STERYL GLYCOSIDES
Structure and nomenclature
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).
ACYLATED STERYL GLYCOSIDES
Structure and nomenclature
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).
