2.3. Isolation and preparation of crude extracts
After having washed, the leaves were cut into small pieces and
dried by sunlight or oven below temperature 40 C. The dried
leaves samples were pulverized into powder form. The dried
powder (0.1 g) was extracted three times with methanol (5 ml
3·) by sonication at 30 min. It was then filtered and the filtrate
was evaporated near to dryness by Kuderna-Danish evaporator.
The extract was passed through the cleanup column
(i.d. =1 cm), which was filled with cotton in the bottom. An
activated silica gel (10 g) soaked with solvent was loaded into
the cleanup column (5 cm), which was then topped with 1.5 cm
of anhydrous sodium sulfate. Five milliliters of solvent were
added to wash the sodium sulfate and the silica gel. The preconcentrated
dried crude extracts, 1 ml of each extract sample,
were then separately transferred into the column, and the vessel
was rinsed twice with 2 ml loaded solvent, which was also
added to the column. Sixty milliliters of loaded solvent were
added to the column and allowed to flow through the column
at a rate of 3–5 ml/min, and the eluent was collected. The collected
eluent from the cleanup procedure was reconcentrated
to 2 ml by using K-D concentrator. Finally the extract (2 ml)
from leaves was filtered through a 0.45 lm Millex HA filter
(Millipore, Molsheim, France) prior to GC–MS analysis
2.3. Isolation and preparation of crude extracts
After having washed, the leaves were cut into small pieces and
dried by sunlight or oven below temperature 40 C. The dried
leaves samples were pulverized into powder form. The dried
powder (0.1 g) was extracted three times with methanol (5 ml
3·) by sonication at 30 min. It was then filtered and the filtrate
was evaporated near to dryness by Kuderna-Danish evaporator.
The extract was passed through the cleanup column
(i.d. =1 cm), which was filled with cotton in the bottom. An
activated silica gel (10 g) soaked with solvent was loaded into
the cleanup column (5 cm), which was then topped with 1.5 cm
of anhydrous sodium sulfate. Five milliliters of solvent were
added to wash the sodium sulfate and the silica gel. The preconcentrated
dried crude extracts, 1 ml of each extract sample,
were then separately transferred into the column, and the vessel
was rinsed twice with 2 ml loaded solvent, which was also
added to the column. Sixty milliliters of loaded solvent were
added to the column and allowed to flow through the column
at a rate of 3–5 ml/min, and the eluent was collected. The collected
eluent from the cleanup procedure was reconcentrated
to 2 ml by using K-D concentrator. Finally the extract (2 ml)
from leaves was filtered through a 0.45 lm Millex HA filter
(Millipore, Molsheim, France) prior to GC–MS analysis
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