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CARDIOVASCULAR JOURNAL OF AFRICA • Vol 24, No 2, March 2013
AFRICA
11
We have researched a product from one such plant species,
consisting solely of the dried and ground pods of the plant
P
glandulosa,
for hypoglycaemic properties.
8
In addition, potent
anti-infective and anti-parasitic compounds have also been
isolated from this plant.
9
In view of the hypoglycaemic effects of
P glandulosa
, as well
as its ability to partially restore the function of pancreatic tissue
and increase cardiomyocyte insulin sensitivity,
7
we set out to
determine the cardiovascular effects of treatment, using a well-
characterised rat model of obesity and pre-diabetes with known
cardiovascular insufficiency and endothelial dysfunction.
10,11
In
addition, using a rat model of high-fat feeding known to develop
hypertension,
12
we determined whether
P
glandulosa
had any
effects on the development of high blood pressure.
Models
1. Diet-induced obesity (DIO)
As described previously,
10
Wistar rats (180–200 g) were randomly
divided into a control and diet group. The DIO group was
fed a diet of normal rat chow supplemented with sucrose and
condensed milk for a basic period of eight weeks. From weeks
eight to 16 the rats were treated with
P
glandulosa
(100 mg/
kg/day) set in jelly/gelatine blocks and given to each one
individually according to the weight of the animal.
8
This was
done to ensure absolute compliance and dose control. The dose
of
P
glandulosa
was calculated as previously described.
8
The diet to induce pre-diabetes in the animals was based
on hyperphagia.
13
Animals were anaesthetised with sodium
pentobarbital (160 mg/kg, intra-peritoneally) before
experimentation. At the time of sacrifice, their body weight
and the weight of the intra-peritoneal fat were noted and trunk
blood was collected for biochemical analyses. For Western blot
analyses, the hearts were removed, immediately snap-frozen in
liquid nitrogen and stored at –80°C.
2. High-fat diet (HFD)
To induce high blood pressure, the rats were fed a diet containing
the following per kg of food: cooking fat 400 g, fructose 100
g, casein 100 g, cholesterol 10 g, and rat chow pellets 390 g.
Blood pressure was monitored on a weekly basis over 16 weeks.
Treatment with
P
glandulosa
(100 mg/kg/day) given in jelly
blocks was either started at the onset of the diet to study the
effect on prevention of the development of hypertension, or after
a period of 12 weeks of the HF diet to study its anti-hypertensive
effects. Rats treated with captopril (50 mg/kg/day) from the onset
of the diet were included as a positive control. All animals were
also placed individually in metabolic cages in order to collect
urine samples.
3. CIRKO mice
A mouse model of animals with a cardiac conditional ablation of
the insulin receptor was used in conjunction with their C57Bl6
littermates.
14
Mice were fed normal chow and treated with
P
glandulosa
at a similar dose to that of the rats for a period of
eight weeks before experimentation.
Methods
Animals had free access to food and water and were kept on a
12-hour day/night cycle in the Central Research Facility of the
Faculty of Health Sciences of the University of Stellenbosch. The
study conformed to the revised South African National Standard
for the Care and Use of Animals for Scientific Purposes (South
African Bureau of Standards, SANS 10386, 2008) and was
registered with the Committee for the use of animals in research
of the University of Stellenbosch – numbers P05/11/013 and
P07/11/020.
The
P glandulosa
plant material was originally obtained from
naturally growing plants. The material was handled according
to a patented and standardised procedure
8
and pre-packed
in capsules for human consumption, which we emptied and
weighed. The voucher specimen was reported previously.
8
Plasma glucose levels were determined in the fasting state.
Blood was obtained via a tail prick and glucose levels were
determined using a conventional glucometer (Cipla MedPro).
Plasma was stored at –80°C in a Snijders Scientific Ultracool
(Tilburg, the Netherlands) and insulin levels were determined
using a coat-a-count assay (Diagnostic Products).
Intra-peritoneal glucose tolerance curves (IPGTTs) were
generated in the animals after an 18-hour fast. Animals were
injected intra-peritoneally with 1 g/kg of a 50% sucrose solution
and blood glucose levels were monitored over a 120-min period.
After removal, the hearts were arrested in ice-cold Krebs
Henseleit (KH) medium (in mM: NaCl 119, NaHCO
3
25, KCl
4.75, KH
2
PO
4
1.2, MgSO
4
.7H
2
O 0.6, Na
2
SO
4
0.6, CaCl
2
.2H
2
O
1.25, glucose 10) and immediately (within 30 sec) mounted onto
the aortic cannula of a perfusion rig. The pulmonary vein was
connected to a second cannula in order to perform perfusions
in the working-heart mode with a preload of 15 cm H
2
O and an
afterload of 100 cm H
2
O, as described previously.
15
The perfusion
medium was continuously gassed with 95% O
2
/5% CO
2
. Hearts
were fitted with a temperature probe and the temperature was
kept constant at 36.5–37°C.
After a stabilisation period of 30 min, rat hearts were
subjected to 35-min regional ischaemia by coronary artery
ligation, followed by reperfusion for one hour, as described
previously.
15
Infarct size was determined according to a well-
established protocol,
15
followed by planimetry, and expressed as
a percentage of the area at risk. Planimetry was performed blind
by a third party.
Mouse hearts were perfused retrogradely, meaning via the
aorta without a connection to the pulmonary vein. After the
30-min stabilisation period, the hearts were subjected to 20-min
normothermic ischaemic cardiac arrest (NICA) by stopping all
perfusion. This was followed by one hour of reperfusion, after
which the infarct development through the whole heart was
determined as described above.
To measure blood pressure, rats were placed in restraining
holders with a dark nose cone to calm them. The restrainers were
placed on a heating pad (32
±
2°C) to warm the rat and maintain
blood flow to the tail. Animals were placed in the restrainers for
at least five minutes before monitoring the blood pressure using
a computerised tail-cuff blood pressure monitor (Kent Scientific
Corporation, Connecticut, USA). Prior to commencement of the
experiment, rats were subjected to the above procedure daily for
at least a week to train the animals for the procedure and to avoid
stress in the rats during experimental determinations.
Animals were placed individually in metabolic cages and the
volume of urine was determined over a period of 24 hours.