1009
Organic solvents such as acetone, ethanol, 0.05 mM Tris-HCl buffer (pH 8), methanol, chloroform
and hexane were used for extraction. Sample (50 g) was dissolved in 500 mL of respective solvents by
sonication for 10 minutes. The extract was filtered through Whatman No.1 liter paper using a vacuum
pump literation and the extraction process was repeated two times. The extract was concentrated under
reduced pressure at 40
o
C, 90 rpm using a rotary evaporator. The yields obtained were kept at -20
o
C for
further analysis. Extraction yields were weighted and calculated using following formula.
Yield (%) =
x100
powder
plant
of
Dry weight
extract
of
Dry weight
Phytochemical study (qualitative analysis)
Phytochemical studies were conducted qualitatively and quantitatively to identify the presence of
bioactive chemical constituents such as alkaloids, lavonoids, terpenoids and steroids, saponins, tannins,
phenolic compounds, coumarins and carbohydrates. Qualitative phytochemical analysis was done
according to the standard protocols [9], while quaitiative phytochemical analysis was done based on
standard proximate measurement [10, 11].
Antioxidant assay using DPPH method
Methanol solution of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical (0.004 %w/v) was prepared and
stored at 10
o
C in the dark. Samples in methanol solution with different concentration of 0.0625 to 4
mg/mL were prepared. Aliquot (0.05 mL) of the sample solution was then added to 5.0 mL of methanolic
DPPH solution. The mixture was shaken vigorously and left to stand for 30 min in the dark. Absorbance
measurements were recorded immediately at 517 nm. The absorbance of the DPPH radical without the
crude extract samples (control) and the reference compound DL-
Į
-tocopherol and gallic acid were
measured. All the determinations were performed in three replicates and the average was counted. The
percentage of inhibition (PI) of the DPPH radical was calculated according to the following formula:
PI (%) =
x100
AC
AT-AC
Where AC = Absorbance of the control and AT = absorbance of the sample.
Analysis of total lipid content
The total amount of oil palm was determined by solvent extraction [12].
Determination of total carotenes, tocopherols and tocotrienols
The analyses of carotenes, tocopherols and tocotrienols were carried out simultaneously by
chromatography analyses with a Shimadzu HPLC (Japan) equipped with a quaternary pump, auto sampler,
degasser, SPD-M20A spectrophotometric detector that was set at 292 and 455 nm and RF-10AXL
fluorescence detector that was set at 290 nm for excitation and 330 nm for emission. Chromatographic
separation of the compounds was achieved at 30
o
C with a normal-phase column (250 x 4.6 mm id; 5 μm
particle size) with a guard column (10 x 4.6 mm) purchased from Merck (Germany). The concentration
