CARDIOVASCULAR JOURNAL OF AFRICA • Vol 23, No 1, February 2012
AFRICA
13
Department, University of KwaZulu-Natal, voucher specimen
no. 685.
Fresh leaves (1 kg) of
S birrea
were air dried at room
temperature (26
±
1°C) for two weeks. They were then milled
in a Waring commercial blender into fine powder, and macer-
ated in 2.5 l of distilled water, with occasional shaking, for 48
hours at room temperature. The powdered leaves were extracted
twice with 2.5 l of distilled water on each occasion, before being
filtered. A rotary evaporator was used to concentrate the aqueous
extract (filtrate)
in vacuo
at 60
±
1°C. Freeze-drying and solvent
elimination of the resulting aqueous filtrate produced 55.5 g
(i.e. 5.55% yield) of a light-brown, powdery, crude leaf extract.
Without any further purification, portions of this extract were
weighed and dissolved in distilled water (at room temperature)
for use on each day of our experiments.
Healthy young adult, treatment-naïve, male and female Wistar
rats (180–200 g,
n
=
40) and New Zealand white albino rabbits
(1.5–3.0 kg,
n
=
6) were used. The animals were kept under
conventional laboratory conditions of temperature, humidity and
light, and allowed free access to food (standard pellet diet) and
tap water. All the animals were fasted for 16 hours, but allowed
free access to drinking water before the commencement of our
experiments.
Each rat was euthanised by halothane inhalation. The rabbits
were anaesthetised by intramuscular administration of a keta-
mine (30 mg/kg body weight) and xylazine (5 mg/kg body
weight) mixture, followed by intravenous injection of 200 mg/kg
body weight of sodium pentobarbital (Euthapent
®
) through the
ear vein. The abdomen and thorax were then opened via midline
incision and the descending thoracic aorta of the rabbits and the
portal vein of the rats were quickly removed.
Rat isolated aortic rings
Isolated descending thoracic aortic ring preparations (3 mm
long) and isolated portal veins were suspended in 30-ml Ugo
Basile organ baths containing Krebs-Henseleit physiological
solution (KHS) of composition (in mM): NaCl, 118; KCl, 4.7;
NaHCO
3
, 25; MgSO
4
, 1.2; CaCl
2
.2H
2
O, 2.52; KH
2
PO
4
.2H
2
O,
1.28; and D-glucose, 5.55 (pH adjusted to 7.4). Calcium-free
KHS was made by adding 0.2 mM ethylenediaminetetra-acetic
acid (EDTA) to the physiological solution. The bathing KHS was
maintained at 35
±
1ºC and aerated continuously with carbogen,
(95% O
2
+
5% CO
2
).
Two smooth muscle preparations from the same animal (one
used as control and the other as SBE and/or reference drug-treat-
ed test preparation) were always set up at a time in order to make
allowances for changes in smooth muscle sensitivity. The tissues
were maintained in 30-ml Ugo Basile organ baths (under physio-
logical conditions) for 45–60 minutes to equilibrate, during
which time the bathing KHS was changed every 15 minutes.
After the equilibration period, graded concentrations of SBE
(50–400 mg/ml) and/or the reference drugs were added to the
bath fluid sequentially at 10-minute intervals. Where necessary,
bath-applied SBE and/or reference drug concentrations were
repeated after washing out the previous SBE (and/or reference
drug concentration/s) four to five times and then allowing the
tissue to rest for five to 10 minutes, or until its tone returned to
baseline. The control smooth muscle tissue preparations were
treated with distilled water (0.5–4 ml only, equivalent to the
volumes of bath-applied SBE or reference drug solution used).
Each test smooth muscle preparation was used for one concen-
tration–response curve only.
Rabbit isolated aortic ring
Each isolated descending thoracic aortic ring preparation was
subjected to the same experimental conditions as for the rat
aortic rings and portal veins described above. In some of the
rabbit aortic ring preparations, the endothelium was removed
mechanically
in situ
by rubbing the luminal surfaces gently six
to eight times with a glass rod.
In order to characterise the involvement of the nitric oxide
pathway prior to the addition of SBE (50–400 mg/ml), some
of the endothelium-intact aortic rings were pre-incubated with
N
G
-nitro-
L
-arginine-methyl-ester (L-NAME, 100
µ
M), a nitric
oxide synthase inhibitor. In addition to verapamil (1–3
µ
g/ml),
an L-type calcium channel blocker, Ca
2+
-free KHS was used to
test the contractile effects of SBE.
The possible role of indomethacin (20
µ
M), a cyclo-oxyge-
nase pathway inhibitor, on the SBE-induced contractile response
was also examined in rabbit aortic ring preparations. The effect
of SBE and/or other reference drugs on the aortic ring prepara-
tions was recorded by means of Ugo Basile’s force–displacement
transducers and pen-writing, two-channel Gemini recorders.
Rat isolated portal vein preparations
Each rat isolated portal vein preparation was suspended in a
30-ml Ugo Basile organ bath containing KHS, and subjected to
the same experimental conditions as for the aortic rings above.
In addition to verapamil (1–3
µ
g/ml), Ca
2+
-free KHS was used to
test the contractile effects of SBE (50–400 mg/ml). The possible
role of prazosin (1–3
µ
g/ml), an
α
1
-adrenergic receptor blocker,
on the SBE-induced contractile responses was also investigated
in some isolated portal vein preparations. Six of the animals used
in this study were pre-treated with reserpine (1 mg/kg subcutane-
ously), a catecholamine blocker, to deplete catecholamines from
the neuronal stores and subsequently block their uptake.
Statistical analysis
Contractile responses to graded concentrations of SBE and/
or reference drugs on the different tissues were calculated as
per-gram increments of maximal contractions (1 g
=
2 cm).
Experimental data were obtained as three to four duplicates,
using data from different animals where applicable, and present-
ed as means [
±
standard error of the means (SEM)] of measure-
ments.
Data from distilled water-treated control smooth muscle
preparations were used as baseline values. In all cases, data
from the SBE- and/or reference drug-treated test smooth muscle
preparations were compared with those from distilled water-
treated controls.
Using GraphPad Prism
®
Version 5.00 (GraphPad
®
Software,
Inc. San Diego, Califonia), the differences between the data
obtained from test and control smooth muscle preparations were
analysed for statistical significance, using Student’s
t
-test, and/
or one-way analysis of variance (ANOVA; 95% confidence
interval) for multiple value comparison, followed by Dunnett’s
post hoc
test. In all cases, values of
p
≤
0.05 were taken to imply
