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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 31, No 2, March/April 2020
AFRICA
83
Flavanol content was determined at 640 nm using a
Spectronic
®
20 GenesysTM photospectrometer (Spectronic
Instruments, Leeds, UK) according to a standard protocol,
33
using the 4-(dimethylamino)-cinnamaldehyde (DAC) reaction.
DAC and rooibos were dissolved in HCl-MeOH (1:3). Catechin
was dissolved in methanol to prepare a 0.05% solution and
this served as standard for the flavanol determinations. For
both flavonol and flavanol determinations, the optimal dilution
factor of rooibos was determined and subsequent analysis was
performed in triplicate.
33
Ex vivo
investigations: aortic ring isometric tension
studies
The thoracic aorta was excised and immediately placed in
ice-cold Krebs Henseleit buffer (KHB, composition 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
.H
2
O 1.25 and glucose 10). All connective
tissue and perivascular fat were removed and the aorta was
cut into 3–4-mm segments and mounted in a 25-ml organ
bath containing oxygenated (95% O
2
and 5% CO
2
) KHB and
maintained at 37°C. The rings were equilibrated for 30 minutes
under a resting tension of 1.5 g. The tension (in grams of
tension) of the aortic ring was recorded with an isometric force
transducer (TRI202PAD, Panlab, ICornellà, BCN, Spain) and
the data were analysed with LabChart 7 software (Dunedin,
New Zealand).
Following the initial equilibration period, aortic rings were
exposed to a first round of contraction (100 nM phenylephrine;
Phe) (Sigma-Aldrich, St Louis, MO, USA) and relaxation (10
μ
M acetylcholine; ACh) (Sigma-Aldrich, St Louis, MO, USA)
in order to establish the functionality of the endothelium.
Following wash-out of the Phe and ACh, the aortic rings were
equilibrated for a further 30 minutes.
The contractile response of the aortic rings was determined at
cumulative concentrations of Phe (100 nM – 1
μ
M). After each
addition of Phe, a plateau response was reached before the addition
of the next dose. At the end of the plateau phase of the final Phe
concentration (1
μ
M), the rings were subjected to cumulative
concentrations of ACh (30 nM – 10
μ
M) to induce relaxation.
The final concentration of ACh resulted in maximum percentage
relaxation of contraction and was the endpoint of the experiment.
The relaxation responses to ACh were expressed as a percentage
of the contraction caused by the final Phe concentration (1
μ
M).
Antioxidant enzyme activity
The activities of superoxide dismutase (SOD) and catalase
(CAT) were determined in liver tissue. Liver tissue homogenates
were prepared in phosphate buffer-containing microcentrifuge
tubes using the Bullet Blender 24 and 0.5-mm zirconium oxide
beads (Next Advance, NY, USA). The supernatant was collected
after centrifugation at 12 000 rpm for 20 minutes and aliquots
were stored at –80°C until the day of analysis.
SOD activity was determined using a commercially available
superoxide dismutase assay kit (Cayman Chemical Company,
Ann Arbor, MI, USA), which measured total SOD (Cu/Zn and
Mn) of mammalian tissue. One unit (U) of SOD was defined
as the amount of enzyme needed to exhibit 50% dismutation
of the superoxide free radical. Tetrazolium salt was used for the
detection of superoxide radicals, and bovine erythrocyte SOD
(Cu/Zn) served as standard.
The protocol by Ellerby and Bredesen was adapted for use in
a 96-well plate to determine CAT activity.
34
In a 96-well clear UV
plate, 5
μ
l diluted sample and 170
μ
l buffer (50 mM potassium
phosphate, pH 7.0) were added, where after 0.1% H
2
O
2
was
added to initiate the reaction. The linear decline in absorbance
was monitored every 30 seconds at 240 nm for five minutes
in a FLUOstar Omega Microplate Reader (BMG Labtech,
Offenburg, Germany). CAT activity (
μ
mole/min/
μ
g protein) was
determined using the molar extinction coefficient of 43.6/M/cm.
Lipid peroxidation
Thiobarbituric acid reactive substances (TBARS) were measured
by spectrophotometric methods using a Labsystems multiscan
MS analyser (AEC Amersham Co, South Africa) according
to a method described previously.
34
Serum samples (200
μ
l)
were mixed with 10
μ
l butylated hydroxytoluene (BHT) (Fluka
Chemie, Switzerland) in ethanol (85%) (Merck Chemicals, South
Africa) and orthophosphoric acid (15 mol/l) (Sigma-Aldrich, St
Louis, MO, USA) buffer at pH 3.6 and vortexed. Thiobarbituric
acid (TBA) (Sigma-Aldrich, St Louis, MO, USA) reagent (25
μ
l)
was added and vortexed again. After incubation at 90°C for 45
minutes in a water bath, the reaction was terminated by placing
the tubes on ice.
TBARS were extracted with n-butanol, saturated NaCl (50
μ
l) was added and the mixture was centrifuged at 12 000 rpm
for one minute. Absorbance was read at 532 nm and values were
expressed in
μ
mol/l of serum.
Supplementary
in vitro
investigations
Adult rat aortic endothelial cell
(AEC) cultures were purchased
from VEC Technologies (Rensselear, New York, USA)
and received in culture. Cell cultures were maintained in a
standard tissue culture incubator (Forma Series II, Thermo
Electron Corporation, Waltham, MA, USA) at an atmospheric
composition of 21% O
2
, 5% CO
2
, 40–60% humidity, and
temperature was maintained at 37°C. The endothelial cell
growth medium (EGM-2, Clonetics, Cambrex Bio Science,
Walkersville, USA) was supplemented with 10% FBS (Highveld
Biological, Lyndhurst, South Africa) and standard endothelial
growth factors [vascular endothelial growth factor (VEGF),
human epidermal growth factor (hEGF), long-chain human
insulin-like growth factor (R3-IGF-1), human fibroblastic
growth factor (hFGF), hydrocortisone, antibiotics (gentamicin
and amphotericin B) and ascorbic acid] according to the
manufacturer’s instructions. Cells were grown to confluency, as
determined by microscopic evaluation and passaging to the next
generation was performed in a 1:2 ratio.
Cells grown to confluency were exposed to 100
μ
M for 24
hours. Nicotine was diluted with phosphate-buffered saline
(PBS). RF was freeze dried in a FreeZone6 (Labconco, Kansas
City, MO, USA) freeze drier to remove the aqueous fraction.
Freeze-dried RF was made up to a 20 mg/ml stock solution in
cell culture medium and further diluted in cell culture medium
to a concentration of 0.015 mg/ml. Cells were co-treated with
nicotine and RF. In all cases, cells were examined for NO
production and necrosis.