PROTOCOL OF
EXTRACTION OF CURCUMA
1)
2)
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5)
Dissolve 5.0 g Curcuma longa in 50 ml of methanol.
Keep this in the dark for about 24 hours and shake vigorously several times a day.
Then, filter the extract using filter paper and funnel.
Dry the extract using the rotavap.
Transfer the dried extract to weighted eppendorf tubes using aliquots of 1 ml of
methanol.
6) Dry the tubes in the concentrator and weigh the tubes again.
7) Keep the tubes in the fridge, protected from light with aluminium foil.
PROTOCOL OF
TLC OF EXTRACT
1)
2)
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5)
Make a stock-solution of 200mg/ml in methanol.
Take 50 µl of this stock-solution (equivalent to 10mg of dried extract).
Dry this 50µl in the concentrator.
Dissolve the residu in 100 µl of methanol.
Spot this volume over the breadth of a TLC-plate and develop in TEFFA ( 5 parts of
toluene, 4 parts of ethyl formate, and 1 part of formic acid) in a saturated chamber.
6) Divide the plate in the bands you can distinguish.
Draw every band with a pencil, and number it.
7) At the left side of the plate, cut off a lane of a few centimeters, to keep as a
reference.
8) Take pictures of this lane in normal light, and in UV-light at 2 different wavelengths
(254 nm and 365 nm).
9) Spray the smaller lane you’ve cut out with Vannilline-Phosphoric acid, to check if
there are any unnoticed bands above the highest noticed band.
After spraying, dry the plate with a hairdryer to remove the Phosphoric acid.
10) Take a picture of this lane, only in normal light. Keep the lane, wrapped in aluminium
foil.
PROTOCOL OF
SEPARATION OF DIFFERENT BANDS
1) Cut out the separate bands and scratch off the silica, containing the curcumacompounds.
2) Put the silica in the appropriate amount of eppendorf tubes (depends on the height
of a band).
3) Add 1 ml of methanol to every tube.
4) Put the tubes in the sonicator.
5) Put the tubes in the centrifuge for 2 minutes at a higher velocity.
6) Take out the supernatants, and transfer them into weighted tubes.
7) Dry the tubes in the concentrator.
8) Weigh the tubes again, and calculate the amount of content of every tube.