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32
Journal of Science and Technology in the Tropics (2010) 6: 31-38
activity [6]. A detailed analysis on the methodologies
employed in the previous studies suggested that
the inconsistent findings on the anti-proliferative
activity could be attributed to differences in the parts
of the plant studied (e.g. leaf, stem, whole plant),
plant extraction procedure, cell lines used and antiproliferation
assay conditions [3].
The diversity of secondary metabolites in plants
is well known. They play a major role in contributing
to the plants’ natural defense mechanism against
predators or diseases caused by microorganisms. As
the stems of P. bleo are heavily covered with sharp
spines that can protect the plant against preys, they
may also be associated with high level of secondary
metabolites of important medicinal properties, such
as anticancer activity. However, previous study has
shown that the stem extracts do not possess significant
anti-proliferative activity against NIH/3T3 and 4T1
cells [6]. In the current study, the same procedure
was employed for the extraction and fractionation of
the leaves of the plant using the same solvents, and
the anti-proliferative activities of the extracts were
assessed using the same cell lines.
The mechanism of cell death caused by oxidative
stress has been studied widely [7]. The generation of
endogenous reactive oxygen or nitrogen species as
a result of oxidative stress can lead to tissue injury
implicated in many diseases. Many antioxidant
compounds are found in higher plants and they have
been shown to possess the ability to quench free
radicals and reactive oxygen species in vitro [8]. In
this study, the in vitro antioxidant property of the leaf
extracts, as well as their ability to limit natural and
oxidant-induced cell death were evaluated.
MATERIALS AND METHODS
Plant Material
The plant cuttings of P. bleo were collected from Taman
Pertanian (Agricultural Garden) of the Universiti
Putra Malaysia (UPM), Selangor, Malaysia. The
voucher specimen (No. ACP 0116) was deposited in
the herbarium of Institut Biosains of UPM.
Preparation of leaf extracts
The leaves of P. bleo were separated, cleaned with
water and dried in a convection oven at 40 o C until
consistent weights were obtained. The dried leaves
were blended into powder form using an electric
blender. A weighed amount (20 g) of the leaf powder
was extracted with methanol (250 mL) using a soxhlet
extractor for four hours. The process was repeated
five times in order to obtain sufficient amount of
the extracts for further testing and the extracts were
combined. The methanol solvent in the combined
extract was removed under reduced pressure at
40 o C using a rotary evaporator and the extract was
further dried using a vacuum concentrator at 40 o C
until a consistent weight was obtained. A portion of
the methanol extract was fractionated by partitioning
into ethyl acetate, t-butanol and water. The solvents
in these fractionated extracts were removed under
reduced pressure at 40 o C using a rotary evaporator.
The ethyl acetate and t-butanol extracts were further
dried to consistent weight using a vacuum concentrator
at 40 o C. The aqueous extract was freeze-dried to a
consistent weight. The yields of the methanol, ethyl
acetate, t-butanol and aqueous extracts were 7.7%,
0.2%, 3.8% and 7.1%, respectively, based on the dry
weight of the leaves.
ABTS radical cation scavenging activity of
P. bleo leaf extracts
The assay is based on the ability of substances with
antioxidant properties to scavenge 2,2’-azinobis-
(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)
radical cations, which are blue-green in colour and
absorb at 734 nm [9]. Any positive reaction will lead
to the decolourisation of the ABTS radical cations.
The methanol extracts of the leaves were dissolved
in 0.01 M phosphate buffered saline (PBS) while the
ethyl acetate and t-butanol extracts were dissolved
in 0.8% (v/v) DMSO. Trolox and vitamin C were
used as positive controls. To perform this assay, the
ABTS solution (715 µL, 5 mM) was mixed with
potassium persulphate solution (285 µL, 2.5 mM)
and the reaction mixture was kept in the dark at room
temperature for 12-16 hours for the generation of
ABTS radical cations.
The ABTS radical cation solution was
appropriately diluted with 0.01 M PBS to give an
absorbance of 0.7 ± 0.2 at 734 nm. To 990 µL of this
diluted ABTS radical cation solution, 10 µL of each
of the concentrations of the leaf extracts, Trolox or
vitamin C was added. The absorbance of the mixture
at 734 nm was recorded at every minute for a period
of 12 minutes. The experiment was repeated five
times for each concentration of the extracts (2.5
to 50 µg/mL for t-butanol extract; 5 to 100 µg/mL
for the other extracts), Trolox (0.25 to 2.5 mM) or
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