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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 32, No 2, March/April 2021
90
AFRICA
Following this, 5 µl of the sample and blank (assay buffer: 50
mM potassium phosphate, 1 mM EDTA, pH 7.0) were assayed
in triplicate, followed by the addition of 215 µl of the cocktail
solution to the wells. The absorbance of NADPH oxidation
in the absence of H
2
O
2
was measured for three minutes with
30-second intervals, at 340 nm. To initiate the reaction, 25 µl of
1.5 mM H
2
O
2
(3.4 µl of 30% stock solution in 20 ml assay buffer)
was added immediately after the first absorbance measurement.
The hydroperoxide-dependent linear NADPH oxidation was
recorded for two to five minutes at 30-second intervals at the
same wavelength. The GPx activity was expressed as µmol
NADPH oxidised/min/mg protein.
Thiobarbituric acid reactive substances
(TBARS) assay
Measurement of TBARS is a widely used assay for the
determination of lipid peroxidation in tissue homogenates
and serves as an indicator of oxidative stress. The assay was
performed according to the modified method of Esterbauer and
Cheeseman.
24
Liver tissue was homogenised in 0.01 mM sodium
phosphate buffer (pH 7.5) containing 1.15% KCl in a Bullet
blender
®
24 (Next Advance, NYC, USA) using 1.6-mm stainless
steel beads, at speed 8 for three minutes and speed 9 for four
minutes, with one-minute rest cycles in-between.
Protein concentrations of the samples were determined using
a BCA assay kit (Sigma Aldrich). The MDA standard solution
(500 µM) was prepared by diluting 1.23 µl of the MDA stock
(125 µM) solution in 10 ml of deiH
2
O and it was serially diluted
in MDA diluent (nmol/ml MDA: 0, 0.322, 0.625, 1.25, 2.5, 5,
10, 25 and 30). Samples and standards (100 µl) were added to
glass test tubes, followed by 100 µl of SDS solution (2%), 2
ml of trichloroacetic acid (TCA) reagent {composition: 10%
TCA, BHT [12.5 mM butylated hydroxytoluene (BHT)/10 ml
TCA solution]} and 2 ml thiobarbituric acid (TBA) (0.67%
w/v) solution on the side of each tube, and the glass tubes were
capped with marbles.
Test tubes were incubated in a water bath at 95°C for one hour
and cooled at room temperature for 10 minutes. Thereafter, the
samples were centrifuged at 3 000 rpm for 15 minutes and the
supernatant was plated (150 µl) in triplicate. The absorbance was
measured at 530 nm using the FLUOstar Omega
®
microplate
reader. TheMDA concentration in the liver was determined using
an MDA standard curve. Thereafter, the results were normalised
using the previously determined protein concentrations and
expressed in µmole MDA equivalents/mg protein.
Statistical analysis
All the results were analysed using GraphPad Prism
®
6. Statistical
analysis was performed using one- or two-way analysis of
variance (ANOVA), followed by the Bonferroni
post hoc
test for
comparison within the groups. The results are expressed as the
mean ± standard error of mean (SEM). A probability of
p
< 0.05
was considered significant.
Results
Food and water intake were measured three times weekly and
body weight was measured once a week during the 10-week
period (Table 2). The HFD group showed a significant increase
in food intake and a significant decrease in mean water intake
relative to the control group. Furthermore, the HFD group
showed a significant increase in mean body weight when
compared to the control group. Blood pressure was measured
from week eight to 10 and the HFD group showed a significant
increase in the mean systolic, diastolic and arterial blood
pressure when compared to the control group. The mean arterial
pressure (MAP) was calculated as follows:
MAP = mean DBP +
1
__
3
(mean SBP – mean DBP)
where DBP is diastolic blood pressure and SBP is systolic blood
pressure.
In week 10, quantitative blood glucose measurements were
obtained in both the control and HFD group after an overnight
fast. At baseline (Fig. 1A), the HFD group showed a significant
increase in blood glucose levels compared to the control (5.56 ±
0.220 vs 4.66 ± 0.113 mmol/l;
p
< 0.01,
n
= 7–8 per group). After
oral administration of the 50% sucrose solution, the HFD group
showed a significant increase in plasma glucose levels at three
(6.13 ± 0.219 vs 4.84 ± 0.341 mmol/l), five (6.59 ± 0.108 vs 5.06 ±
0.229 mmol/l), 10 (7.31 ± 0.437 vs 5.89 ± 0.245 mmol/l), 15 (7.63
± 0.359 vs 5.51 ± 0.362 mmol/l), 20 (7.44 ± 0.270 vs 6.40 ± 0.229
mmol/l) and 25 minutes (7.04 ± 0.248 vs 6.31 ± 0.212 mmol/l;
p
< 0.05,
n
= 7–8 per group), compared to the control group
(Fig. 1A). No significant differences were observed between the
control and HFD groups from 30 to 120 minutes.
Additionally, according to the area under the curve (AUC,
Fig. 1B) analysis, the HFD group presented with a significant
increase in blood glucose levels when compared to the control
group (716 ± 15.4 vs 635 ± 31.2 arbitrary units,
p
< 0.05,
n
=
7–8 per group). The untreated control and HFD animals showed
normal urinary glucose levels during the 16-week period (
n
= 8
per group, Table 2).
The results in Table 3 represent biometric and blood pressure
measurements during and after the 16-week period. The HFD
animals showed a significant increase in food intake and a
significant decrease in water intake compared to the control
animals. Furthermore, the HFD animals showed a significant
increase in leptin levels, and IP fat, liver and absolute body weight
when compared to the control animals. Additionally, the HFD
group showed a significant increase in the mean systolic, diastolic
and arterial blood pressure compared to the control group,
Table 2. Mean food and water intake per rat per day together with the
mean body weight, and systolic, diastolic and arterial blood pressure of
the HFD versus control groups before treatment with the GRT extract
Parameters
Control
HFD
Sample size
(
n
/group)
Mean food intake (g)
17.18 ± 0.272 20.58 ± 0.429**** 20–28
Mean water intake (ml)
20.48 ± 0.445 13.60 ± 0.229**** 20–28
Mean body weight (g)
246.00 ± 4.404 274.10 ± 4.886*** 20–28
Mean systolic blood pressure
(mmHg)
125.00 ± 1.720 139.00± 2.460****
10
Mean diastolic blood
pressure (mmHg)
84.48 ± 1.171 93.95 ± 1.226****
10
Mean arterial pressure (mmHg) 99.94 ± 1.480 111.70 ± 2.240****
10
Urinary glucose (mmol/l)
Normal
Normal
10
All data are expressed as mean ± SEM, ****
p
< 0.0001 HFD versus control,
***
p
< 0.001 HFD versus control,
n
= 20–28 per group, except urinary glucose,
mean systolic and diastolic blood pressure and mean arterial pressure,
n
= 10 per
group.