Extraction Method #2 Fractionated precipitation: Dried plant of 6g is extracted with Methanol at a mass-volume ratio of 1:10 After extraction, the methanolic solution is split into 3 parts. The first part of the solution is mixed with half the volume of acetone while the second part is mixed with same volume of acetone and the last part is mixed with twice the volume of acetone. The resulting solution is then filtered and the residue is removed. The residue is washed with the same ratio of methanol and acetone as step 1 and 3 respectively. The washing solution is then added to the filtrate. The solutions are then evaporated. The crude extracts are redissolved in methanol at the same mass-volume ratio of 1:10. Step 2-8 is repeated 2 more times.

Extraction Method #2 cont. Solvent-solvent distribution: After precipitation, the crude extracts are dissolved in 50% aqueous methanol. Hexane is added to the solution to extract the non- polar compounds in the precipitate. The aqueous methanol phase (bottom) is separated and then evaporated to dryness. The resulting residue is dissolved in pure methanol. The methanolic solution is mixed with aluminum oxide and the suspension was evaporated to dryness with a rotary evaporator.

Extraction Method #2 cont. Chromatography: The alumina is eluted with a hybrid of Dichloromethane- 96% Ethanol solution of ratio 9:1 and 8:2. A cotton wool of mass of 0.2 g was placed at the bottom of the column to prevent alumina from flowing out g (subject to experimental changes) of Alumina is mixed with the eluent. The mixture of alumina and eluent was stirred and poured into the column until is 75% full. The bands in the mobile phases are collected in different beakers for further tests.

Extraction Method #2 cont. Purification of Ecdysteroids: For further purification, the ecdysteriods are separated by reversed-phase chromatography In reversed-phase chromatography, octyl silane is used as the stationary phase (being non-polar) instead of silica/alumina. A cotton wool of mass of 0.2 g was placed at the bottom of the column to prevent alumina from flowing out g (subject to experimental changes) Octyl Silane is mixed with the eluent. To control the flow of the mobile phase, a vacuum will be used at the outlet.

Extraction Method #2 cont. Different concentrations of methanol are used as eluents in this chromatography. Stepwise gradient elution is used with an increase of 5% of methanol content in each step. Different bands of ecdysteroids will be formed on the stationary phase. The specific hormone, 20-hydroxyecdysone is isolated with methanol of 35-40% purity. The compounds will be used against the bioassay.