Steryl glycoside analysis

STERYL GLYCOSIDE

ANALYSIS

Steryl glycosides are present in vegetal extracts in free form but may be also prepared from their acylated forms by mild saponification. If both forms are needed, the saponification step is omitted. When the other glycolipids and the phospholipids present in extracts must be preserved for further analysis, it is recommended to extract vegetal tissues according to specific and efficient procedures (hot alcohols).

On a very small scale, steryl glycosides are analyzed after a separation by TLC, on a larger scale column chromatography is used. Both procedures may be followed by precise quantification by HPLC of the intact molecules or by determination of the sterol and sugar moieties.

Pure standard compounds may be obtained from Matreya

Procedure

Extraction:

Vegetal tissues are preferably homogenized in hot 80% isopropanol or ethanol for some minutes to inactivate hydrolytic enzymes. After filtration, the residue is extracted with chloroform/methanol mixture (2/1, v/v). After concentration of the combined extracts under vacuum, the lipid residue is taken up in chloroform and purified by phase partition after addition of  methanol and water (Folch’s wash).
Alternatively, inactivation of hydrolytic enzymes may be obtained by treatment of minced tissues for 1 min in a microwave oven at high power (about 500 W) followed by a classical extraction with chloroform/methanol. 

Saponification of the extracts:

To prepare large quantities of steryl glycosides (from free and acylated species), lipid extracts are deacylated (saponified) with 0.1 M KOH in methanol for about 30 min at 40°C. The reaction is stopped by neutralization with 4M HCl and the lipids are purified by phase partition as above. The chloroform phase is evaporated under vacuum and the neutral lipids including steryl glycosides are dissolved in hexane (if present, the complex glycosphingolipids are not soluble in hexane).

TLC separation:

Lipids dissolved in  hexane are submitted to TLC on silica gel G60 plates with the mixture chloroform/methanol/conc. ammonia (40/10/2, v/v) as developing solvent. The lipid spots are visualized under UV after spraying with primuline. If sterol glycosides are present, a fluorescent spot with a Rf of about 0.46 is observed. 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:

Steryl glycosides may be isolated from a complex lipid extract by a chromatographic procedure involving an acetone elution from a silicic acid column as already described (to see: click here). The glycolipid fraction (not including acylated steryl glycosides) is evaporated and readily submitted to TLC or other separation procedures.

When large quantities of steryl glycosides have to be prepared, it is more efficient to use the following procedure adapted to lipid extracts which are previously saponified.

Procedure

Dissolve the crude lipid extract in 1 ml (or more) of hexane and pour the solution on the top of a silicic acid column  previously equilibrated with hexane (keep the silicic acid to lipid weight ratio at about 80 or higher).
The solvent volumes are given as an approximated indication for a 1 g silicic acid column. 

1. Elute pigments with 10 ml of hexane  
2. Elute fatty acids and sterols with 20 ml of hexane/diethyl ether (1/1, v/v)
3. Wash with 5 ml chloroform
4. Elute the steryl glycoside fraction with 10 ml of chloroform/methanol (95/5, v/v).

If all glycolipids have to be collected, elute with 20 ml of acetone/methanol (9/1, v/v) in the step 4. 

Separation and quantification by HPLC:

It is recommended to isolate previously a glycolipid fraction with a  small column of silicic acid (see above) and to saponify that fraction. Thus, acylated steryl glycosides and glycoglycerolipids are removed and do not interfere with the fractions of interest. In plant extracts, two fractions are remaining, steryl glycosides and ceramide monohexosides (equivalent to the cerebrosides of animal tissues).  

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.
Detection: 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. 

Steryl glycosides are eluted at 8-9 min and ceramide monohexosides at 10-11 min. A detection limit of about 0.5 mg for steryl glycosides 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).