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Stroke is the second leading cause of death worldwide, affecting about 240 people a day in South Africa and leaving survivors with residual disabilities. At the moment, there is no clinically approved neuroprotective product for stroke but the consumption of plant polyphenols has been suggested to offer some protection against stroke. In this study, we investigated the effects of long-term consumption of fermented rooibos herbal tea (FRHT) on ischemia/reperfusion (I/R)-induced brain injury in adult male Wistar rats. FRHT was administered to the animals ad libitum for 7 weeks prior to the induction of ischemic injury via a 20-minute bilateral occlusion of the common carotid arteries (BCCAO) followed by reperfusion for 24, 96 and 168 hours respectively. Neurobehavioural deficits, brain oedema, blood-brain barrier (BBB) damage, apoptosis, lipid peroxidation and total antioxidant capacity were subsequently evaluated using standard methods. Our results showed that long-term consumption of FRHT by Wistar rats significantly reduced brain oedema and neuronal apoptosis, but did not attenuate BBB damage following cerebral ischemia. Analysis of whole-brain homogenates showed significantly reduced lipid peroxidation levels, increased total antioxidant capacity and resulted in improved neurobehavioural outcomes in FRHT-treated rats when compared with untreated animals. Taken together, our results tend to suggest that continuous consumption of FRHT could confer some protection against ischemic brain injury (IBI) and is therefore highly recommended for patients with stroke-predisposing conditions. © 2017, Nencki Institute of Experimental Biology. All rights reserved.
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© 2017 by Acta Neurobiologiae Experimentalis
Long‑term consumption of fermented rooibos
herbal tea oers neuroprotection against
ischemic brain injury in rats
Olusiji Akinrinmade, Sylvester Omoruyi, Daneel Dietrich, and Okobi Ekpo*
Department of Medical Biosciences, University of the Western Cape, Bellville, South Africa,
* Email: oekpo@uwc.ac.za
Stroke is the second leading cause of death worldwide, affecting about 240 people aday in South Africa and leaving survivors
with residual disabilities. At the moment, there is no clinically approved neuroprotective product for stroke but the consumption
of plant polyphenols has been suggested to offer some protection against stroke. In this study, we investigated the effects
of long‑term consumption of fermented rooibos herbal tea (FRHT) on ischemia/reperfusion (I/R)‑induced brain injury in adult
male Wistar rats. FRHT was administered to the animals ad libitum for 7weeks prior to the induction of ischemic injury via
a20‑minute bilateral occlusion of the common carotid arteries (BCCAO) followed by reperfusion for 24, 96 and 168hours
respectively. Neurobehavioural deficits, brain oedema, blood‑brain barrier (BBB) damage, apoptosis, lipid peroxidation
and total antioxidant capacity were subsequently evaluated using standard methods. Our results showed that long‑term
consumption of FRHT by Wistar rats significantly reduced brain oedema and neuronal apoptosis, but did not attenuate BBB
damage following cerebral ischemia. Analysis of whole‑brain homogenates showed significantly reduced lipid peroxidation
levels, increased total antioxidant capacity and resulted in improved neurobehavioural outcomes in FRHT‑treated rats
when compared with untreated animals. Taken together, our results tend to suggest that continuous consumption of FRHT
could confer some protection against ischemic brain injury (IBI) and is therefore highly recommended for patients with
stroke‑predisposing conditions.
Key words: apoptosis, brain oedema, cerebral ischemia, fermented rooibos herbal tea, lipid peroxidation, total antioxidant
capacity
INTRODUCTION
Recent statistics from the Heart and Stroke
Foundation, South Africa (HSFSA) show that about 130
heart attacks and 240 stroke episodes occur daily in
South Africa, which implies that about 10 people will
suffer a stroke and 5 people will suffer a heart attack
every passing hour (HSFSA 2013). The burden of stroke
does not only lie in its high mortality but also in its high
morbidity rate as up to 50% of stroke survivors often
become chronically disabled (Wilkinson et al. 1997).
Although most of the medications approved for the
treatment of stroke worldwide (e.g. alteplase (rt‑PA),
reteplase, tenecteplase, anistreplase, streptokinase
and urokinase) are known to restore blood flow
(Duggal and Harger 2011), some have been found to
be neurotoxic, to disrupt neurovascular matrix and to
increase the risk of intracerebral hemorrhage (ICH)
(Wang et al. 2003). Even compounds that showed
promising neuroprotective activity in experimental
brain ischemia models have failed to successfully
translate in clinical human trials (Ziemka‑Nalecz and
Zalewska 2014), with the result that there is hitherto
no clinically effective neuroprotective drug licensed
for stroke (Macrae 2011).
Oxidative stress is considered a major contributing
factor in cerebral I/R‑induced injury (Liu 2015).
A stroke usually produces oxidative stress leading to
production of reactive oxygen species (ROS) and other
free radicals, mainly due to low‑oxygen inhibition of
cellular respiration, inflammation and excitotoxicity
(Pradeep et al. 2012). Free radicals are known to
attack important intracellular and extracellular
macromolecules in the body such as DNA, RNA,
carbohydrate moieties, unsaturated lipids, proteins and
micronutrients, leading to cell damage and homeostatic
disruption (Lobo et al. 2010). To protect against free
radical damage, the body has a defense system of
Correspondence should be addressed to O. Ekpo
Email: oekpo@uwc.ac.za
Received 19 February 2016, accepted 16 March 2017
Research paper
Acta Neurobiol Exp 2017, 77: 94–105
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Rooibos tea protects rat brains from ischemic injury 95Acta Neurobiol Exp 2017, 77: 94–105
endogenous antioxidants which can be boosted with
intake of antioxidant‑rich foods and supplements.
There is increasing scientific interest in the
potential health benefits and possible neuroprotective
effects of antioxidant‑rich food substances and
beverages in literature. Some studies have focused on
the effects of these substances when consumed prior to
the onset of an IBI. Rooibos tea, a very popular beverage
in South Africa, is known to contain such polyphenol
antioxidants as the monomeric flavonoids aspalathin,
chrysoeriol, isoorientin, isoquercitrin, isovitexin,
luteolin, nothofagin, orientin, quercetin, rutin and
vitexin (Erickson 2003). It has since gained increasing
popularity among international consumers due to
its many acclaimed health benefits (Mahomoodally
2013). Another study has shown that rooibos tea
could prevent DNA damage and inflammation via
its anti‑oxidative activity (Baba et al. 2009). A study
by Inanami and others (1995) found that rooibos tea
could protect against age‑related changes in the brains
of rats compared to the controls, mainly due to its
ability to prevent the age‑related accumulation of lipid
peroxides in the brain.
Recent animal studies have shown that rooibos
tea has potent antioxidant, antimutagenic,
immune‑modulating and chemopreventive effects (Van
der Merwe et al. 2006, Ichiyama et al. 2007, Marnewick
et al. 2011). Another study showed that high intake
of rooibos tea resulted in significant reductions in
lipid peroxidation, low density lipoprotein (LDL)
cholesterol, triglycerides and an increase in high
density lipoprotein (HDL) cholesterol levels, suggesting
that rooibos tea could lower the risks of cardiovascular
and degenerative diseases (Marnewick et al. 2011).
Previous studies have reported the cardioprotective
(Dludla et al. 2014) as well as hepatoprotective (Ulicná
et al. 2003) properties of FRHT following experimental
injury and ischemia. However, information on the
neuroprotective effects of FRHT is scanty in literature,
but maternal consumption of pomegranate juice
was reported to be neuroprotective for the neonatal
brain (Loren et al. 2005), probably through the effects
of its bioactive compounds. Rooibos tea has a rare
source of the dietary dihydrochalcones, aspalathin
and nothofagin (McKay and Blumberg 2007) which
could cross the BBB like other flavonoid compounds
(Youdim et al. 2003, Rashid et al. 2014) to possibly exert
neuroprotective effects against IBI.
The present study therefore aims to determine
the neuroprotective effects of FRHT on brain oedema,
apoptosis, neurologic deficits, BBB damage, lipid
peroxidation, and total antioxidant capacity following
IBI induced by bilateral common carotid artery
occlusion (BCCAO).
EXPERIMENTAL PROCEDURE
Ethical considerations
Ethical guidelines as specied by the Animal Research
Ethics Committee (AREC) of the University of the Western
Cape, Bellville, South Africa were followed, with assigned
ethics and project registration numbers: 13/10/94 and
ScR1Rc2013/07/18 respectively.
Animals
Fifty (50) healthy male Wistar rats with an average
weight of 250 g were procured from the University of
Stellenbosch animal facility in Cape Town, South Africa
and maintained at the Animal House of the University of
the Western Cape, Bellville, South Africa, under standard
laboratory conditions of temperature (25±2°C), humidity
(50±15%) and 12 hours light‑dark cycle. Animals were
acclimatized for two weeks and fed on standard rat chow
and tap water ad libitum and received humane care in
accordance with National Institutes of Health (NIH) Guide
for Care and Use of Laboratory Animals.
Experimental design and grouping
Animals were randomly separated into four groups –
Control‑sham (C‑SHAM) group (15 rats), Rooibos‑sham
(R‑SHAM) group (5 rats), Rooibos+ischemia (R+I) group
(15 rats) and the ISCHEMIA group (15 rats). All animals
in the respective groups were used for the assessment
of brain oedema, BBB integrity, histological and
immunohistochemistry (IHC) assessment of apoptosis
and neurochemical studies (NS) of lipid peroxidation,
oxygen radical antioxidant capacity assay (ORAC) and
ferric reducing antioxidant power (FRAP). All rats in
the C‑SHAM and ISCHEMIA groups received food and
tap water ad libitum throughout the study while rats
in the R‑SHAM and R+I groups had daily access to food
and FRHT ad libitum for 7 weeks prior to BCCAO or
sham surgery and were sacrificed at the end of 24‑hour,
96‑hour and 168‑hourpost‑BCCAO periods respectively,
depending on the experimental protocol.
Rooibos tea preparation
Aqueous extracts of FRHT (generous gift
from Rooibos Ltd; Clanwilliam, South Africa) at
a concentration of 2 g/100 ml FRHT was used
throughout this study as this concentration has
been reported to be routine for tea‑making purposes
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96 O. Akinrinmade et al. Acta Neurobiol Exp 2017, 77: 94–105
(Marnewick et al. 2003, Pantsi et al. 2011). Fresh
preparations were made every day and administered
to the experimental rats ad libitum (Opuwari and
Monsees 2014).
Bilateral common carotid artery occlusion
(BCCAO) surgery
Animal weights were recorded prior to a 20‑minute
BCCAO with a 4‑0 silk suture as previously reported
(Speetzen et al. 2013). Isourane was used for anesthesia
and surgery occurred in an aseptic environment. The
animals were maintained under Isourane anesthesia
throughout the occlusion period until reperfusion. The
analgesic – Meloxicam was administered after surgery
to prevent post‑surgical pain and distress. Rectal
temperature of the animals was monitored during and
after surgery and maintained at about 37°C by a heating
pad and Infrared Lamp in the recovery cage. In the
sham groups (C‑SHAM and R‑SHAM), the same surgical
procedures were followed but BCCAO was not done.
Physiologic parameters
As previously described by Ord and others (2012) and
while under anesthesia, physiological parameters were
measured prior to, during and after the occlusion of
the right and left common carotid arteries. Systolic and
diastolic blood pressure and heart rate were measured by
the CODA non‑invasive tail cu blood pressure monitor
(Kent Scientic, USA), which uses the volume pressure
technology. Values from each animal were determined
from the means of a minimum of 3 separate blood
pressure and heart rate measurements. Temperature
was monitored using a rectal thermometer and was
maintained at about 37°C using a heating pad and infrared
heating lamp.
Brain oedema assessment
Twenty four hours after reperfusion, rats were
sacriced under deep anesthesia (Sodium pentobarbital,
150 mg/bw i.p) and decapitated. The brains were
harvested and the cerebellum, pons, and olfactory bulbs
removed and weighed immediately (wet weight (WW)).
Brain sections were then placed in an oven (Memmert,
Germany), dehydrated at 105°C for 48 hours and reweighed
(dry weight (DW)). Brain oedema was estimated as the
dierence in percentage of brain water and calculated
with the formula below (Bigdeli et al. 2007).
Brain water content (BWC)=[(WW−DW)/WW]×100.
Blood‑brain barrier assessment
Extravasation of plasma contents into the brain tissue
occurs when there is disruption of BBB integrity (Klohs
et al. 2009). In this study, BBB integrity was evaluated as
Evans Blue (EB) (Sigma Aldrich, USA) extravasation into
the brain parenchyma as previously described (Bigdeli et
al. 2007). Briey, 4 ml/kg of 2% EB solution in PBS was
injected into tail vein of each rat an hour before sacrice.
The brains were weighed, homogenized in 1:10 w/v PBS
and an equal volume of 60% trichloroacetic acid (TCA)
(Sigma Aldrich, USA) before centrifugation at 1000×g for
30 minutes at 4°C. Readings were then taken at 610 nm
using a POLAR star omega spectrophotometer (BMG
Labtech, Ortenberg, Germany) and a standard curve was
used to determine EB levels in each brain sample.
Histological and immunohistological studies
Rats were sacrificed 24 hours, 4 days and 7 days
after BCCAO with an overdose of Sodium pentobarbital
injection (150 mg/bw i.p). The thoracic cavity was opened
and the rat perfused transcardially with a 300 ml of cold
Phosphate buffered saline (PBS). A pale color of the liver
was indicative of a successful perfusion after which, the
rats were decapitated and the brains removed, weighed
and bisected along the mid‑sagittal plane. The right
hemisphere was fixed in 4% paraformaldehyde in 0.1 M
phosphate buffer (pH 7.4) for 24 hours for histological
and immunohistochemical analysis. The left hemisphere
was also quickly immersed in 1:10 w/v 0.1 M cold PBS
solution and stored at −80°C to be used for biochemical
analyses (Zhang et al. 2012). After fixation of tissues,
brains were processed in a Leica‑2125 automatic tissue
processor (Leica, Germany) to prepare specimens for
sectioning, hematoxylin and eosin (H & E) as well
as cresyl violet staining. A Terminal dUTP Nick‑End
Labeling (TUNEL) assay was also conducted using the in
situ DNA Fragmentation Assay Kit (BioVision, U.S.A.) for
assessment of apoptosis following the manufacturer’s
instruction.
Morphologic and morphometric studies
Morphological and morphometric analysis was
carried out on the H & E, cresyl violet and TUNEL‑stained
cornus ammonis 1 (CA1) region of the hippocampus on
images captured at a magnication of ×400 using the
Zeiss Primo Vert microscope (Zeiss, Germany). In order to
cover the area of interest at ×400, three captured images
were used with each measuring 1159.4×869.57 µm. Viable
pyramidal neurons of the hippocampus were identied
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Rooibos tea protects rat brains from ischemic injury 97Acta Neurobiol Exp 2017, 77: 94–105
as those exhibiting clear purple cytoplasmic staining
with visible nuclei and nucleoli, while ischemic (dead)
cells were identied as showing shrunken perikarya,
triangular shapes and mostly exhibiting dark‑stained
nuclei. The data obtained was expressed as percentages
of viable or TUNEL‑positive cells in total number of cells.
Quantication of viable and TUNEL positive cells was
done using the NIH Image analysis software (Image J)
(Onken et al. 2012).
Neurochemical assays
Homogenization of tissues
Brain tissues stored at −80°C were thawed and
homogenized in 10 times (w/v) 0.1M PBS (pH 7.4) in
a Teon glass homogenizer for two periods of 10 seconds
each. The homogenate was then centrifuged at 15,000 rpm
in a microcentrifuge at 4°C for 10 minutes. The supernatant
was collected and transferred into newly marked Eppendorf
tubes for further analysis using dierent biochemical
assays (Ahmed et al. 2014).
Lipid peroxidation assay
Malondialdehyde (MDA) is produced as a by‑product
of the reaction of superoxide (O2‑) and hydroxyl (OH)
radicals with unsaturated lipid (Ozkul et al. 2007). MDA
levels in tissues could indicate the severity of lipid
peroxidation (LPO) (Serteser et al. 2002). In this study,
LPO assessment was done as previously described by
Wills (1966). Briefly, 100 μl of supernatant was mixed with
12.50 μl of cold ethanol, 100 μl of 0.2 M ortho‑phosphoric
acid and 12.50 μl of 0.67% TBA (Sigma Aldrich, USA). The
reaction mixture was then heated at 90°C for 45 minutes,
cooled for 2 minutes before 1000 μl of n‑butanol and
100 μl of saturated sodium chloride (NaCl) was added.
The mixture was centrifuged at 12,000 rpm at 4°C for
2 minutes before spectrophotometric reading at 532 nm.
The results were expressed as µmol of MDA per g of wet
brain tissue.
Oxygen radical absorbance capacity (ORAC)
The ORAC assay is one of the most accepted methods
for measuring the activity and amount of antioxidants
present in biological samples (Cao et al. 1998). Briefly,
2,2’‑azobis‑2‑methyl‑propanimidamide, dihydrochloride
(AAPH) and fluorescein (Sigma Aldrich, USA) were used
as the free radical producing system and fluorescent
molecule respectively. The oxidation of fluorescein was
measured by initiating a reaction following the addition
of 50 µl of AAPH to a mixture of 138 µl of fluorescein
and 12 µl of sample in a 96‑well black plate and the
fluorescence read for 2 hours at every 5 minutes interval
at an emission and excitation wavelength of 530 nm and
485 nm using the Fluoroskan Ascent fluorescent plate
reader (Thermo Fisher Scientific, Waltham, MA, USA).
A standard curve was prepared from a 500 µM stock
solution of Trolox, an artificial Vitamin E (Prior et al.
2003). The results obtained were expressed as µM Trolox
equivalent (TE)/g of wet brain tissue.
Ferric reducing antioxidant power (FRAP) assay
The FRAP assay uses an oxidation/reduction
reaction to measure the ability of antioxidants in
a sample to reduce ferric tripyridyltriazine (Fe3+
TPTZ) to a ferrous form (Fe2+) which has an intense blue
color and can be quantified spectrophotometrically
(Ndhlala et al. 2010). In this study, a mixture of 30 ml
acetate buffer (300 mM, pH 3.6), 3 ml TPTZ (10 mM in
100 mMHCl), and 3 ml FeCl3·6 H2O (20 mM) was used to
prepare the FRAP reagent, from which 300 µl was added
to 10 µl of the sample and incubated for 30 minutes in
an oven (Memmert, Germany) at 37°C and then read
at a wavelength of 593 nm in the Multiskan Spectrum
automated plate reader (Thermo Fisher Scientific,
Waltham, USA). A serial dilution was prepared from
a stock solution of Ascorbic acid for the preparation of
a standard curve and expressed as FRAP mg per g of wet
brain tissue (Vakili et al. 2014).
Open eld neurobehavioral test
The open eld (OF) test is a commonly used
neurobehavioral assessment tool that provides
simultaneous measurement of locomotion and anxiety
in laboratory animals (Kendigelen et al. 2012). The
apparatus used for the OF assessment in this study
involved a square plexi glass box (72×72×20 cm), with
a digital camera (Samsung HMX‑F90, South Korea)
mounted directly above it. The open‑eld arena was
divided into 16 equal squares, via a 4×4 grid, to assist in
data analysis and animals were tested individually. Each
session lasted 10 minutes in a single run after which
the rat was returned into its home cage and the OF box
cleaned with 70% ethanol before testing the next rat. The
Smart video tracking software version 3.0, from Panlab
Harvard Apparatus (Massachusetts, USA) was used to
measure the locomotor activity of each experimental rat
by extracting the total distance traveled in the OF arena.
As a measure of anxiety, the total distance traveled in the
12 squares near the walls was compared with the distance
traveled in the 4 squares at the center of the arena. All
analysis was done by “blind” observers.
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98 O. Akinrinmade et al. Acta Neurobiol Exp 2017, 77: 94–105
RESULTS
Eects of FRHT on brain oedema
Twenty four hours after reperfusion, brain water
content was assessed to evaluate BCCAO‑induced
brain oedema. Hemispheric brain water content was
signicantly higher (p<0.05) in the ISCHEMIA group
(77.80%±0.27) when compared with the C‑SHAM group
(76.68%±0.36) (Fig. 1). No signicant increase in brain
water content was observed in the R+I group compared to
the C‑SHAM group (77.17%±0.11 and 76.68%±0.36).
Eects of FRHT on the blood‑brain barrier (BBB)
Assessment of BBB integrity after 20 minutes BCCAO
was done by examining the extravasation of injected
Evans blue solution from systemic circulation into the
brain tissue 4 days after surgery. The BBB integrity
appeared to have been compromised in the ISCHEMIA
group as the Evans blue content in the brain specimens
was higher than in the C‑SHAM group (0.6880 µg/g±0.22
vs. 0.9189 µg/g±0.34) (Fig. 2); this dierence was however
not statistically signicant. On the other hand, it does
appear that pre‑treatment with FRHT for 7 weeks did
ameliorate the impairment of BBB integrity as Evans blue
extravasation values were similar to the C‑SHAM group
(0.7341 µg/g±0.25 and 0.6880 µg/g±0.22).
Eects of FRHT on lipid peroxidation
Seven days after BCCAO, the level of lipid peroxidation
was determined by measuring malondialdehyde (MDA)
levels in the cerebrum. Results show that the level of
MDA in the cerebral hemispheres of rats in the ISCHEMIA
group was signicantly higher (0.029 µmol/g±0.0014)
when compared to the C‑SHAM (0.017 µmol/g±0.0014),
and R‑SHAM groups (0.014 µmol/g±0.003) (Fig. 3).
Whereas 7 weeks administration of FRHT to the R+I
group prior to the induction of BCCAO resulted in
signicantly lower levels of MDA in the cerebral
hemispheres when compared to the ISCHEMIA group
(0.017 µmol/g±0.0008 vs. 0.029 µmol/g±0.0014) to around
sham levels (0.014 µmol/g±0.003 vs. 0.017 µmol/g±0.0014).
Eects of FRHT on oxygen radical absorbance
capacity (ORAC)
Assessment of total antioxidant capacity is very
important in our understanding of how antioxidants
protect against reactive oxygen species (ROS). In the human
body, peroxyl radicals are the most abundant free radicals,
which make the measurement of antioxidant capacity
against peroxyl‑radical even more biologically relevant.
In this study, measurement of antioxidant capacity against
peroxyl‑radical in the R‑SHAM and R+I groups showed that
ORACROO values were signicantly higher (36.68 µmol/g±1.98
and 35.16 µmol/g±1.62 respectively) when compared to the
ISCHEMIA group (22.26 µmol/g±3.22) (Fig. 4).
Eects of FRHT on ferric reducing antioxidant
power (FRAP)
The FRAP assay (a measure of the ability of compounds
to neutralize free radicals by acting as an electron donor)
was done. Measurement of FRAP levels in the C‑SHAM and
R‑SHAM groups presented a higher signicant dierence
Fig.1. Eects of FRHT on cerebral oedema in Wistar rats, 24hours after
BCCAO and sham surgery. The ISCHEMIA group had asignicantly higher
percentage brain water content compared with the C‑SHAM and R+I
groups. Data is presented as means ±S.E.Ms, n=5, (*)=signicant dierence
at p<0.05; wbt=wet brain tissue.
Fig. 2. Blood brain barrier integrity assessment using the Evans blue
extravasation method. No signicant dierences were observed. Data
presented as mean ±S.E.M, n=5, wbt=wet brain tissue.
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Rooibos tea protects rat brains from ischemic injury 99Acta Neurobiol Exp 2017, 77: 94–105
(2.36±0.16 and 1.93±0.37 respectively) when compared to
the ISCHEMIA group (0.63±0.11). Similarly, the FRAP values
for samples from the R+I group were signicantly higher
than the ISCHEMIA group (2.12±0.16 vs. 0.63±0.11) (Fig. 5).
Eects of FRHT on neuronal loss
Hematoxylin and eosin staining
Seven days after 20 minutes BCCAO or sham surgery,
neuronal damage in the CA1 region of the hippocampus
was evaluated by staining serial sagittal sections of the
right cerebral hemispheres with hematoxylin and eosin
stains. No histopathological changes were seen in the
C‑SHAM and R‑SHAM groups (Figs 6A and 6B) whereas
the ISCHEMIA group showed marked ischemic neuronal
damage with cells exhibiting triangular shapes and
deeper staining due to the condensation of cytoplasm and
karyoplasm (Fig. 6C). These changes were less frequent in
the R+I group (Fig. 6D).
Cresyl violet/nissl staining
Representative photomicrographs of cresyl violet/nissl
staining of rat brain sections showed neurodegenerative
changes in the cyto‑architecture of the CA1 region of the
hippocampus in the ISCHEMIA group 7 days post‑BCCAO.
These changes were identied by the presence of shrunken
and darkly stained neurons (Fig. 6G). Quantitative analysis
showed a signicant decrease in the amount of viable
hippocampal neurons in the CA1 region of rats in the
ISCHEMIA group when compared with the C‑SHAM and
R‑SHAM groups (Fig. 7A). However, following 7 weeks of
pre‑treatment with FRHT, there were signicantly more
cells in the R+I group than in the ISCHEMIA group. The
R+I group shows more viable cells in the CA1 region of the
hippocampus (Fig. 6H), almost similar to the control groups
(Figs 6E and 6F).
Immunohistochemical staining
Terminal deoxynucleotidyltransferase‑mediated UTP
nick end labeling (TUNEL)‑positive cells were identied
as dark or light‑brown color or with dark brown granules
in the cell nucleus. The percentage rate of apoptosis
(%RA) in the hippocampal CA1 region was calculated as
RA=100×(number of apoptotic neurons/total number of
neurons). In this study, the C‑SHAM and R‑SHAM groups
had fewer (8.12%±2.6 and 6.21%±2.47) TUNEL‑positive
cells respectively in the hippocampal CA1 region (Figs 6I,
6J, and 7B) compared to the ISCHEMIA group with more
TUNEL‑positive cells (46.72%±12.8). The R+I group also had
more TUNEL‑positive cells (36.54%±12.2) compared to the
C‑SHAM and R‑SHAM groups but the dierence was not
statistically signicantly.
Eects of FRHT on neurobehaviour
The open eld test (OFT) is one of the most widely used
measures of animal neurobehavioral decits. Measures of
total distance travelled and rearing events are used as an
index of exploratory activity (Lever et al. 2006). In this study,
we used a 10‑minute OFT to investigate neurobehavioural
decits on days 1, 4, and 7 after BCCAO. Results obtained
Fig.3. Eects of FRHT on cerebral lipid peroxidation in Wistar rats, 7days
after BCCAO. MDA levels in the ISCHEMIA group were signicantly higher
compared to the C‑SHAM, R‑SHAM and R+I groups. Data is presented
as mean ±S.E.M, n=5, wbt=wet brain tissue; (*)=signicant dierence at
P<0.001.
Fig.4. Eects of 7weeks intake of FRAP on peroxyl radical absorbance
capacity (ORACROO) in the cerebral hemispheres of rats 7 days after
BCCAO. Values for the ISCHEMIA group were signicantly lower
compared with the R‑SHAM and R+I groups respectively. Data is
presented as mean ±S.E.M, n=5; (*)=signicant dierence at P<0.01;
wbt=wet brain tissue.
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100 O. Akinrinmade et al. Acta Neurobiol Exp 2017, 77: 94–105
showed that rats in the R+I and ISCHEMIA groups generally
travelled less distance (low locomotor activity) and had
fewer rearing events (exploration) compared with the
C‑SHAM group (Figs 8D and 8E).
DISCUSSION
The clinical manifestations of ischemia
reperfusion‑induced brain injury are diverse and may
include brain oedema formation, BBB disruption,
neuronal loss, oxidative stress, inammation, neurological
decits, etc. some of which have been observed in animal
experiments. Knowland and others (2014) reported that
the high mortality often associated with ischemic stroke
could be linked to BBB damage, one major reason the
maintenance of BBB integrity is crucial to the management
of ischemic stroke (Zhang et al. 2013). BBB disruption may
occur spontaneously during acute stroke or following
reperfusion therapy (Nguyen et al. 2013), and the resulting
oxidative damage to brain capillary endothelial cells and
basement membrane causes damage to the blood‑brain
barrier and results in cerebral oedema (Cunningham et al.
2005). Such oedema is characterized by the pathological
accumulation of uid in brain tissue and often results in
the expansion of brain tissue volume (Ito et al. 1979, Kahle
et al. 2009, Bansal et al. 2013).
After a stroke episode, damage to tight junction
proteins often results in increased BBB permeability, brain
oedema and leukocyte inltration (Andjelkovic and Keep
2016). Reports on the potential neuroprotective eects of
polyphenols against ischemia‑induced BBB disruption (and
Fig. 5. Eects of 7 weeks intake of FRHT on ferric reducing antioxidant
power (FRAP) in the cerebral hemispheres of Wistar rats, 7 days after
BCCAO. The ISCHEMIA groups showed signicantly lower FRAP levels
compared to the R+I and SHAM groups. Values are presented as mean
±S.E.M, n=5; (**, ***, ****)=signicant dierences at P<0.01, P<0.001, and
P<0.0001 respectively; wbt=wet brain tissue.
Fig.6. Eects of 7weeks intake of FRHT on neuronal loss in the CA1 region of the hippocampus after BCCAO or SHAM surgery. Images A, E and I contain
inserts with the hippocampal regions of interest indicated in white rectangular outlines. FigsA–D represent H and E stained sections, with the ISCHEMIA
group showing numerous shrunken CA1 pyramidal cells with condensed and deeply stained nuclei (white arrows). FigsE–H show staining with cresyl violet
while FigsI–L represent TUNEL staining (for apoptosis).
9_766_Akinrinmade_v4.indd 100 30/03/17 22:24
Rooibos tea protects rat brains from ischemic injury 101Acta Neurobiol Exp 2017, 77: 94–105
brain oedema) are scarce in literature, and the associated
cellular and molecular mechanisms are poorly understood
(Panickar and Anderson 2011). The present study showed that
pre‑treatment with FRHT resulted in signicantly reduced
brain oedema (Fig. 1) as well as reduced BBB permeability
though not statistically signicant (Fig. 2), possibly due to the
actions of the rich polyphenol contents in FRHT as previously
reported for other teas (Lee et al. 2004, ArunaDevi et al. 2010).
Tea polyphenols (Xue et al. 2013), Lycium barbarum extracts
(Yang et al. 2012), dietary olive leaf extract (Mohagheghi et
al. 2011) and lavender extract (Rabiei and Raeian‑Kopaei
2014) have all been shown to potentially protect the BBB
against ischemic damage albeit via poorly understood
mechanisms of action. We suggest that the observed BBB
disruption and brain oedema protection eects of FRHT
could have occurred either due to attenuation of oxidative
stress damage to capillary endothelial cells and basement
membrane (Panickar et al. 2013, Krueger et al. 2015), reversal
of the ischemia‑induced reduction in the expression of the
tight junction proteins claudin‑5, occludin and ZO‑1 as seen
with green tea polyphenols (Liu et al. 2013) or modulation of
paracellular permeability to prevent disruption of the tight
junctions between brain microvascular endothelial cells
(Yang et al. 2016).
I/R‑induced injury is known to be associated with
neuronal death (Woodru et al. 2011, Baron et al. 2014).
Following an ischemic stroke, the brain loses as many
neurons as it does in almost 3.6 years of normal aging
(Saver 2006). Our study showed that FRHT pre‑treatment
signicantly attenuated I/R‑induced neuronal death
in the hippocampal CA1 region (Fig. 7), consistent with
previous ndings (Abe et al. 1995, Koponen et al. 2000,
Wang et al. 2005, Nikonenko et al. 2009) possibly through
its antioxidant properties.
The role of oxidative stress in the pathophysiology of
ischemic stroke has been well documented (Ozkul et al.
2007, Manzanero et al. 2013, Cichoń et al. 2015). Cellular
damage during and after I/R‑induced brain injury has been
suggested to be due to oxidative damage caused by harmful
free radicals (e.g. superoxide anions (O2) (Kinuta et al.
1989, Tsai et al. 2014). Results from this study showed that
MDA levels increased markedly in the ISCHEMIA group,
indicative of lipid peroxidation but were maintained at
physiologic values in the R+I group (Fig. 3). This could be
due to the antioxidant properties of FRHT mediated by
its polyphenolic compounds, particularly aspalathin, as
previously reported (Inanami et al. 1995, Fukasawa et al.
2009, Marnewick et al. 2011, Awoniyi et al. 2012, Hong et
al. 2014). The low levels of ORAC and FRAP (indicative of
the high levels of oxidative stress) observed in this study
for the ISCHEMIC group appeared to have been modulated
by long‑term pre‑treatment with FRHT. Similar ndings by
Cao and colleagues (1998), Vergely and others (1998), Jung
and colleagues (2011), and Akinmoladun and others (2015),
all partly support the involvement of the antioxidant
actions of FRHT.
The severity of neurologic impairment after I/R‑induced
brain injury has previously been correlated with
histomorphological ndings (Furlan et al. 1996, Hong et al.
2000, Schiavon et al. 2014) and the present study showed
similar relations. Rats in the R+I group generally showed
better neurologic outcomes and less histomorphological
damage when compared to the ISCHEMIA group (Fig. 6 vs.
Fig. 8) probably due to the benecial eects of rooibos tea.
This is in line with ndings from a study by Inanami and
others (1995) which showed that rooibos tea could prevent
age‑related changes in the brain. Another study by Dal‑Pan
and colleagues (2017) showed that high polyphenol
concentrations in berries could prevent neuropathological
damage and cognitive impairment in an animal model of
Alzheimer’s disease. In our study, only changes that took
place within the rst 7 days post‑surgery were reported
although neuronal death and associated neurobehavioural
decits are known to occur months after I/R‑induced
Fig. 7. Neuronal survival (CV staining) and percentage rate of apoptosis
(%RA) (TUNEL staining) in the CA1 region of the hippocampus 7days after
BCCAO or sham surgery. (A) ISCHEMIA group had the least viable cells.
(B) ISCHEMIA group had the highest (%RA). Data is expressed as mean
±SEM; n=5 rats per group. Signicant dierence for 7A: ****P < 0.0001
vs. control‑sham, ***P < 0.001 vs. R‑sham and **P<0.01 vs. R+I); for 7B:
*P<0.05 R‑sham vs. ISCHEMIA.
9_766_Akinrinmade_v4.indd 101 30/03/17 22:24
102 O. Akinrinmade et al. Acta Neurobiol Exp 2017, 77: 94–105
injury (Shin et al. 2010). The potential role of FRHT and
other botanicals in post‑stroke recovery especially in terms
of improvements in cognitive and motor impairments,
require further investigation.
CONCLUSION
Our results provide some evidence of multifactorial
neuroprotection against I/R‑induced injury, conferred
by long‑term consumption of FRHT. A possible reason for
this eect is the action of the many polyphenols in FRHT
in modulating the oxidative stress and neuroinammatory
pathways which cause neuronal death, brain oedema and
BBB disruption. The ndings from this study will contribute
to existing information on the role of botanicals in stroke
prevention and management.
STATISTICAL ANALYSIS
Results were compared using one‑way analysis of
variance (ANOVA) test. Tukey’s post‑hoc test was conducted
for further comparison among groups if a statistically
signicant dierence was obtained. A two‑way ANOVA
followed by Fisher’s protected least signicance dierence
(post‑hoc LSD) test was used for analysis of open eld
measurements. Values were expressed as means ±standard
error of mean (SEM). P<0.05 was considered as statistically
signicant.
ACKNOWLEDGEMENT AND FUNDING
The authors would like to thank the sta of the Oxidative
Stress Laboratory at the Cape Peninsula University of
Technology, South Africa as well as Mr. Noel Markgraa
at the University of Stellenbosch Animal Facility, for their
technical assistance. This study was also supported by
a grant from the National Research Foundation, South
Africa (Grant ID: 89018).
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... In addition, of all included studies, Akinrinmade et al. [41], Chang et al. [28], Demir et al. [37] and Kosari-Nasab et al. [32] performed blinding of the evaluation method, which may increase the accuracy and reliance of extracted results. Finally, only Chen et al. [33] did not inform about the ethic code register. ...
... Also promoted vasodilatation and consequent enhanced the residual cerebral blood flow. Akinrinmade et al. [41] Stroke No effects on energy expenditure evaluated by locomotor activity (open field test). ...
... Pre-treatment with tea polyphenols was able to reduce brain edema and ameliorate BBB elevated permeability on ischemic injury due to attenuation of oxidative damage and reduction in the expression of the tight junction proteins claudin-5, occluding and ZO-1 [41]. Other studies showed that administration of resveratrol or quercetin induced inhibition of NF-κB, TNF-α, Il-6, and Il-1β expression in the hippocampus of rats with depression-like behavior via Akt/GSK3β signaling pathway [35,37,55]. ...
Article
ackground: Noncommunicable diseases (NCDs) lead to drastic metabolic alterations with associated energy balance and body weight changes, two related physiological processes regulated by the brain. Polyphenol-based treatments for NCDs have emerged as a promising therapy, which seems to involve the energy balance modulation. However, it remains unclear what the most effective polyphenols-based treatment is to attenuate adverse effects in the energy balance of NCDs. Objectives: This systematic review aimed to evaluate the literature on the metabolic and neurological effects of polyphenols-based treatment in rodent models of NCDs. Methods: Literature search was carried out in the following databases: CINAHL, Medline/PubMed, SCOPUS, and Web of Science. For title and abstract screening, original papers with polyphenols exposure in rodents were selected. For full-text screening, studies with models of NCDs that reported metabolic and neurological outcomes when treated with polyphenols were selected for inclusion in this review. Results: 23 articles, using individual compound (11 articles) or polyphenols extracts (12 articles), were included in this review: 5 articles using tea polyphenols, 12 articles using grape-derived polyphenols, 3 articles using the polyphenol quercetin, and 3 articles using other polyphenol sources. Most results agree on the beneficial effect of polyphenols in attenuating alterations in energy balance and body weight. Such effects were associated with neuroprotective responses in different brain areas including hippocampus and hypothalamus. Conclusion: In conclusion, this review shows that the treatment with polyphenols, especially resveratrol or quercetin, attenuates the adverse effects of NCDs on energy balance and are associated with neuroprotective effects. Keywords: Energy balance; neuroprotection; noncommunicable disease; polyphenols; rodents; systematic review.
... In addition, of all included studies, Akinrinmade et al. [41], Chang et al. [28], Demir et al. [37] and Kosari-Nasab et al. [32] performed blinding of the evaluation method, which may increase the accuracy and reliance of extracted results. Finally, only Chen et al. [33] did not inform about the ethic code register. ...
... Also promoted vasodilatation and consequent enhanced the residual cerebral blood flow. Akinrinmade et al. [41] Stroke No effects on energy expenditure evaluated by locomotor activity (open field test). ...
... Pre-treatment with tea polyphenols was able to reduce brain edema and ameliorate BBB elevated permeability on ischemic injury due to attenuation of oxidative damage and reduction in the expression of the tight junction proteins claudin-5, occluding and ZO-1 [41]. Other studies showed that administration of resveratrol or quercetin induced inhibition of NF-κB, TNF-α, Il-6, and Il-1β expression in the hippocampus of rats with depression-like behavior via Akt/GSK3β signaling pathway [35,37,55]. ...
Article
Background: Noncommunicable diseases (NCDs) lead to drastic metabolic alterations with associated energy balance and body weight changes, two related physiological processes regulated by the brain. Polyphenol-based treatments for NCDs have emerged as a promising therapy, which seems to involve the energy balance modulation. However, it remains unclear what the most effective polyphenols-based treatment is to attenuate adverse effects in the energy balance of NCDs. Objectives: This systematic review aimed to evaluate the literature on the metabolic and neurological effects of polyphenols-based treatment in rodent models of NCDs. Methods: Literature search was carried out in the following databases: CINAHL, Medline/PubMed, SCOPUS, and Web of Science. For title and abstract screening, original papers with polyphenols exposure in rodents were selected. For full-text screening, studies with models of NCDs that reported metabolic and neurological outcomes when treated with polyphenols were selected for inclusion in this review. Results: 23 articles, using individual compound (11 articles) or polyphenols extracts (12 articles), were included in this review: 5 articles using tea polyphenols, 12 articles using grape-derived polyphenols, 3 articles using the polyphenol quercetin, and 3 articles using other polyphenol sources. Most results agree on the beneficial effect of polyphenols in attenuating alterations in energy balance and body weight. Such effects were associated with neuroprotective responses in different brain areas including hippocampus and hypothalamus. Conclusion: In conclusion, this review shows that the treatment with polyphenols, especially resveratrol or quercetin, attenuates the adverse effects of NCDs on energy balance and are associated with neuroprotective effects.
... Alongside these findings, other evidence from laboratory models suggests inter-relationships between Rooibos ingestion and improvements in spatial memory, [56] reduced brain oedema and neuronal apoptosis, [57] reductions in esophageal papilloma size, [58] antispasmodic effects, [59,60] bronchodilation, [60] and chemoprotection [61,62]. ...
... This research suggests promise for conditions such as allergic rhinitis [22] but further investigations are needed. Additionally, its effects on bone health [51,52], cognitive health [56,57] and potential hepatoprotective effects [12,31,53] warrant further exploration in the form of human trials. Recently, a review of experimental evidence [68] concluded that blood glucose levels were significantly lower in diabetic rodent models treated with rooibos extracts providing phenolic compounds. ...
Article
Full-text available
An expanse of research has investigated the effects of black and green teas in relation to aspects of health. Rooibos tea, also known as Red bush is derived from the South African Cape fynbos plant, Aspalathus linearis, and is caffeine free, naturally sweet and abundant in polyphenols. Evidence related to the health aspects of drinking Rooibos tea is advancing, but does not appear to have been collated. Therefore, we aimed to examine the health effects of Rooibos tea through a systematic review of the literature. A PUBMED search was undertaken (2000 up to June 2020) for human and laboratory studies investigating the efficacy of Rooibos in relation to health. Seven human studies and 49 laboratory studies were identified. Overall Rooibos tea consumption seems to benefit the lipid and redox profiles of those at risk of cardiovascular disease. It also appears to possess other promising ‘general’ effects on glycaemic control, bone, liver, cognitive and respiratory health. Ongoing research using standardised interventions is now needed to help formulate congruent conclusions that are relevant to public health.
... One such extract, rooibos, Aspalathus linearis L. (Burm.f.) R. Dahlgren (Leguminosae) is a plant indigenous to South Africaʼs fynbos area. Rooibos has shown to improve antioxidant status [8] and cardiovascular health [9], reduce I/R-I in control hearts [10], lower blood glucose [11], and improve lipid profile [12]. Rooibos contains various bioactive phenolic compounds such as aspalathin (C21H24O11), a C-linked dihydrochalcone glucoside unique to rooibos and implicated as one of the main contributors of its therapeutic potential [13]. ...
... In a recent publication, GRT reduced hepatosteatosis in a highfat diet rat model [25]. Rooibos has also been implicated to improve lipid profiles in rats [8] and humans [9]. In the present study, GRT had no toxic effect on biometric parameters and did not induce morphological changes in the heart (▶ Table 1). ...
Article
Diabetic patients develop ischemic heart disease and strokes more readily. Following an ischemic event, restoration of blood flow increases oxidative stress resulting in myocardial damage, termed ischemia/reperfusion injury. Aspalathus linearis (rooibos), rich in the antioxidant phenolic compound aspalathin, has been implicated as cardioprotective against ischemia/reperfusion injury with undefined mechanism in control rats. Primarily, the therapeutic potential of Afriplex green rooibos extract to prevent ischemia/reperfusion injury in cardiovascular disease-compromised rats was investigated. Additionally, Afriplex Green rooibos extract's cardioprotective signaling on metabolic markers and stress markers was determined using western blotting. Three hundred male Wistar rats received either 16-wk standard diet or high-caloric diet. During the final 6 wk, half received 60 mg/kg/day Afriplex green rooibos extract, containing 12.48% aspalathin. High-caloric diet increased body weight, body fat, fasting serum triglycerides, and homeostatic model assessment of insulin resistance - indicative of prediabetes. High-caloric diet rats had increased heart mass, infarct size, and decreased heart function. Afriplex green rooibos extract treatment for 6 wk lowered pre-ischemic heart rate, reduced infarct size, and improved heart function pre- and post-ischemia, without significantly affecting biometric parameters. Stabilized high-caloric diet hearts had decreased insulin independence via adenosine monophosphate activated kinase and increased inflammation (p38 mitogen-activated protein kinase), whereas Afriplex green rooibos extract treatment decreased insulin dependence (protein kinase B) and conferred anti-inflammatory effect. After 20 min ischemia, high-caloric diet hearts had upregulated ataxia-telangiectasia mutated kinase decreased insulin independence, and downregulated insulin dependence and glycogen synthase kinase 3 β inhibition. In contrast, Afriplex green rooibos extract supplementation downregulated insulin independence and inhibited extracellular signal-regulated kinase 1 and 2. During reperfusion, all protective signaling was decreased in high-caloric diet, while Afriplex green rooibos extract supplementation reduced oxidative stress (c-Jun N-terminal kinases 1 and 2) and inflammation. Taken together, Afriplex green rooibos extract supplementation for 6 wk preconditioned cardiovascular disease-compromised rat hearts against ischemia/reperfusion injury by lowering inflammation, oxidative stress, and heart rate.
... Current results are in line with reports identifying fermented rooibos as a neuroprotective agent in animal models, where it was shown to decrease lipid peroxides and confering protection against ischemic brain injury in rats. 55,56 No comparative data on neuroprotective effects of fermented vs. unfermented rooibos is available. In our opinion, such a comparison between differences in composition and differences in physiological effect, may contribute to a better understanding and highlight from a mechanistic point of view, the in vivo observed effects in relation with the central nervous system. ...
Article
South African rooibos (Aspalathus linearis) tea is globally consumed for its health benefits and caffeine free nature, but no information is available on the neuroprotective capacity of (unfermented) green rooibos....
... Obserwowano także ograniczenie niekorzystnych zmian, wywoływanych stresem oksydacyjnym w mózgach szczurów, którym podawano napary. Wyniki badana sugerują, że napary z czerwonokrzewu mają protekcyjne działanie w stosunku do uszkodzeń mózgu, spowodowanych niedokrwieniem [67]. ...
Article
Full-text available
Aspalathus linearis (Burman f.) R. Dahlgren is a shrub of the Fabaceae family, growing endemically in southern Africa. Aspalathus linearis has transformed from a wild plant to a cultivated crop. Now it is commercially available as fermented (red) rooibos and the less common unfermented (green) rooibos. This article aimed to review the literature on the redbush with a focus on its health-promoting properties. Numerous laboratory investigations of extracts and infusions of A. linearis have shown that rooibos is a rich source of biologically active compounds, especially polyphenols. The active compounds present in redbush infusions and extracts include flavonoids, phenolic acids, and minute amounts of procyanidins, coumarins, lignans, phenylethanoids, and phenylpropanoids. The characteristic active compounds of rooibos are the C-glucosyl dihydrochalcone - aspalathin and notofagin. An important active compound found in the infusion is also PPAG (Z-2-(beta-D-glucopyranosyloxy)-3-phenylpropenoic acid), which significantly determines the sensory quality of the infusion and its hypoglycaemic properties. Rooibos is also a source of minerals. Studies in both experimental models and healthy volunteers have confirmed the multiple effects of the redbush. The antioxidant properties of A. linearis were especially well studied. The greater antioxidant activity has been reported for aspalathin-rich green rooibos. The consumption of redbush infusions has been reported to have beneficial effects on the parameters like total plasma antioxidant capacity, lipid peroxidation index, and blood glutathione levels. Rooibos has also been shown to alleviate the inflammatory process induced by lipopolysaccharides or that associated with colitis. This is underlined by reduced expression of pro-inflammatory factors, as well as antioxidant properties. Redbush may be used in supporting the treatment of glucose and lipid metabolism disorders, endocrine disorders, and metabolic syndrome. It has been proven to stimulate pancreatic cells to secrete insulin, regulate the expression of genes responsible for glucose metabolism, and affect the amount of glucose transporter protein type 4 (GLUT-4). Other health properties of rooibos may include antimutagenic and hepatoprotective properties and acceleration of skin healing. The bioavailability of redbush flavonoids and the interaction of rooibos ingredients with drugs were also investigated. Studying the mechanism of absorption of flavonoids contained in the redbush and their interactions with drugs will allow determining the appropriate dosage of the extracts. Precise recognition of the chemical composition and health-promoting properties of the redbush will enable its utilization as pharmacotherapy support.
... Due to the popularity of Aspalanthus linearis worldwide, several studies providing preclinical and clinical analysis of extracts, fractions and even those extracts enriched with aspalathin, are on the increase. Some examples of such studies are those by (1) Sasaki et al., 2018 investigating the role of rooibos in diabetic rodents using an extract with high levels of PPAG; (2) histological and immunohistological studies focusing on modulatory effects linked to neurological inflammation; (3) using various neurochemical assays, are the interest areas explored by Akinrinmade et al. (2017); (4) Marnewick et al. (2011) analyzing the effects on those patients at higher risk of cardiovascular diseases and the oxidized glutathione (GSSG) measurements were taken together with blood pressure measurements, amongst the top evaluations conducted; (5) previous work determining lipid peroxidation, blood biomarker analysis for oxidative stress management and comprehensive analysis of clinical pathological markers (Marnewick et al., 2011;Marnewick et al., 2009) and (6) protective antioxidant effects of rooibos when used with other products such as red palm oil in terms of liver function in rats (Ajuwon et al., 2013). ...
Article
As the use of medicinal plants continues to gain popularity worldwide, there is dire need for herbal medicines to be guaranteed in their safety and efficacy. South Africa has a largely under-explored medical flora scientifically due to the vast number of plant species that are consumed for medicinal purposes by the public, creating an urgent need to better define plants with therapeutic effects. To meet these aims, a combination of high-throughput analytical techniques that are sensitive and versatile are used for the standardization and authentication of commercialized natural products as part of quality control regimes. These methods also serve to highlight the role of analytical methods in providing accurate and reliable information pertaining to the biochemicals of medicinal plants in those species whose chemistry remains partially understood or poorly characterized. The review highlights the advancements made in analytical technology for the assessment of biochemical profiles, biomarker compounds and quality control in chosen commercialized products, produced from indigenous South African species. We also summarize studies on the phytochemistry, pharmacology, clinical trials and available patents associated with some of South Africa's medicinal plants where commercialization has occurred or is imminent. This was achieved through a detailed literature search using web-based database searches including Google Scholar, Scopus and Web of Science (WoS) as well as ethnobotanical literature on South African medicinal plants. Bibliometric analysis was performed on the data mined from WoS. It is clear that future advancements and further development of the natural products industry in South Africa will benefit from a diverse range of technological approaches.
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Various surgical interventions into the head and neck vasculature differentially impact nature, localization and dynamics of ischemic changes in the brain structures. In this regard, our review aims to compare the time course of basic cellular and molecular mechanisms. Selection of a animals model should be done along the following criteria: development of oxidative stress in brain cells, blood-brain barrier breakdown, glial activation and neuroinflammation, alterations in angiogenesis; reproducibility of the model.
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Rooibos tea, brewed using Aspalathus linearis leaves, is a popular South African herbal infusion, but its everyday intake is not fully described in terms of the neuropsychopharmacological outcomes. The cell-protective activity of A. linearis is connected with the ability of reducing glycaemia, inflammation as well as oxidative stress. It was already shown that “fermented” rooibos herbal tea (FRHT), which is rich in phenolic compounds, improves the cognitive performance of rats in the water maze and impacts dopaminergic striatal transmission. The present research was taken to extend the knowledge about the feasible behavioural and neurochemical implications of sustained oral FRHT consumption. We hypothesized that it might affect brain amino acid content and thus induce behaviour and neuroprotection. FRHT of different leaf to water ratios (1:100, 2:100 and 4:100), analysed by chromatographic methods as regards their flavonoid characteristics, were given to rats as only liquid for 3 months. Their behaviour was evaluated in the hole-board test (HBT). Brain amino acids concentration was analysed in the striatum, hippocampus and prefrontal cortex by HPLC-ECD. The rats drinking rooibos tea presented increased motor activity defined as time spent on moving in the HBT. Their exploration measured by head-dipping and rearing was enhanced. Longer time of the testing-box central zone occupation indicated to reduction in anxiety-related behaviour. Excitatory amino acids (aspartate and glutamate) content was decreased in the striatum of animals drinking the infusions whereas taurine level was increased both in the striatum and hippocampus. In conclusion we suggest that long-term FRHT intake affects exploration and anxiety-related behaviour of the rats as well as exerts biochemical outcomes in the brain that support the neuroprotective impact of rooibos tea.
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Ethnopharmacological relevance: Everyday use of the herbal tea rooibos, produced from Aspalathus linearis (Brum.f) Dahlg. (Fabaceae) is customary in South Africa, a continuation of its historical use by indigenous people. Although evidence of its traditional indications is anecdotal, rooibos tea is regarded as a general health tea. Aims of the study: Available contemporary research indicates to broad cell protective activity of rooibos focusing on its antioxidative, anti-inflammatory, anti-hyperglycaemic and antithrombotic features affecting metabolic syndrome, cardiovascular risk and neuroprotection. Nevertheless little is known about its impact on brain functions. The present experiment aimed to evaluate the possible behavioural and neurochemical effects of long-term oral administration of "fermented" rooibos herbal tea (FRHT) infusions to adult male Sprague-Dawley rats. Materials and methods: Infusions, prepared using 1, 2 and 4 g of "fermented" (oxidised) A. linearis leaves for 100 ml of hot water, were characterised in terms of flavonoid content by ultra-high and high performance liquid chromatography (UHPLC-qTOF-MS, HPLC-DAD) and administered to rats as sole drinking fluid for 12 weeks. Spatial memory behaviour was assessed in a modified version of the Morris water maze. Dopamine, noradrenaline, serotonin and their metabolite levels (DOPAC, 3-MT, HVA, MHPG, 5-HIAA) were quantified in prefrontal cortex, hippocampus and striatum by HPLC-ECD. Body weight and blood glucose level were additionally estimated. Results: All FRHT-treated rats showed improvement of long-term spatial memory defined as increased number of crossings over the previous platform position in SE quadrant of the water maze. It was not accompanied by excessive motor activity. Striatal dopamine and its metabolite 3-MT (3-methoxytyramine) levels were increased in treated rats. There were no differences in body weight gain between control and treated animals but blood glucose level was significantly lower in the latter ones. Conclusion: The improvement of long-term memory in FRHT-treated rats and stimulating impact of FRHT on their dopaminergic striatal transmission support the wellness enhancing effect of rooibos tea, contributing to a better understanding of the neurological background of traditional habitual consumption of this herbal tea.
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No effective preventive treatment is available for age-related cognitive decline and Alzheimer's disease (AD). Epidemiological studies indicate that a diet rich in fruit is associated with cognitive improvement. It was thus proposed that high polyphenol concentrations found in berries can prevent cognitive impairment associated with aging and AD. Therefore, the Neurophenols project aimed at investigating the effects of a polyphenolic extract from blueberries and grapes (PEBG) in the triple-transgenic (3xTg-AD) mouse model of AD, which develops AD neuropathological markers, including amyloid-β plaques and neurofibrillary tangles, leading to memory deficits. In this study, 12-month-old 3xTg-AD and NonTg mice were fed a diet supplemented with standardized PEBG (500 or 2500mg/kg) for 4 months (n=15-20/group). A cognitive evaluation with the novel object recognition test was performed at 15 months of age and mice were sacrificed at 16 months of age. We observed that PEBG supplementation with doses of 500 or 2500mg/kg prevented the decrease in novel object recognition observed in both 15-month-old 3xTg-AD mice and NonTg mice fed a control diet. Although PEBG treatment did not reduce Aβ and tau pathologies, it prevented the decrease in mature BDNF observed in 16-month-old 3xTg-AD mice. Finally, plasma concentrations of phenolic metabolites, such as dihydroxyphenyl valerolactone, a microbial metabolite of epicatechin, positively correlated with memory performances in supplemented mice. The improvement in object recognition observed in 3xTg-AD mice after PEBG administration supports the consumption of polyphenols-rich extracts to prevent memory impairment associated with age-related disease, without significant effects on classical AD neuropathology.
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Global cerebral ischemia (GCI) usually occurs after cardiac arrest and severe shock due to lack of blood flow and oxygen delivery to the brain. Some areas of the brain, such as hippocampus, are more susceptible to injury than the others following GCI. Several animal models have been developed to study the pathophysiology of GCI. In this chapter, we describe the most widely used protocols in assessing the neurological function and hippocampus-dependent learning and memory deficit following GCI.
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Poststroke depression, the second most serious psychosomatic complication after brain stroke, leads to delay of the rehabilitation process and is associated with an increased disability and cognitive impairment along with increase in term mortality. Research into the biochemical changes in depression is still insufficiently described. The aim of our study was therefore to evaluate the possible association between plasma protein oxidative/nitrative damages and the development of poststroke depression. We evaluated oxidative/nitrative modifications of specific proteins by measurement of 3-nitrotyrosine and carbonyl groups levels using ELISA test. Additionally, we checked differences in proteins thiol groups by spectrophotometric assay based on reaction between DTNB and thiols. We also evaluated catalase activity in erythrocytes measured as ability to decompose H2O2. Correlation analysis was performed using Spearman’s rank. We observed significant ( P < 0.001 ) differences in all oxidative/nitrative stress parameters in brain stroke patients compared to healthy group. Our research shows that oxidative damage of proteins is correlated with the degree of poststroke depression, while nitrative changes do not show any relationship. We demonstrate a positive correlation between the concentration of carbonyl groups and the Geriatric Depression Scale and a negative correlation between the degree of depression and the concentration of -SH groups or catalase activity.
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This study was to investigate the protective effects of tea polyphenols on the blood-brain barrier (BBB) of rats with global cerebral ischemia/reperfusion (GCIR) injury. Sprague Dawley rats underwent four-vessel occlusion to construct the model of GCIR. Half an hour before complete occlusion, they were treated with tea polyphenols (TP) (6.4%; 100 or 200 mg/kg) via tail intravenous injection. 24 h after reperfusion, BBB permeability was evaluated by measuring brain water content (BWC) and residual amount of Evan's blue dye in cerebral tissue. In addition to this, MMP-9 and collagen IV protein expression in cerebral tissue were also detected using immunohistochemistry. ANOVA and SNK-q were used to do statistical analysis. Statistical significance was considered at P < 0.05. Compared to the untreated, the TP-treated rats had significantly decreased BWC (P < 0.05), decreased residual amount of Evan's blue dye in cerebral tissue (P < 0.05), down-regulated MMP-9 (P < 0.05) and up-regulated collagen IV expression in brain tissue (P < 0.05). It can be concluded from these findings that TP may reduce the MMP-9 mediated collagen IV degradation caused by GCIR to protect the BBB.
Chapter
Ischemic and hemorrhagic stroke cause marked blood–brain barrier (BBB) and neurovascular unit dysfunction in humans and in animal models. The tight junctions (TJs) that link the cerebral endothelial cells are critical for BBB function and they undergo marked modifications after stroke. Those changes contribute to increased BBB permeability, brain edema, and leukocyte infiltration, all hallmarks of stroke. This review describes the types of TJ modifications that occur after stroke (e.g., protein phosphorylation, altered protein/protein interactions, protein relocation, and degradation), the underlying mechanisms and potential therapeutic interventions. While the effects of stroke on the BBB have long been considered as secondary to neuronal injury, increasing evidence stresses the importance of neurovascular injury in stroke and that BBB damage may be a cause rather than a result of stroke-induced parenchymal damage.
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Adropin is a peptide encoded by the energy homeostasis associated gene (Enho) and plays a critical role in the regulation of lipid metabolism, insulin sensitivity, and endothelial function. Little is known of the effects of adropin in the brain and whether this peptide modulates ischemia-induced blood-brain barrier (BBB) injury. Here, we used an in vitro BBB model of rat brain microvascular endothelial cells (RBE4) and hypothesized that adropin would reduce endothelial permeability during ischemic conditions. To mimic ischemic conditions in vitro, RBE4 cell monolayers were subjected to 16h hypoxia/low glucose (HLG). This resulted in a significant increase in paracellular permeability to FITC-labeled dextran (40kDa), a dramatic upregulation of vascular endothelial growth factor (VEGF), and the loss of junction proteins occludin and VE-cadherin. Notably, HLG also significantly decreased Enho expression and adropin levels. Treatment of RBE4 cells with synthetic adropin (1, 10 and 100ng/ml) concentration-dependently reduced endothelial permeability after HLG, but this was not mediated through protection to junction proteins or through reduced levels of VEGF. We found that HLG dramatically increased myosin light chain 2 (MLC2) phosphorylation in RBE4 cells, which was significantly reduced by adropin treatment. We also found that HLG significantly increased Rho-associated kinase (ROCK) activity, a critical upstream effector of MLC2 phosphorylation, and that adropin treatment attenuated that effect. These data indicate that treatment with adropin reduces endothelial cell permeability after HLG insult by inhibition of the ROCK-MLC2 signaling pathway. These promising findings suggest that adropin protects against endothelial barrier dysfunction during ischemic conditions.
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Oxidative stress is considered a major contributing factor in cerebral ischemia/reperfusion injury. Phloretin, a dihydrochalcone belonging to the flavonoid family, is particularly rich in apples and apple-derived products. A large body of evidence demonstrates that phloretin exhibits anti-oxidant properties, and phloretin has potential implications for treating oxidative stress injuries in cerebral ischemia/reperfusion. Therefore, the neuroprotective and antioxidant effects of phloretin against ischemia/reperfusion injury, as well as related probable mechanisms, were investigated. The cerebral ischemic/reperfusion injury model was reproduced in male Sprague-Dawley rats through middle cerebral artery occlusion. At 24h after reperfusion, neurological score, infarct volume, and brain water content were assessed. Oxidative stress was evaluated by superoxide dismutases (SOD), glutathione (GSH), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) levels. Nrf2 expression was measured by RT-PCR and western blot. Consequently, results showed that phloretin pretreatment for 14days significantly reduced infarct volume and brain edema, and ameliorated neurological scores in focal cerebral ischemia/reperfusion rats. SOD, GSH and GSH-Px activities were greatly decreased, and MDA levels significantly increased after ischemia/reperfusion injury. However, phloretin pretreatment dramatically suppressed these oxidative stress processes. Furthermore, phloretin upregulated Nrf2 mRNA and protein expression of in ischemia/reperfusion brain tissue. Taken together, phloretin exhibited neuroprotective effects in cerebral ischemia/reperfusion, and the mechanisms are associated with oxidative stress inhibition and Nrf2 defense pathway activation. Copyright © 2015 Elsevier B.V. All rights reserved.
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The pathophysiology of stroke is characterized by biochemical and physical alterations in the brain. Modulation of such aberrations by therapeutic agents affords insights into their mechanism of action. Incontrovertible evidences that oxidative stress is involved in the pathophysiology of neurologic disorders have brought antioxidative compounds, especially plant phytochemicals, under increasing focus as potential remedies for the prevention and management of neurodegenerative diseases. Kolaviron, a biflavonoid complex isolated from Garcinia kola Heckel (Guttiferae) was evaluated for neuroprotectivity in brains of male Wistar rats submitted to bilateral common carotid artery occlusion-induced global ischemia/reperfusion injury (I/R). Animals were divided into six groups: sham treated, vehicle (I/R), 50 mg/kg kolaviron + I/R, 100 mg/kg kolaviron + I/R, 200 mg/kg kolaviron + I/R and quercetin (20 mg/kg i.p.) + I/R. The common carotid arteries were occluded for 30 min followed by 2 h of reperfusion. Relative brain weight and brain water content were determined and oxidative stress and neurochemical markers were also evaluated. I/R caused significant decreases in glutathione level and the activities of enzymic antioxidants, the sodium pump and acetylcholinesterase while significant increases were recorded in relative brain weight, brain water content, lipid peroxidation and the activities of glutamine synthetase and myeloperoxidase. There was a remarkable ablation of I/R induced oxidative stress, neurochemical aberrations and brain edema in animals pretreated with kolaviron. The results suggested that the protection afforded by kolaviron probably involved regulation of redox and electrolyte homeostasis as well as anti-inflammatory and antiexcitotoxic mechanisms.