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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 31, No 2, March/April 2020
82
AFRICA
A seven-week rooibos treatment protocol was shown to
protect against ischaemia/reperfusion injury in isolated perfused
rat hearts.
19
Furthermore, in a rat model of chronic rooibos
consumption, no adverse effects were found.
20
However, despite
these promising results, studies into the cardioprotective effects
of rooibos, both fermented and unfermented, remain limited,
with investigations into the effects of rooibos on the vascular
endothelium, in particular, lacking.
In addition to controlling circadian rhythms, the hormone
melatonin has been shown to be a versatile biological signalling
molecule,
21
involved in many physiological processes in humans
and animals, including blood pressure control and the scavenging
of free radicals.
22,23
In addition to the pineal gland, melatonin is
secreted from a variety of organs (regarded as non-endocrine
organs) and tissues, including the retina, Harder’s glands,
gastroenteric mucous membrane, megakaryocytes, platelets,
lymphocytes, bone marrow and the skin, but at lower and varying
rates.
24,25
Under experimental conditions, chronic melatonin
administration was demonstrated to be cardioprotective,
which can be attributed to its free-radical scavenging and
antioxidant properties.
26,27
Melatonin has also been suggested
to be atheroprotective and may slow the progression of
atherosclerotic development.
28
Melatonin has been shown to
act as a vasoconstrictor in the caudal artery and a vasorelaxant
in the mesenteric artery and aorta.
25
In addition, melatonin
treatment has not been associated with any toxic effects.
29
In view of the above, this study aimed to address a considerable
knowledge gap related to the putative beneficial effects of
rooibos on nicotine-induced vascular injury and oxidative stress.
It is of particular interest and importance to investigate whether
medicinal plants such as rooibos may protect the vascular
endothelium by countering the harmful effects of increased
ROS production associated with nicotine exposure and restoring
the release of NO. Melatonin was included in the study as it is
known to be a potent antioxidant and cardioprotective molecule,
hence, it served as a control for rooibos.
Methods
Ethics approval was received from Stellenbosch University;
project number SU-ACUM12-00041. Experiments were
conducted according 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).
A total of 90 adult male Wistar rats, weighing between
220 and 310 g at the start of the study, were housed in the
central animal facility of the Faculty of Medicine and Health
Sciences, Stellenbosch University. Animals were housed at room
temperature (23°C
±
2°C) under normal 12-hour light and
12-hour dark cycles with free access to rat chow and fluids, and
allowed to adapt to laboratory conditions for seven days prior
to the start of treatment. Animals were randomly assigned to
treatment groups of 10 rats per group in order to prevent bias,
and individually caged in order to monitor fluid intake. The
experimental rats were weighed daily.
Nicotine [(-)-nicotine, Sigma-Aldrich, St. Louis, MO, USA]
was dissolved in sterile 0.9% physiological saline and injected
subcutaneously. Physiological saline (0.9%) served as the vehicle
control for nicotine and was also injected subcutaneously.
Rooibos (2% fermented and unfermented) was a gift from the
Promec Unit of the South African Medical Research Council
and was prepared according to a standard laboratory protocol.
30
Rooibos solution served as the drinking fluid in the cages
housing the rats assigned to the rooibos experimental groups.
Melatonin (Sigma-Aldrich, St. Louis, MO, USA) was dissolved
in 1 ml absolute ethanol and then added to the drinking water
at a final concentration of 0.05% (v/v) ethanol with melatonin,
as previously described.
27
Fresh melatonin preparations were
supplied on a daily basis and rat fluid intake was monitored
daily to ensure that the correct concentration of melatonin
was received. The melatonin solution served as the drinking
fluid in the cages housing the rats assigned to the melatonin
experimental groups. See Table 1 for the treatment groups, as
well as their abbreviations, used in the remainder of the text.
At the end of the six-week treatment period, the rats
were fasted overnight and euthanised with an overdose of
sodium pentobarbital (160 mg/kg) by means of intra-peritoneal
injection. Blood was collected and allowed to clot on ice for 30
minutes, after which it was centrifuged at 1 200 g for 10 minutes
at 4°C and the serum was aspirated. Liver tissue was excised,
rinsed in saline solution, blotted dry and snap frozen in liquid
nitrogen. Serum and snap-frozen liver tissue were then stored
at –80°C for subsequent analysis. The aorta was excised and
immediately used for vascular contraction/relaxation studies.
Biochemical analysis of rooibos
The soluble solid content of the rooibos preparation was
determined gravimetrically (six repetitions each) after drying 1-ml
aliquots at 70°C for 24 hours, and these were subsequently placed
in a desiccator for 24 hours. Total polyphenol content and analysis
for known flavonoid compounds were determined by the ARC
Infruitec-Nietvoorbij, Post-Harvest Wine Technology Division,
Stellenbosch, South Africa. The total polyphenol content was
determined using the Folin-Ciocalteu’s phenol reagent, as
described by Arthur
et al
.
31
The absorbance was read at 765 nm
and expressed as mg gallic equivalents per mg soluble solids.
Analysis for known flavonoid compounds was determined
according to an established HPLC method.
32
Flavonol content
was spectrophotometrically determined using a Spectronic
®
20
GenesysTM photospectrometer (Spectronic Instruments, Leeds,
UK) at 360 nm, according to a standard protocol,
33
utilising
quercetin as standard. Both quercetin and rooibos were diluted
in 95% ethanol.
Table 1.Treatment groups and abbreviations
Group
Abbreviation
Control groups (
n
= 10/group)
Saline vehicle control
Veh control
Drinking control (tap water)
Water control
Melatonin 4 mg/kg bw/day
Mel
2% fermented rooibos
RF
2% unfermented rooibos
RUF
Treatment groups (
n
= 10/group)
Nicotine 5 mg/kg bw/day
Nicotine
Nicotine 5 mg/kg bw/day + melatonin 4 mg/kg bw/day
NMel
Nicotine 5 mg/kg bw/day + 2% fermented rooibos
NRF
Nicotine 5 mg/kg bw/day + 2% unfermented rooibos
NRUF
bw: body weight.