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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 32, No 2, March/April 2021
88
AFRICA
Animals were randomly divided into five experimental groups
(
n
= 8–10 per group) and fed either a control or high-fat diet
(HFD) for a period of 16 weeks. The age-matched control group
received standard Epol
TM
[Epol (Pty) Ltd, Worcester, Western
Cape, RSA] rat chow composed of: fat 4.8 g/100 g, protein 17.1%,
carbohydrates 34.6%, sugar 6.6 g/100 g and energy 1 272 kJ/100
g. The HFD group however received a diet composed of: fat 27.9
g/100 g, cholesterol 6.4 mg/100 g, protein 14.6%, carbohydrates
29.5%, sugar 13.3 g/100 g, fructose 11 g/100 g and energy 1 823
kJ/100 g. The HFD diet was specifically adapted to induce high
blood pressure together with obesity.
19
The rat chow and HFD
compositions were analysed by Microchem Laboratory (Pty) Ltd
in Maitland, Cape Town. Food and fluid intake were measured
three times weekly and the animals were weighed once a week.
The good manufacturing practice (GMP)-certified GRT
extract was prepared by Afriplex (Pty) Ltd (Paarl, South Africa)
and it was kindly given as a donation. It was supplied with the
total polyphenolic composition analysis
19
(Table 1).
A total of 10 rats per group in both the HFD and control
groups were treated with 60 mg/kg/day of GRT extract from
week 11 to week 16 of the diet period. An additional HFD group
was included, which served as a positive control (
n
= 8) for blood
pressure effects and these rats were treated with captopril (50 mg/
kg/day), a well-known ACE inhibitor and anti-hypertensive drug.
The administered GRT extract dose was deduced from
available literature on similar extracts
9,20
as there is currently
no evidence on the exact recommended dosage of this specific
product. The captopril dosage was previously used in our
laboratory as a positive control in the same rat model.
21
The
GRT extract and captopril treatments were prepared in the form
of strawberry jelly/gelatine blocks and were given to each animal
individually according to their body weight. The untreated
groups (without GRT extract or captopril supplementation)
were given jelly/gelatine blocks to normalise for the effect the
additional sugar content present in the jelly/gelatine might have.
An oral sucrose tolerance test (OSTT) were performed in
the week before commencement of treatment (week 10) and
again a week before sacrifice (week 15) in both the controls
and HFD groups. The animals were fasted overnight with free
access to drinking water. Blood glucose levels were determined,
using a handheld Glucoplus
TM
glucometer, from a drop of blood
collected after a tail prick with a lancet at the tail tip. Following
measurement of baseline (0 minutes, fasting level) glucose levels,
the animals were gavaged with 50% sucrose solution (1 g/kg) and
the disappearance of glucose in the blood was monitored for
two hours. After the OSTT procedure, the animals were left to
recover for a week from this metabolic insult, prior to sacrifice.
The blood pressure of each animal was measured using a
CODA
®
non-invasive blood pressure acquisition system (Kent
Scientific), which utilises a volume pressure-recording (VPR)
tail-cuff system to measure the blood volume of the tail. Prior to
the actual study, the animals were acclimatised to the apparatus
for a period of two weeks. Blood pressure was then monitored on
a weekly basis for 16 weeks, and baseline levels were determined
for two weeks prior to treatment.
Glucose present in the urine was determined in weeks 10 and
16 of the study. Animals were individually placed in metabolic
cages for a 24-hour period while having
ad libitum
access to food
and water. Urine was collected in a plastic measuring cylinder
attached to each cage. Glucose levels were determined using a
Test-it™ 10 dipstick.
At the time of sacrifice, the animals were weighed and
euthanised with an overdose of sodium pentobarbital (Eutha-
naze 160 mg/kg, intra-peritoneally). Following this, fasting or
non-fasting blood was collected from all the animals, transferred
to vacutainer tubes (SGVac) and allowed to clot at room
temperature (25°C). After 30 minutes, the blood was centrifuged
at 1 200 ×
g
for 10 minutes at 4°C. Thereafter, the serum was
collected and stored at –80°C for biochemical analysis.
The liver and the intra-peritoneal (IP) fat were removed,
rinsed, weighed, snap-frozen in liquid nitrogen and stored at
–80°C for downstream experimental procedures. Additionally,
the thoracic aorta was gently excised from the thoracic cavity with
the perivascular adipose tissue (PVAT) intact. Then one aortic
ring per animal was cut in half and one piece was immediately
used for vascular contraction/relaxation experimentation, while
the rest of the tissue was stored in liquid nitrogen for Western
blot analyses.
Vascular contraction/relaxation of the aortic rings was
performed to determine the endothelial function of the animals.
A total of 10 aortae were used for each experimental group,
except for the captopril group (
n
= 8). The noradrenergic agonist
phenylephrine (1 mM; 0.002g Phe in 10 ml 0.9% saline) and the
endothelium-dependant nitrogen oxide (NO)-releasing agent
acetylcholine (10 mM; 0.0182 g in 10 ml 0.9% saline) stocks were
prepared. Acetylcholine (10 mM), labelled stock A, was serially
diluted to make stock B (1 mM; 1 ml stock A in 9 ml 0.9% saline),
and finally stock C (100 µM; 1 ml stock B in 9 ml 0.9% saline).
The aortic ring (3–4 mm) was mounted onto two stainless
steel hooks and slowly submerged in the organ bath (AD
Instruments, Bella Vista, New South Wales, Australia) filled with
Krebs-Henseleit buffer (KHB composition in mM: 119 NaCl, 25
NaHCO
3
, 4.75 KCl, 1.2 KH
2
PO
4
, 0.6 MgSO
4
.7H
2
O, 0.6 Na
2
SO
4
,
1.25 CaCl
2
.H
2
O and 10 glucose) at 37°C and gassed with 95% O
2
and 5% CO
2
. The tension was slowly adjusted to 0.2 g and the
preparation was initially stabilised for a period of 40 minutes,
changing the buffer after every 10 minutes while gradually
increasing the tension to 1.5 g. After 40 minutes, the KHB was
changed and adjusted to exactly 25 ml.
Thereafter, 100 nM phenylephrine (2.5 µl of 1 mM stock)
was added to induce maximal contraction, followed by 10 µM
acetylcholine (25 µl of stock A) to induce at least 70% relaxation.
The organ bath was rinsed three times with pre-warmed KHB to
Table 1. High-performance liquid chromatography (HPLC)
analysis of the GRT extract used in the study
Compound (g compound/100 g soluble solids)
HPLC analysis
Phenylpyruvic acid-2-
O-
glucoside (PPAG)
0.423265
Aspalathin
12.78348
Nothofagin
1.974419
Isoorientin
1.427281
Orientin
1.255839
Ferulic acid
nq
Vitexin
0.338513
Isovitexin
0.298022
Quercetin-3-robinobioside
1.040565
Hyperoside
0.398773
Rutin
0.496034
Isoquercitin
0.572251852
Nq, not quantifiable.