl glycosides are eluted at 5-6 min. A detection limit of about 0.5 mg is easily obtained. A calibration curve is run in injecting known amounts of standards (2 to 100 mg) dissolved in chloroform.

For other details, see Sugawara T et al (Lipids 1999, 34, 1231).