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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 30, No 2, March/April 2019
96
AFRICA
leaves are used in a popular beverage, commonly known as
‘rooibos tea’.
21
Rooibos does not contain caffeine or other
stimulants, and has a high concentration of potent antioxidants,
vitamins and minerals.
22
Its antioxidant-driven cardio-protective
properties are well established and substantiated by scientific
research.
20,23,24
These have, however, been poorly investigated
in the context of ART-induced oxidative stress, even though a
couple of studies have shown favourable effects of rooibos on
suppressing HIV-binding to MT-4 cells.
25,26
The aims of this
study, therefore, were to examine the effects of treatment with
the first-line FDC drug on blood lipid levels and oxidative
stress markers, myocardial ischaemic tolerance and vascular
endothelial function in male Wistar rats, and furthermore, to
determine whether co-treatment with an aqueous rooibos extract
exerts protective effects.
Methods
Approval for theuseof maleWistar rats for this studywas obtained
from the Research Ethics Committee for Animal Care and Use,
Stellenbosch University (Protocol #: SU-ACUD14-00021). Rats
were housed in the animal facility at Stellenbosch University.
They were handled according to the institutional ethical
guidelines and the Revised South African National Standard
for the Care and Use of Animals for Scientific Purposes (South
African Bureau of Standards, SANS 10386, 2008).
The animals had
ad libitum
access to standard rat chow and
fluids (water or rooibos). They were housed in groups of three
rats per cage under 12 hours light and 12 hours dark. The rats
were weighed and assessed for hair loss and other signs of stress
on a weekly basis.
The study made use of a total of 100 rats. They were
randomly assigned into four treatment groups: (1) control (25
rats): gavaged with tap water and received tap water to drink; (2)
rooibos (25 rats): gavaged with tap water and received rooibos to
drink; (3) antiretroviral therapy (ART) control (22 rats): gavaged
with ART and received tap water to drink; ART + rooibos (24
rats): gavaged with ART and received rooibos to drink.
A total of 65 rats were used for isolated heart perfusion
studies and aortic ring isometric tension studies. Of these, 34
hearts were used to determine functional recovery by inducing
global ischaemia and 31 hearts to determine infarct size through
the induction of regional ischaemia. Twenty-two rats were fasted
overnight and blood serum was collected from the thoracic cavity
after sacrifice. A total of four rats were lost to gavage during the
treatment period. The total treatment period was nine weeks of
ART, rooibos or combination treatment.
ART drug preparation and treatment
Each FDC tablet [Odimune, Cipla MedPro (Pty) Ltd, Bellville,
Western Cape, SA], containing the daily dose of active ingredients
for an average human weighing 70 kg, was crushed and the
human:rat dosage conversion was calculated at six-fold the
human dosage, according to previously published guidelines.
27
The dose per rat was calculated weekly according to the average
total body mass of the rats per cage (human daily dose: 600 mg
EFV, 200 mg FTC and 300 mg TDF). The calculated dose of
crushed powder was suspended in 1 ml tap water/rat/day and
vortexed thoroughly. It was administered to the rats daily via oral
gavage by a qualified laboratory animal husbandry professional.
Vehicle control rats were gavaged with an equal volume of
ART-free tap water.
Rooibos preparation and treatment
The rooibos leaves were mixed with freshly boiled tap water, left
to steep for half an hour and then filtered to yield an aqueous
extract of a final concentration of 2% (w/v), according to a
previously published method.
28
The rooibos mixture was placed
in light-resistant drinking bottles in the animal cages and
substituted for the drinking water. The volume of the rooibos
solution remaining in the bottles was measured weekly to
calculate the amount of fluid consumed, and replenished with
a fresh batch weekly (800–1 000 ml/week). The same was done
with the normal drinking water of the control and ART groups.
Liquid chromatography mass spectrometry analysis was
conducted on the rooibos infusions after applying different
storage methods (fresh, fridge for one week or frozen at –20°C
for more than one week). It showed no differences in polyphenol
levels and the composition for all was comparable to that of
rooibos infusions used in previously published data.
29
The rats
were treated from four weeks of age for nine weeks, according to
previously published rooibos treatment protocols in rats.
20
Rat euthanasia, organ harvesting and blood collection
Rats were euthanised via an intraperitoneal injection of
sodium pentobarbitone (160 mg/kg), conducted by a researcher
authorised by the South African Veterinary Council (SAVC).
Upon disappearance of the pedal reflex, a thoracotomy was
performed to quickly remove the heart. The heart and thoracic
aorta were carefully excised and placed in ice cold Krebs-
Henseleit buffer (KHB) containing, in mM: NaCl 119; NaHCO
3
24.9; KCl 4.74; KH
2
PO
4
1.19; MgSO
4
0.6; NaSO
4
0.59; CaCl
2
1.25; glucose 10. After excision of the heart, blood was collected
from the chest cavity and placed in blood collection tubes on ice.
Blood samples were centrifuged at 2 000 ×
g
for 15 minutes
at 4°C, after which the serum and/ or plasma was removed and
stored at –80°C. Serum and plasma were subsequently analysed
at the Division of Chemical Pathology, University of Cape Town,
for triglyceride (TG), phospholipid (PL) and thiobarbituric acid-
reactive substance (TBARS) levels.
Lipid and TBARS analyses
Serum TG and PL levels were determined using commercially
available enzymatic colorimetric kits [Wako LabAssay
TM
TG
(290-63701) and PL (296-63801), Wako Chemicals, GmbH,
Germany] and read on a microplate reader (Spectra-Max Plus
384 with SoftMax Pro 4.8 data-acquisition and analysis software:
Separations, Cape Town, SA). In principle, performing the assay
involved pipetting the triglyceride standard and samples into
a microtitre plate (Greiner: Merck, South Africa), adding an
enzymatic colour reagent to each well and incubating at room
temperature for 30 minutes before reading the absorbance at
600 nm.
The concentrations of TBARS were determined
spectrophotometrically according to the method of Jentzsch
et al.
,
30
and calculated using the appropriate molar extinction