Pharmacognosy - practical training
Practice No.2
Principle: Anthraquinone glycosides are hydrolyzed to anthraquinone aglycones.
These ones are separated from reaction mixture with organic solvent. The redcolored salts of anthraquinone aglycones are created in the presence of alkaline
solution (Bornträger reaction). These products can be used for TLC presence proof
or colorimetric determination.
H
H
OH
O
O
OH
O
O
O
O
O
OH
O
O
O
tautomeric forms
Part A: Colorimetric determination of anthraglycosides
Powdered drug (Rhei radix - 50.0 mg, Frangulae cortex - 12.5mg) is refluxed on a
water bath in a 50 ml flask with 1.0 ml conc.hydrochloric acid and 7.5 ml conc.
acetic acid for 15 minutes. 30 ml of chloroform is added to the cool solution and
then the sample is refluxed for 15 minutes again. The cool mixture is filtered through
cotton wool into a separatory funnel (250 ml volume). Cotton wool is placed back
into a flask and 30 ml chloroform is added. Solution is refluxed for 10 minutes. Cool
solution is filtered through cotton wool into the same separatory funnel as before. The
cotton wool is washed 3 times with 10 ml chloroform. 12 ml conc.natrium hydroxide
solution is then added slowly to the solution in the funnel. Following, 24 ml of
natrium hydroxide in
ammonia solution is added.
This mixture is shaken for 5
minutes. Upper (water) layer is separated in a 100 ml flask. Lower (chloroform)
layer is shaken again with 20 ml natrium hydroxide in ammonia solution for 3
minutes. The upper layer is separated and added to the previous water sample. The
lower layer is once more time shaken in the same way as before. All collected water
layers are refluxed 2 hrs on the heat plate. Then, the cool solution is filtered. The
cotton wool is washed with a small amount of natrium hydroxide in ammonia solution.
The complete water sample is poured into a 100 ml volumetric flask and the same
ammonia-natrium hydroxide solution is added up to 100 ml volume exact. The
absorbance of this sample is measured immediately at 530 nm. The ammonianatrium hydroxide solution is as a blank sample used. Contents of anthraglycosides
is from calibration curve calculated in %.
Part B: TLC analysis of Frangulae cortex
About 0.2 g of powdered drug is hydrolyzed with 10 ml of 0.5% hydrochloric acid
under reflux for 10 minutes on the water bath. 20 ml of chloroform is added to a
cool reaction solution and once more refluxed for 10 minutes. The warm extract is
filtered in a separatory funnel through cotton wool and the upper phase is placed
back in the flask together with filter (cotton wool). 20 ml of chloroform is added in
this flask and refluxed for 10 minutes. The cool solution is filtered in the separatory
funnel to the previous extract. The water phase (upper) is removed. The chloroform
phase is evaporated in vacuo to small volume and chromatographed on Silufol plate
together with
the
standard
substance.
Elution
ethylacetate (45:15). After drying the plate is detected
hydroxide
mixture: petroletherby
ethanolic
kalium
solution (0.5 mol/l), dried in drying-oven at 105 C. The corresponing
spots are evaluated.
Part C: Identity tests of anthraglycoside drugs
ALOE
Thin layer chromatography (TLC): About 0.2 g of powdered drug is extracted in a
flask with boiling methanol during 1 minute. The cool solution is then filtered in a
tube. From this sample the TLC is carried on Silufol plate in a chromatographic
chamber in an eluent: ethylacetate-methanol-water (100:17:13). After total drying the
plate is detected by 10 % kalium hydroxide solution in methanol. In the visible light
the yellow spot of barbaloine is present, their florescence in UV at 366 nm is also
yellow.
FRANGULAE CORTEX
1. Powdered drug is treated with 1 drop of 6.5 % kalium hydroxide solution. The
powder color turns to dark red.
2. About 0.5 g of powdered drug is heated with 5 ml conc. hydrochloric acid in a
flask for 1 minute. The cooled solution is filtered and then shaken with 10 ml
of benzene. 5 ml of benzene (upper) layer is shaken separately with 5 ml of 15 %
ammonia solution. The ammonia layer becomes rose color.
SENNAE FOLIUM
0.3 g of powdered drug is heated with 10 ml of 0.5 mol/l kalium hydroxide solution
and 1 ml 3 % hydrogen peroxide solution for 1 minute. Warm solution is filtered.
5 ml of this solution is treated with conc. acetic acid and is shaken with 10 ml of
benzene. 5 ml of benzene layer (upper) is filtered and again shaken with 3 ml of
15% ammonia solution. Lower phase has a reddish color (oxymethylanthraquinones).
RHEI RADIX
1. In a microsublimation apparatus is a small amount of drug heated while the yellow
substance in upper part of the apparatus is condensed. After air cooling this of
apparatus a part of the substance is treated with 1 drop of 6.5 % kalium hydroxide
solution. The mixture becomes red.
2. About 0.1 g of powdered drug is heated with 10 ml 0.1 mol/l kalium
hydroxide solution for 1 minute. The cool solution is filtered, then acidified with 11
% hydrochloric acid (reagent lakmus paper detection). This mixture is shaken with
20 ml of diethylether. Separated upper layer is shaken with 5 ml 10 % ammonia
solution. Lower aqueous phase is reddish (emodine), upper one is yellow
(chrysophanic acid).