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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 31, No 2, March/April 2020
84
AFRICA
Flow cytometric analysis: NO production was measured
by 4,5-diaminofluorescin-2 diacetate (DAF-2/DA) fluorescence
(Calbiochem, San Diego, CA, USA) according to a
previously established protocol.
36,37
Diethylamine NONOate
diethylammonium salt (DEA/NO) served as positive control.
Propidium iodide (PI, Sigma-Aldrich, St Louis, MO, USA) was
used to determine necrosis,
38
and osmotic stress-induced cell
injury served as a positive control.
Statistical analysis
All data are expressed as mean
±
standard error of the mean
(SEM). When comparisons between two groups were made,
an unpaired
t
-test was performed. For multiple comparisons,
the ANOVA (two-way where appropriate), followed by the
Bonferroni correction, was applied. A
p
-value
<
0.05 was
considered significant. All data were analysed using GraphPad
Prism
®
5 software (GraphPad Software, San Diego, CA, USA).
All aortic ring isometric tension data are expressed as the
percentage contraction from a resting tension of 1.5 g or
percentage relaxation of maximum contraction, respectively. For
in vitro
investigations, controls were adjusted to 100% and values
are expressed as a percentage of the controls.
Results
Biochemical analysis of rooibos
RUF had a significantly higher soluble solid content and total
polyphenolic content compared to RF, while the daily total
phenolic intake of the RUF treatment groups (2% RUF, and
2% RUF and 5 mg/kg bw/day nicotine co-treatment) was also
significantly higher than that of the RF treatment groups (2%
RF, and 2% RF and 5 mg/kg bw/day nicotine co-treatment)
(Table 2).
RF had a significantly higher flavonol content than RUF.
The daily flavonol intake of the RF treatment groups was also
significantly higher than that of the RUF treatment groups
(Table 2), while RUF had a significantly higher flavanol content
than RF. The daily flavanol intake of the RUF treatment groups
was significantly higher than that of the RF treatment groups
(Table 2). Values of known flavonoid compounds, as determined
by HPLC analysis, are given in Table 3.
Ex vivo
investigations: aortic ring isometric tension
studies
The vascular function of all treatment groups was assessed by
means of aortic ring isometric tension studies. The experimental
protocol consisted of cumulative additions of Phe and ACh
to test the functionality of the endothelium. Aortic rings from
the nicotine-treated rats showed a significant pro-contractile
response to Phe administration when compared to the saline
vehicle control (Fig. 1A), with E
max
values of 131.3
±
17.33%
(nicotine) vs 102.9
±
4.99% (vehicle control), but Phe had
no significant effect on relaxation (Fig. 1B). Aortic rings
from Mel-treated rats (E
max
value of 78.06
±
7.39%) showed a
significant anti-contractile response to Phe administration when
compared to the water control, RF and RUF treatment groups
(Fig. 2A) (E
max
values of 110.9
±
10.64, 112.9
±
9.67 and 108.3
±
8.11%, respectively). Aortic rings from Mel, RF and RUF
treatment rats (E
max
values of 86.62
±
4.5, 70.84
±
6.91 and
79.94
±
7.01%, respectively) showed a significant pro-relaxation
response to ACh administration when compared to the water
control group (E
max
value of 63.28
±
4.03%) (Fig. 2B).
Aortic rings from NMel, NRF and NRUF treatment rats
(E
max
values of 84.64
±
6.67, 109.2
±
9.87 and 110.2
±
6.29%,
respectively) showed a significant anti-contractile response to
Phe administration when compared to the nicotine-treated group
(E
max
value of 131.3
±
17.33%). Additionally, aortic rings from
NMel-treated rats also showed a significant anti-contractile
response to Phe administration when compared to the NRF-
and NRUF-treated groups (Fig. 3A). Aortic rings from NMel-
and NRF-treated rats (E
max
values of 93.11
±
3.28 and 89.60
±
5.96%, respectively) showed a significant pro-relaxation response
Table 2. Soluble solid, total polyphenolic, flavonol and flavanol content of 2% fermented and 2% unfermented rooibos
Variables
2% RF
2% RUF
NRF
NRUF
Soluble solids (mg/ml)
3.50
±
0.22
4.60
±
0.40
#
Total phenolic content (mg gallic acid/mg soluble solids)
0.16
±
0.01
0.23
±
0.03
#
Daily total phenolic intake (mg gallic acid equivalents/day/100 g bw)
5.17
±
0.28
9.43
±
0.46
#
4.86
±
0.31
8.07
±
0.26
#
Flavonol content (mg quercetin equivalents/mg soluble solids)
0.36
±
0.02
@
0.18
±
0.02
Daily flavonol intake (mg quercetin equivalents/ day/100 g bw)
1.11
±
0.06
@
0.74
±
0.04
1.04
±
0.07
@
0.63
±
0.02
Flavanol content (mg catechin equivalents/mg soluble solids)
0.05
±
0.00
0.10
±
0.01
#
Daily flavanol intake (mg catechin equivalents/ day/100 g bw)
0.10
±
0.01
0.37
±
0.02
#
0.09
±
0.01
0.32
±
0.01
#
bw: body weight; NRF: nicotine 5 mg/kg bw/day + 2% RF co-treatment; NRUF: nicotine 5 mg/kg bw/day + 2% RUF co-treatment.
#
p
<
0.05 vs 2% RF treatment groups;
@
p
<
0.05 vs 2% RUF treatment groups;
n
= 5–6.
Table 3. HPLC quantification of flavonoids in 2% fermented
and 2% unfermented rooibos consumed by rats
2% fermented rooibos
2% unfermented rooibos
Flavonoid compounds
% of soluble
solids
Daily intake
(mg/100 g
bw)
% of soluble
solids
Daily intake
(mg/100 g
bw)
Phenylpyruvic
acid-2-O-glucoside
(PPAG)
0.391
±
0.03 0.124
±
0.01 0.361
±
0.04 0.148
±
0.01
Aspalathin
0.221
±
0.01 0.070
±
0.01 8.907
±
1.05 3.645
±
0.18
Nothofagin
0.051
±
0.01 0.016
±
0.00 1.311
±
0.15 0.537
±
0.03
Isoorientin
0.933
±
0.06 0.295
±
0.02 1.478
±
0.17 0.605
±
0.03
Orientin
0.842
±
0.05 0.266
±
0.01 1.132
±
0.13 0.463
±
0.02
Ferulic acid
0.055
±
0.01 0.017
±
0.00
not detected
Quercetin-3-
robinobioside
0.562
±
0.04 0.178
±
0.01 0.395
±
0.05 0.162
±
0.01
Vitexin
0.165
±
0.01 0.052
±
0.01 0.168
±
0.02 0.069
±
0.01
Hyperoside
0.156
±
0.01 0.050
±
0.01 0.066
±
0.01 0.027
±
0.01
Rutin
0.047
±
0.01 0.015
±
0.00 0.313
±
0.04 0.128
±
0.01
Isovitexin
0.168
±
0.01 0.053
±
0.01 0.224
±
0.03 0.091
±
0.01
Isoquercitrin
0.106
±
0.01 0.034
±
0.01 0.099
±
0.01 0.041
±
0.01
Luteolin-7-glucoside 0.024
±
0.01 0.008
±
0.00 0.031
±
0.01 0.013
±
0.00
bw: body weight;
n
= 5–6.