Antioxidants are radical scavengers which protect the human
body against free radicals that cause pathological conditions such as
ischemia, anaemia, asthma, arthritis, inflammation, neurodegeneration,
ageing process and perhaps dementias (Polterait, 1997). Oxygen radicals
induce oxidative stress that is believed to be a primary factor in various
diseases as well as normal process of ageing (Aust et al., 1993;
Stohls, 1995). Several studies have described the antioxidant properties
of medicinal plants, foods and beverages which are rich in phenolic compounds
(which are known to serve as antioxidants (Brown and Rice-Evans, 1998;
Krings and Berger, 2001). The importance of antioxidants in human health
has become increasingly clear due to spectacular advances in understanding
the mechanisms of their reaction oxidants (Bergman et al., 2003).
The present study investigates three plants used in Botswana herbal medicine,
for their potential to scavenge free radicals and as a consequence, may
be considered as effective sources for combating oxidative damage.
MATERIALS AND METHODS
Chemicals and other materials: 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) (Sigma), positive
controls: (Quercetin, (-) epicatechin, L-ascorbic acid), Silica 60 coated
aluminium thin layer chromatography plates (Merck Si gel of thickness
200 µm). All other chemicals used were of analytical grade.
Plants and sample preparation Colophospermum mopane
(J. Kirk ex Benth) J. Leonard: Colophospermum mopane (Caesalpiniaceae) is a shrub
or medium sized to tall deciduous tree occurring in almost pure stands
in hot, low lying areas, often on alluvial or lime rich soils. It bears
flattened, oval indehiscent pods (fruit pods).
The seeds of Colophospermum mopane are used in traditional
medicine to treat asthma attacks and facilitate wound healing. Dried seeds
of Colophospermum mopane were collected from Seolwane village in
Tswapong region in Eastern Botswana. The outer coating of the seeds was
removed and discarded. The remaining inner seed component was crushed
into a sticky course material. This crushed material was extracted independently
from the following solvents; methanol, chloroform, water in a soxhlet
apparatus for 8 h. Extracts of chloroform and methanol were evaporated
to dryness and concentrated using rotary evaporator. Water extract was
concentrated using a freeze drier.
Ozoroa paniculosa (Sond) R. Fern and A. Fern: Ozoroa paniculosa (Anacardiaceae) is a small to medium-sized
deciduous tree with distribution ranging from northern South Africa through
Eastern Botswana to Zimbabwe. It often occurs on rocky hillsides. It is
characterised by elliptic, grey to blue-green (above), silvery to silky
leaves (below). Leaves have a broadly tapering apex with a bristle-like
tip. This plant fruits a drupe, elliptic or kidney shaped, initially green
with a small reddish brown spots ripening black and wrinkled.
The roots of Ozoroa paniculosa are used by the Batswapong
tribe to treat an inflammed uterus and are believed to enhance female
Whole root of Ozoroa paniculosa was harvested from
Tswapong hills in Eastern Botswana under the supervision of a traditional
healer. The roots were chopped into small slices and sun-dried. The dried
roots were then crushed into course powder using mortar and pestle. The
powdered material was extracted in either water, methanol, ethylacetate
or chloroform in a soxhlet apparatus for 8 h. Water extract was concentrated
using freeze drier while the other extracts were concentrated to dryness
under reduced pressure.
Cucumis metuliferus (E. Mey. ex Naud): Cucumis metuliferus (Cucurbitaceae) is an annual
climber with angled stems and stiff brown hairs. The young fruit is dark
green with mottled light green spots. As it ripens it becomes bright orange
with very sharp spines that can injure the skin. The species has a wide
scanty distribution from South Africa to tropical Africa.
Whole fruit of ripe Cucumis metuliferus is eaten
and is believed to be not only nutritious but has medicinal properties
as well. Some segments of the society in Botswana believe it is good for
diabetic patients. Ripe fruits of C. metuliferus were collected
from Glen valley, Gaborone. The whole fruit was crushed and both the pulp
and skin were extracted exhaustively in soxhlet using water, methanol,
petroleum ether and chloroform. Water extracts were concentrated to dryness
using a freeze drier whilst extracts containing organic solvents were
concentrated using a rota evaporator. All samples were stored in the fridge
until ready for testing.
Botanical identification: The plant specimen were authenticated by comparison with
Herbarium specimen at the National Herbarium and Gallery, Gaborone, where
voucher specimen have been deposited.
Antioxidant assays Qualitative scavenging activity on
DPPH radical: One milligram of each extract was weighed into a small sample
tube and 5 mL of methanol added. The mixture was vortexed. Then 100 µL
of each of the mixture was spotted drop by drop (followed by intermittent
drying) onto a silica coated TLC plate (200 µm thickness), about 20 mm
away from the bottom of the plate. The point of the spot was clearly labelled
and the plate was dried in air and developed in a tank containing the
mobile phase (Ethylacetate: Formic acid: Water 85:15:10). This was done
to screen for the presence of any phytochemicals with free radical scavenging
property. After development the plate was allowed to dry and viewed UV
light 254 and 365 nm. The absorbing and fluorescent bands were marked.
The plate was then sprayed with Diphenyl-picrylhydrazyl (DPPH) reagent
in methanol (0.2%). After this, the plate was left to dry and the bleaching
produced on the plate was noted. The DPPH reagent in this case was used
to detect the presence of antioxidants.
Quantitative scavenging activity on DPPH radical: The free radical scavenging activity of the crude extracts
was measured by the 2, 2-diphenyl-1-picryl-hydrazyl (DPPH) method proposed
by Brand-Williams et al. (1995). Briefly, a 0.1 mM solution of
DPPH in methanol was prepared and 1.0 mL of this solution was added to
0.5 mL of samples in different concentrations. After 20 min, the absorbance
was measured at 525 nm. The DPPH radical scavenging activity was calculated
according to the following equation:
DPPH. scavenging activity (%)
= [(A0-A1)/A0] x 100%
where, A0 was the absorbance of the blank (i.e.,
no sample, DPPH solution only) and A1 was the absorbance in
the presence of test extract.
RESULTS AND DISCUSSION
Qualitative screening of antioxidant activity in shown is
As can be shown from Fig.
1, at 50 µg mL-1 and above, both methanol and water extracts
from Ozoroa paniculosa exhibited good scavenging activity (90%)
comparable to the control compounds L-ascorbic acid
||Qualitative screening of antioxidant
|++: Low antioxidant response, +++:
Moderate antioxidant response, ++++: Strong antioxidant response
activity of Ozoroa paniculosa root crude extracts
scavenging activity of Colophospermum mopane seeds crude
(91%) and epicatechin (92%). At 100 µg mL-1 and
above, the scavenging activity of the ethyl acetate extract from O.
paniculosa roots reached a similar magnitude of activity (90%). The
order of free radical scavenging potency for the tested extracts from
O. paniculosa roots was as follows: methanolic extract>water extract>ethyl
acetate extract. This perhaps indicates that the free radical scavenging
principles are polar. Between 50-100 µg mL-1
radical scavenging activity of whole fruit extracts of Cucumis
the free radical scavenging potencies of water and methanol
extracts from Colophospermum mopane seeds were <=70%. However,
above 100 µg mL-1, both water and methanolic extracts of C.
mopane exhibited scavenging activity >=70% (Fig.
2). Chloroform extract of C. mopane seeds showed poor scavenging
activity at all tested concentrations. All extracts of C. metuliferus
showed poor free radical scavenging activity (30%) at all tested concentrations
3). Both water and methanol extracts of C. metuliferus exhibited
equal potency. These findings show limited antioxidant potential of extracts
from whole fruits of C. metuliferus.
The various antioxidant potencies exhibited by the studied
plants may at least in part, explain why these plants are used to treat
inflammatory disorders such as gout and asthma. The common link between
free oxidant radicals and inflammatory reactions has been well established
(Mongelli et al., 1997; Wang et al., 1999). The antioxidant
capacities shown by the tested extracts in this study may lend credence
to the use of O. paniculosa and C. mopane as anti-inflammatory
and antioxidant agents in folk medicine.
Traditional healers for selection of the studied plants.
Funding for this study was provided by the Botswana College of Agriculture.