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Kenyan purple tea anthocyanins ability to cross the blood brain barrier and reinforce brain antioxidant capacity in mice

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Studies on antioxidants as neuroprotective agents have been hampered by the impermeability of the blood brain barrier (BBB) to many compounds. However, previous studies have shown that a group of tea flavonoids, the catechins, are brain permeable and neuroprotective. Despite this remarkable observation, there exist no data on the bioavailability and pharmacological benefits of tea anthocyanins (ACNs) in the brain tissue. This study investigated the ability of Kenyan purple tea ACNs to cross the BBB and boost the brain antioxidant capacity. Mice were orally administered with purified and characterized Kenyan purple tea ACNs or a combination of Kenyan purple tea ACNs and coenzyme-Q10 at a dose of 200 mg/kg body weight in an experiment that lasted for 15 days. Twenty-four hours post the last dosage of antioxidants, CO2 was used to euthanize the mice after which the brain was excised and used for various biochemical analyses. Brain extracts were analysed by high-performance liquid chromatography for ACN metabolites and spectrophotometry for cellular glutathione (GSH). Kenyan purple tea ACNs significantly (P < 0.05) raised brain GSH levels implying boost in brain antioxidant capacity. However, co-administration of both antioxidants caused a reduction of these beneficial effects implying a negative interaction. Notably, ACN metabolites were detected in brain tissue of ACN-fed mice. Our results constitute the first demonstration that Kenyan purple tea ACNs can cross the BBB reinforcing the brain's antioxidant capacity. Hence, the need to study them as suitable candidates for dietary supplements that could support antioxidant capacity in the brain and have potential to provide neuroprotection in neurodegenerative conditions.
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Kenyan purple tea anthocyanins ability to
cross the blood brain barrier and reinforce
brain antioxidant capacity in mice
Khalid Rashid
1,2
, Francis Nyamu Wachira
2,3
, James Nyariki Nyabuga
1
,
Bernard Wanyonyi
4
, Grace Murilla
4
, Alfred Orina Isaac
1,5
1
Biochemistry and Molecular Biology Department, Egerton University, Egerton, Kenya,
2
Tea and Health
Department, Tea Research Foundation of Kenya, Kericho, Kenya,
3
Programs Department, Association for
Strengthening Agricultural Research in Eastern and Central Africa, Entebbe, Uganda,
4
Pharmacology and
Chemotherapy Division, Trypanosomosis Research Centre, Kenya Agricultural Research Institute, Kikuyu,
Kenya,
5
Department of Pharmaceutical Sciences and Technology, Technical University of Kenya, Nairobi, Kenya
Studies on antioxidants as neuroprotective agents have been hampered by the impermeability of the
blood brain barrier (BBB) to many compounds. However, previous studies have shown that a group of
tea flavonoids, the catechins, are brain permeable and neuroprotective. Despite this remarkable
observation, there exist no data on the b ioavailability and pharmacological benefits of tea anthocyanins
(ACNs) in the brain tissue. This study investigated the ability of Kenyan purple tea ACNs to cross the
BBB and boost the brain antioxidant capacity. Mice were orally administered with purified and
characterized Kenyan purple tea ACNs or a combination of Kenyan purple tea ACNs and coenzyme-
Q
10
at a dose of 200 mg/kg body weight in an experiment that lasted for 15 days. Twenty-four hours
post the last dosage of anti oxidants, CO
2
was used to euthanize the mice after which the brain was
excised and used for various biochemical analyses. Brain extracts were analysed by high-performance
liquid chromatography for ACN metabolites and spectrophotometry for cellular glutathione (GSH).
Kenyan purple tea ACNs significantly (P < 0.05) raised brain GSH levels implying boost in brain
antioxidant capacity. However, co-administration of both antioxidants caused a reduction of these
beneficial effects implying a negative interaction. Notably, ACN metabolites were detected in brain
tissue of ACN-fed mice. Our results constitute the first demonstration t hat Kenyan purple tea ACNs can
cro ss the BBB reinforcing the brains antioxidant capacity. Hence, the need to study them as suitable
candidates for dietary supplements that could support antioxidant capacity in the brain and have
potential to provide neuroprotection in neurodegenerative conditions.
Keywords: Anthocyanins, Blood brain barrier, Coenzym e-Q
10
, Glutathione, Kenyan purple tea
Introduction
Tea, an evergreen plant native to China and belonging
to t he family Theaceae, is one of the most commonly
consumed beverages in the world and is manufactured
from the young tender leaves of the plant Camellia
sinensis.
1
The chemical composition of tea is
complex and includes polyphenols, amino acids,
carbohydrates, proteins, chlorophyll, volatile com-
pounds, minerals, trace elements, and alkaloids such
as caffeine, theophylline, and theobromine. Among
these, polyphenols constitutes the main bioactive mol-
ecules in tea.
1
For centuries, the Chinese have used tea beverage
to treat a myriad of diseases.
2
This pharmacological
value of tea heavily relies upon its antioxidative prop-
erties kn own to surpass that of major antioxidants
such as vitamins C and E
3
and other synthetic antiox-
idants such as butylated hydroxyl anisole and buty-
lated hydroxytoluene.
4
The ability of tea to
scavenge for free radicals is associated with the pos-
session of a phenolic hydroxyl group attached to the
flavan-3-ol structure of these compounds.
5
However, the importance of polyphenols in enh an-
cing resistance to oxidative stress goes beyond
simple radical scavenging activity and is also due to
amplified activity of most detoxifying enzymes such
as glutathione peroxidase (GPx) and glutathione
reductase (GR).
6
Indeed, as a result of their free
Correspondence to: Alfred Orina Isaac, Department of Pharmaceutical
Sciences and Technology, Technical University of Kenya, PO Box
52428-00200, Nairobi, Kenya. Email: orinaisaac@hotmail.com
©W.S.Maney&SonLtd2014
DOI 1 0.1179/1476830513Y.00 00000081
Nutritional Neuroscience 2014 VOL. 17 NO. 4178
radical quenching strengths, tea polyphenols have
widely been credited with therapeutic action against
free radical-mediated diseases.
5
The last decade has witnessed increased interests in
the use of tea polyphenols as neuroprotective agents.
This is because previous studies have shown phyto-
chemicals present in tea such as epigallocatechin
gallate (EGCG), which is the major and most active
component of green tea catechins and epicatechin
(EC) metabolites including epicatechin glucuronide
and 3
-O-methylated epicatechin glucuronide, formed
after oral ingestion of EC by rats, can cross the
blood brain barrier (BBB) protecting nerve cells
from reactive oxygen species (ROS)-induced cell
death.
6
Other neuroprotective properties associated
with tea polyphenols include preventing loss of dopa-
minergic neurons and preservation of striatal levels of
dopamine, decreased expression of neuronal nitric
oxide synthase, inhibition of pro-apoptotic genes,
and protection against beta-amyloid-induced neuro-
toxicity strongly suggesting that tea polyphenols have
potential application in the treatment of neurodegen-
erative disorders such as Alzheimers and Parkinsons
disease.
7
However, compared with other flavonoid groups
from tea such as the catechins, little is known about
the bioavailability and pharmacological benefits of
tea anthocyanins (ACNs). This is despite the fact
that ACNs from other sources have been associated
with a broad spectrum of health benefits including car-
diovascular, neurological, urinary tract, and ocular
protection as well as anti-carcinogenic, anti-diabetic,
anti-ageing, antioxidant, and anti-inflammatory prop-
erties.
8
We therefore initiated a study to investigate the
ability of ACNs from Kenyan purple tea cultivars to
cross the BBB and offer neuroprotection through its
known antioxidant capacity.
Nevertheless, it has now been widely established
that isolated individual antioxidants do not explain
the observed health benefits of diets, implying that
interactions between antioxidants may yield positive
synergistic effects.
9
Indeed, synergistic effects of
various ACNs have been demonstrated in black-
current and wine grape.
10
Therefore, the health
benefits of a diet rich in phytochemicals is attributed
to the complex mixture of phytochemicals present in
it, an observation which clearly suggest that to
improve their nutrition and health, consumers should
consume antioxidants from diverse sources.
However, only a very limited number of studies have
investigated combinations of purified ACN extracts
with other chemical components of food. Thus, this
study aimed to evaluate the synergistic, additive, or
antagonistic types of interactions manifested by puri-
fied tea ACN extracts and coenzyme Q
10
(Co-Q
10
).
Co-Q
10
, or ubiquinone, is an endogenously
synthesized lipid, which shuttles electrons from com-
plexes I and II to complex III (ubiquinol cytochrome
c oxidase) of the electron transport chain.
Intracellular synthesis that occurs in the inner mito-
chondrial membrane via the mevalonate pathway is
the major source of Co-Q
10
, although small amounts
can be obtained from the diet.
11
Co-Q
10
is an essential
cofactor involved in mitochondrial oxidative phos-
phorylation and when reduced, it is a powerful antiox-
idant that prevents oxidative damage by free radicals
including oxidation of lipids within the mitochondrial
membrane.
12
More importantly, Co-Q
10
crosses the
BBB exerting a multitude of neuroprotective effects
in the brain and protecting against pathophysiology
associated with neurodegenerative disorders,
12
hence
the inclusion of this nutraceutical in this study.
Materials and methods
Tea samples
Purple tea used to extract ACNs was obtained from
the Tea Research Foundation of Kenya, Timbilil
Estate in Kericho (latitude 0°22
S, longitude 35°21
E,
altitude 2180 m a.m.s.l.). ACNs were extracted from
the purple tea variety TRFK 306. Young tender
shoots comprising of two leaves plus a bud were har-
vested, dried using a microwave, and pulverized with
a grinder into fine powder.
Extraction and purification of ACNs
Extraction and purification of tea ACNs was carried
out as described elsewhere.
13
Five grams of powdered
leaves of purple Camellia sinensis were weighed into
250 ml conical flasks covered with foil to prevent
photodegradation and mixed with 50 ml methanol/
formic acid at a ratio of 99 /1 volume/volume (v/v).
The sample was magnetically stirred for 4 hours at
room temperature at a speed of 900 revolutions per
minute (rpm). The resultant solution was filtered and
methanol and formic acid were removed using a
rotary evaporator (Buchi Rotavapour R-300, Flawil,
Switzerland) at 35°C under vacuum, and the residue
was reconstituted to 10 ml with distilled water. The
extract was then passed through a membrane filter
0.45 μM and kept at 4°C for analysis.
The tea extracts were passed through reverse phase
C18 solid phase extraction (SUPELCO, SPE, Sigma-
Aldrich, Pennsylvania, PA, USA) cartridges (Sigma-
Aldrich, Pennsylvania, PA, USA) previously activated
with acidified methanol (10% HCl/methanol v/v).
ACNs were adsorbed into the column while sugars,
acids, and other water-soluble compounds were
washed out using 0.01% HCl in distilled water. The
ACNs were recovered using acidified methanol (10%
formic acid/methanol v/v). The cartridges were
washed with ethyl acetate (Fischer Scientific,
Loughborough, UK) to remove phenolic compounds
Rashid et al. Kenyan purple tea ACNs to cross the BBB and boost the brain antioxidant capacity
Nutritional Neuroscience 2014
VOL. 17 NO. 4 179
other than ACNs. The purified extracts were then
stored at 10°C until further analysis.
Lyophilization of ACN extract
Prior to the lyophilization process, methanol and
formic acid were removed using a rotary evaporator
at 35°C under vacuum and the residue was reconsti-
tuted with distilled water. Pre-freezing of the extract
was done before being placed on the drying accessory.
A volume of 200 ml of the ACN sample were placed in
dehydration flasks and rapidly frozen by spinning the
round bottom flasks in a dry ice-acetone bath.
Temperature and pressure of the lyophilizer were
allowed to reach appropriate levels of 40°C and
100 × 10
3
M Bar, respectively, before the freeze
drying process was initiated. Lyophilization was
done using a Modulyo freeze dryer (Edwards,
Crawley, UK) producing a free-flowing powder that
was weighed and stored in airtight containers at
room temperature until use.
High-performance liquid chromatography
analysis of ACNs
Qualitative and quantitative analyses of the tea extract
and ACN profiles of purple tea variety TRFK/306
were carried out in triplicate by high-performance
liquid chromatography (HPLC) as described else-
where.
13
Briefly, 1 ml of theACN sample was pipetted
into separate tubes and diluted to 2 ml with mobile
phase A solution (87:3:10 water/acetonitrile/formic
acid v/v/v), filtered and loaded into 2 ml vials.
A Shimadzu LC 20 AT HPLC fitted with a SIL 20A
autosample and a SPD-20 UVvisible detector with
a class LC10 chromatography workstation with UV
detection at 520 nm were used for analysis of the pre-
pared samples. A Luna TM 5 μM, C18, 25 cm ×
4.6 mm internal diameter (Phenomenex, Torrance,
CA, USA) column fitted with a Rheodyne pre-
column filter of 7335 model was used. Mobile phase
solutions were filtered through a 0.45 μm nitrocellu-
lose filter on a membrane filter disk and degassed
before injection into the HPLC system.
Gradient elution was employed for analysis using the
following solvent: the eluents were mobile phase A
(water/acetonitrile/formic acid at a ratio of 87/3/10
v/v/v) and mobile phase B (100% HPLC grade aceto-
nitrile). The flow rate of the mobile phase was set at
1ml/minute, column temperature at 35 ± 0.5°C, and
injection volume at 20 μl. Chromatographic conditions
were set as follows: 3% B in A at the time of injection, at
45 minutes 25% B in A, at 46 minutes 30% B in A, and
at 47 minutes 3% B in A. The conditions were set at 3%
B for 10 minutes before the next injection to allow for
equilibration.
Identification of individual anthocyanidins was
carried out by comparing the retention times from
sample chromatographs and absorbances of
unknown peaks with the peaks obtained from the indi-
vidual and mixed standards under similar conditions.
The standards used were cyanidin-3-O-galactoside,
cyanidin-3-O-glucoside, cyanidin chloride, delphinidin
chloride, petunidin chloride, pelargonidin chloride,
and malvidin chloride purchased from Sigma
Aldrich, (London, UK). Quantification of ACNs
was performed at 520 nm using external ACN stan-
dards each with its determined calibration curve. The
identified individual ACN content calculated on a
dry matter basis was determined by the formula:
A
sample
A
intercept

× V × d × 100
Slope
anthocyanin
× m × DM
where
A
sample
is the peak of the individual ACN in the test
sample
A
intercept
is the peak area at the point the individual
ACN calibration line intercepts the y-axis
Slope
anthocyanin
is the individual ACN calibration line
slope
V is the sample extraction volume
d is the dilution factor
m is the mass, in grams of the sample test portion
DM is the dry matter content in percentage
Experimental animals
All experimental protocols and procedures involving
use of mice as experimental animals adhered to rules
and regulations approved by Institutional Animal
Care and Use Committee (IACUC) of
Trypanosomiasis Research Centre of Kenya
Agricultural Research Institute (KARI-TRC)
Muguga, Kenya and Egerton University as well as
the National Regulations of the Kenya Veterinary
Association. A total of 15, 8-week-old female adult
healthy Swiss white mice weighing between 21 and
30 g were obtained from the TRC breeding colony
and used in all experiments. The animals were
housed in standard mice cages at a temperature of
2128°C and were provided ad libitum access to
water and standard mice cubes (Unga Feeds Ltd,
Nairobi, Kenya) with wood-chippings provided as
the bedding material. All mice were treated with
0.02 ml of Ivermectin (Ivermectin
®
, Anupco, Suffolk,
England) injected subcutaneously to each mouse to
eradicate endoparasites and ectoparasites infestation.
After 2 weeks of acclimatization, mice were randomly
selected and divided into two groups and appropriate
controls were used for this experiment. Group 1 was
supplemented with Kenyan purple tea ACNs, while
group 2 was supplemente d with a combination of
Kenyan purple tea ACNs and Co-Q
10
. Note that we
did not have a group on Co-Q
10
alone because the
Rashid et al. Kenyan purple t ea ACNs to cross the BBB and boost the brain antioxidant capacity
Nutritional Neuroscience 2014
VOL. 17 NO. 4180
antioxidant abilities of Co-Q
10
are well known and this
study was focused on determining if Co-Q
10
will boost
the beneficial effects of ACNs. The test antioxidants
were administered orally at a dosage of 200 mg/kg
body weight for 14 days after every second day using
a gavage needle. Twenty-four hours post the last
dosage of antioxidants, carbon dioxide was used to
euthanize the mice after which the brain was excised,
snap frozen in dry ice, and stored in liquid nitrogen
until analysis.
Packed cell volume and body weight
At 1 week interval, blood was taken from each mouse
by tail snip into 100 μl micro-haematocrit capillary
tubes for packed cell volume (PCV) determination.
14
After blood collection, the capillary tubes were
sealed with plasticin at one end and centrifuged in a
haematocrit centrifuge (Hawksley H, Lancing, UK)
at 10 000 rpm for 5 minutes. PCV was then read
using a micro-haematocrit reader and expressed as a
percentage (%) of the total blood volume. Body
weight of each mouse was determined every 2 days
using the analytical electronic balance (Mettler
PM34, DoltaRange
®
, Mumbai, India).
Brain sample preparation
Snap-frozen whole brains were homogenized on ice
water (4°C) in 0.5 ml of 0.25 M sucrose, 5 mM
Hepes-Tris, pH 7.4, with protease inhibitor cocktail
to a final concentration of 10% (w/v). The homogen-
ates were aliquoted into 1.5 ml microfuge tubes to
avoid repeated freezethaw process and stored in
liquid nitrogen until analysis
Glutathione assay
Glutathione (GSH) assay was performed as described
in a previous experiment
15
with slight modifications.
A volume of 50 μl of brain homogenates were mixed
with 50 μl solution containing sulphosalicylic acid
(SSA, 5% w/v) and 0.25 mM ethylenediaminetetraa-
cetic acid (EDTA) and the mixture centrifuged at
8000 × g for 10 minutes at 4°C. A volume of
200 μmol/l of GSH standard solution was prepared
in 0.5% SSA and serial dilutions made using the
same solution (0.5% SSA) to final concentrations of
100, 50, 25, 12.5, 6.25, 3.13, and 1.56 μmol/l.
Ellmans reagent (5,5
-dithiobis (2-nitrobenzoic acid)
(DTNB)) was prepared by dissolving in 0.1 M potass-
ium phosphate buffer with 5 mM EDTA disodium
salt, pH 7.5 (KPE buffer) to a final concentration of
0.6 mg/ml. A volume of 25 μl of each standard were
loaded on a 96-well microtitre plate to wells BHin
columns 1, 2, and 3 followed by 25 μl of the sample
to the remaining wells in triplicate. To each well,
100 μl of fresh ly prepared DTNB was then added
and the absorbance measured at 405 nm at intervals
of 30 seconds using a multi-detection microtitre plate
reader (Bio-Tek Synergy HT, Winooski, VT, USA).
HPLC analysis of brain homogenates for
detection of ACNs
A volume of 600 μl methanol/formic acid (99/1) was
mixed with 600 μl brain homogenate in a 1.5 ml micro-
fuge tube and the mixture centrifuged at a speed of
5000 g for 10 minutes. One millilitre of the supernatant
was then pipetted into a separate tube and HPLC
analysis of the samples carried out as described in
Section 2.1.4. High-performance liquid chromato-
graphy analysis of anthocyanins.
Data analysis
Data were analysed using Prism Graph pad version 5.0
and a P value of <0.05 considered to be statistically
significant. Significance of difference between means
for PCV and GSH was determined by one-way analy-
sis of variance and Tukeys post hoc test was performed
to evaluate differences among group means. Graphs
were plotted to show the trend of the various response
variables. The data are expressed as the mean ± stan-
dard error of the mean.
Results
Purple tea ACN profile
Following lyophilization of the ACN extracts, a free-
flowing powder that was bright red in colour with a
characteristic smell of fresh berries was produced.
ACN profiling of Kenyan purple tea revealed the pres-
ence of anthocyanidins; cyanidin, peonidin, pelargoni-
din, delphinidin, and malvidin (Fig. 1). The
anthocyanidins profile revealed cyanidin as the most
abundant (1755.60 μg/ml) and delphinidin was least
(122.85 μg/ml) abundant (Table 1).
ACN in brain tissue
HPLC analysis of ACNs in brain tissue was carried to
identify intact and/or metabolized ACNs. ACN
metabolites, vividly absent from animals not sup-
plemented with ACNs and having very close retention
times to individual intact ACNs (Table 2), were
detected in brain tissue of animals from the various
ACN groups (Fig. 2).
Effects of tea ACNs and Co-Q
10
on mice
Clinical symptoms and survival
Animals supplemented with ACNs and Co-Q
10
were
marked with hyperactivity from the onset of Co-Q
10
supplementation to the last day of the experiment.
No clinical signs were detectable in the ACNs only
groups signifying that the tea polyphenols were well
tolerated in the experimental animals. One animal
supplemented with both test antioxidants died 9 day
post start of antioxidant administration (DPSAA).
Rashid et al. Kenyan purple tea ACNs to cross the BBB and boost the brain antioxidant capacity
Nutritional Neuroscience 2014
VOL. 17 NO. 4 181
PCV and body weight
PCV levels and body weight parameters were
measured prior to the start of the experiment to estab-
lish baseline data (Table 3). There was a gradual
increase in PCV levels of the untreated animals
throughout the experiment rising from 55.2 ± 0.49 to
58.4 ± 2.50%. Experimental animals supplemented
with ACNs only and ACNs and Co-Q
10
had a rather
steady increase in PCV rising from 53.4 ± 1.03 to
53.4 ± 1.36% 3 days prior to antioxidant adminis-
tration to 58.6 ± 1.08 and 59.25 ± 2.03% on the sixth
DPSAA, respectively. This was followed by a steady
decrease in PCV levels to the last day of the exper-
iment, reaching 53.2 ± 1.93 and 55 ± 0.71% in ACNs
only and ACNs and Co-Q
10
groups, respectively.
However, these fluctuations did not portray a statisti-
cal significant difference (P > 0.05) between the differ-
ent groups analysed.
Mice receiving both antioxidants showed a signifi-
cant decrease in mean body weight changes during
the experimental period (P > 0.05) (Fig. 3). The
decrease in weight commenced immediately after
start of antioxidant administration, falling from
26.76 ± 0.77 to 23.30 ± 1.67 g by the seventh
DPSAA after which a marginal increase was observed
reaching 23.45 ± 1.82 g. Animals supplemented with
ACNs lost weight consistently from the first DPSAA
to the last day of the experiment, dropping from
27.88 ± 1.60 to 25.76 ± 1.69 g. Untreated animals
registered an unsteady but gradual increase in mean
body weight rising from 24.64 ± 0.59 to 26.3 ±
0.50 g by the end of the experimental period.
However, no significant differences in mean body
weight were recorded between untreated animals and
animals supplemented with ACNs (P > 0.05).
The effect of ACNs and Co-Q
10
on GSH levels
Level of GSH in brain tissue of mice supplemented
with either one or both antioxidant supplements is pre-
sented in Fig. 4. Results indicate that supplementing
experimental animals with Kenyan purple tea ACNs
Figure 1 A representative HPLC chromatogram of un-aerated (green) tea from a Kenyan purple tea variety clone TRFK 306.
Table 1 Concentration of ACNs (μg/ml) in non-aerated
(green) tea derived from Kenyan purple leaf coloured variety
TRFK 306 by HPLC
Individual ACNs/anthocyanidins Concentrations (μg/ml)
Cyanidin-3-O-galactoside 139.25
Cyanidin-3-O-glucoside 50.26
Delphinidin 122.85
Cyanidin 1755.60
Pelargonidin 840.08
Peonidin 371.36
Malvidin 304.83
Total ACN content 3584.23
Table 2 Retention times in minutes of the detected
metabolites against intact individual ACNs
Chromatogram B retention times in
minutes of the detected metabolites
Possible
metabolites of
23.321 Cyanidin-21.580
Pelargonidin-24.671
25.227 Pelargonidin-24.671
Peonidin-25.630
27.114 Malvidin-28.931
Chromatogram C retention times in
minutes of the detected metabolites
Possible
metabolites of
15.334 Delphinidin-17.250
21.272 Cyanidin-21.580
28.287 Malvidin-28.931
Rashid et al. Kenyan purple t ea ACNs to cross the BBB and boost the brain antioxidant capacity
Nutritional Neuroscience 2014
VOL. 17 NO. 4182
significantly boosted the levels of endogenous total
GSH levels in the brain tissue (2.22 ± 0.18 μM) as
compared with the controls (1.27 ± 0.12 μM)
(P = 0.0006). However, when both test antioxidants
were provided, there was a decline in total GSH
levels to amounts below even the level recorded in
healthy animals not supplemented with antioxidants
(0.97 ± 0.06 μ M).
Discussion
Polyphenols and other phytochemicals in plants must
cross the BBB to be able to exert their beneficial effects
in the central nervous system (CNS). Otherwise, the
multitude of health benefits associated with these fla-
vonoids such as strong antioxidant and anti-inflamma-
tory properties would be excluded from the brain and
the CNS in general. Despite their proven potential
health benefits, studies on the bioavailability of
ACNs in the brain tissue have received much less atten-
tion than those for other flavonoids in tea. Most
studies have concentrated on the bioavailability and
neuroprotective effects of catechins, the most abun-
dant polyphenols in tea. EGCG, the most potent
member in the catechin family has previously been
shown to cross the BBB protecting against neuronal
death in a multitude of neurological diseases.
7
Despite this remarkable observation, there exist no
data on the bioavailability of Kenyan purple tea
ACNs. Hence, this study endeavoured to evaluate the
bioavailability of purple tea ACNs in the brain tissue
of mice and their ability to enhance antioxidant
capacity in this vital organ.
The amplification of endogenous antioxidants in the
brain tissue of animals supplemented with ACNs
strongly hinted the presence of these flavonoids in
Figure 2 Representative HPLC chromatogram of brain homogenates from animals not supplemented with antioxidants (A),
animal supplemented with ACNs and Co-Q
10
(B), and animal supplemented with ACNs only (C). Chromatograms B and C show
presence of possible ACNs metabolites in the brain tissue indicated by arrows.
Table 3 Changes in PCV levels of mice supplemented with
ACNs, ACNs and Co-Q
10
, or water only
Days post start of
antioxidant
administration
PCV levels in %
Controls ACNs
ACNs and
Co-Q
10
3 55.2 ± 0.49 53.4 ± 1.03 53.40 ± 1.36
6 57.4 ± 0.51 58.6 ± 1.08 59.25 ± 2.03
12 58.4 ± 2.50 53.2 ± 1.93 55.00 ± 0.71
Figure 3 Changes in body weight of mice supplemented
with ACNs, ACNs and Co-Q
10
, or water only.
Rashid et al. Kenyan purple tea ACNs to cross the BBB and boost the brain antioxidant capacity
Nutritional Neuroscience 2014
VOL. 17 NO. 4 183
the brain, since compounds are known to act directly at
the sites where they localize. The presence of these fla-
vonoids in the brain was later confirmed by the detec-
tion of metabolites in ACN-fed animals but absent in
the placebo group. However, it was not possible to
detect intact ACNs from the brain homogenates.
Considering that approximately two-thirds of ACNs
are highly biotransformed and end up as methylated
and glucuronidated metabolites,
16
absence of intact
ACNs in the brain homogenates was not surprising.
Although the study reports for the first time that
ACNs from Kenyan purple tea cultivars are bioavail-
able in the brain, several other authors have previously
reported the presence of ACNs from other plant
sources in the brain and the CNS in general.
Previously, it was reported that ACNs from blueberries
are able to transverse the BBB and their concentrations
correlated with cognitive performance.
17
A separate
study concurred with this finding and reported the
presence of intact ACNs in the eyes and the brain
tissue of blueberry-fed pigs.
18
Findings from the
present study provide new information on the ability
of Kenyan purple tea ACNs to cross the BBB enhan-
cing the brains antioxidant capacity.
Our results also indicate that oral intake of Kenyan
purple tea ACNs markedly increased brain GSH levels
suggesting that this nutraceutical had a sparing effect
on endogenous antioxidants. Indeed, ACNs from a
wide array of sources have been shown in several
instances to up-regulate endogenous antioxidant
levels. A previous study working with the same
variety of purple tea reported an increase in cellular
GSH content in cells exposed to ACNs.
13
Red mixed
berry juice rich in ACNs has been shown to decrease
oxidative DNA damage, increase GSH levels and
GSH status in healthy human volunteers.
19
ACNs
have also been shown to protect against GSH
depletion by activating GSH-related enzymes such as
GR and GPx with concomitant increase in antioxi-
dant capacity and decreased vulnerability of cells to
oxidative stress.
6
The mechanisms by which ACNs
are thought to exhibit these effects include quenching
of ROS, chelating of metals known to participate in
reactions that result in the vicious formation of reac-
tive moieties, and by the activation of antioxidant
response element upstream of genes that are involved
in antioxidation and detoxification.
20
Our results on PCV, body weight, and brain gluta-
thione strongly suggest negative interactions between
Kenyan purple tea ACNs and Co-Q
10
. An antagonisti-
c effect between the two antioxidants was unexpected
as it was thought that the nutraceuticals would have
a more pronounced effect in combination rather than
in isolation. It is not clear whether this was a mere
coincidence. However, ACNs and other substances
known to lower cholesterol levels or prevent its absorp-
tion are expected to have adverse effects on non-sterol
compounds such as Co-Q
10
.
21
The negative inter-
actions have also been observed while employing
other lipid-lowering substances such as hydroxymethly
glutarly CoA (HMG-CoA) competitive inhibitors
known as statins.
23
In fact, several authors have
reported the ability of Co-Q
10
to reverse the detrimen-
tal side effects associated with statins including muscle
myopathy and rhabdomyolysis.
22
Further studies will
be critical to determine if indeed purple tea ACNs
could nullify the beneficial effects of Co-Q
10
sup-
plements or vice versa.
Finally, the HPLC quantification of ACNs in the
Kenyan purple tea cultivars revealed three times
higher concentrations than those established by a pre-
vious study.
13
Moreover, anthocyanidin fractions
identified by using pure anthocyanidin standards
revealed different patterns from those established by
a previous experiment despite using the same purple
tea variety in both experiments.
13
However, tea culti-
vars used in this experiment were obtained from the
Kericho station as opposed to the cultivars used by
in the previous experiment,
13
which were obtained
from the Kangaita substation in Kirinyaga (0°26
S
and 37°15
E, elevation 2020 m a.m.s.l.). Kericho and
Kangaita stations have different altitudes and tea
growing conditions. The different environmental con-
ditions including temperature, humidity, mineral
nutrient availability, light intensity among others
could result in markedly different flavonoid profiles.
Indeed, thes e findings are a direct corroboration of a
previous study which reported that the amount of fla-
vonoids present in plants is markedly seasonal and
depends on climate conditions.
23
Therefore, environ-
mental differences between the two geographical
regions may have contributed to the different ACN
concentrations and profiles observed between the
two tea cultivars. Indeed, higher altitudes are generally
Figure 4 Total brain GSH levels in mice supplemented with
ACNs, ACNs and Co-Q
10
, or water only.
#
P < 0.05, statistically
significant versus untreated group.
Rashid et al. Kenyan purple t ea ACNs to cross the BBB and boost the brain antioxidant capacity
Nutritional Neuroscience 2014
VOL. 17 NO. 4184
considered advantageous and are associated with
higher ACN concentrations in plants than their
lower counterparts.
23
In conclusion, findings from this study provide com-
pelling evidence that Kenyan purple tea ACNs are
able to cross the BBB and exert their physiological
effects in this organ by up-regulating endogenous anti-
oxidant reserves. We therefore recommend the study of
this nutraceutical as a suitable candidate for consider-
ation as dietary supplements to modulate conditio ns
associated with oxidative stress in the brain such as
Alzheimers and Parkinsons disease, amyotrophic
lateral sclerosis and multiple sclerosis. Indeed, one of
our ongoing studies is investigating the neuroprotec-
tive effects of ACNs and Co-Q
10
in a mouse model
simulating brain damage associated with human
African trypanosomiasis.
Acknowledgment
We thank the National Council for Science and
Technology, Tea Research Foundation of Kenya,
and Egerton University for funding this work.
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Rashid et al. Kenyan purple tea ACNs to cross the BBB and boost the brain antioxidant capacity
Nutritional Neuroscience 2014
VOL. 17 NO. 4 185
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Preprint
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Raspberries are rich in phenolic phytochemicals. To study the health benefits of raspberries, four fresh raspberry varieties (Heritage, Kiwigold, Goldie, and Anne) were evaluated for total antioxidant and antiproliferative activities. The total amount of phenolics and flavonoids for each of the four raspberry varieties was determined. The Heritage raspberry variety had the highest total phenolic content (512.7 +/- 4.7 mg/100 g of raspberry) of the varieties measured followed by Kiwigold (451.1 +/- 4.5 mg/100 g of raspberry), Goldie (427.5 +/- 7.5 mg/100 g of raspberry), and Anne (359.2 +/- 3.4 mg/100 g of raspberry). Similarly, the Heritage raspberry variety contained the highest total flavonoids (103.4 +/- 2.0 mg/100 g of raspberry) of the varieties tested, followed by Kiwigold (87.3 +/- 1.8 mg/100 g of raspberry), Goldie (84.2 +/- 1.8 mg/100 g of raspberry), and Anne (63.5 +/- 0.7 mg/100 g of raspberry). The color of the raspberry juice correlated well to the total phenolic, flavonoid, and anthocyanin contents of the raspberry. Heritage had the highest a/b ratio and the darkest colored juice, and the Anne variety showed the lowest phytochemical content and the palest color. Heritage raspberry variety had the highest total antioxidant activity, followed by Kiwigold and Goldie, and the Anne raspberry variety had the lowest antioxidant activity of the varieties tested. The proliferation of HepG(2) human liver cancer cells was significantly inhibited in a dose-dependent manner after exposure to the raspberry extracts. The extract equivalent to 50 mg of Goldie, Heritage, and Kiwigold fruit inhibited the proliferation of those cells by 89.4 +/- 0.1, 88 +/- 0.2, and 87.6 +/- 1.0%, respectively. Anne had the lowest antiproliferative activity of the varieties measured but still exhibited a significant inhibition of 70.3 +/- 1.2% with an extract equivalent to 50 mg of fruit. The antioxidant activity of the raspberry was directly related to the total amount of phenolics and flavonoids found in the raspberry (p < 0.01). No relationship was found between antiproliferative activity and the total amount of phenolics/flavonoids found in the same raspberry (p > 0.05).
Article
The antioxidant capacity of individual anthocyanins is well established. Less information is available however, about the relative contribution to which specific anthocyanins in a complex mixture affect total antioxidant capacity in different soft fruit sources; especially those that share a similar pathway for anthocyanin synthesis. The objectives of this work were to compare the antioxidant capacity of two different soft fruits, blackcurrant and grape, which share similarities in anthocyanin biosynthetic pathways but are composed of distinctly different anthocyanin profiles. Anthocyanin composition profiles of grape and blackcurrant were characterized by High Performance Liquid Chromatography/Mass Spectrometry (HPLC). ORAC (Oxygen Radical Absorbance Capacity) and ABTS (2,2’-azinobis 3-ethylbenzothiazoline- 6-sulfonic acid) assays were used for antioxidant activity quantification. An anthocyanin antioxidant capacity index (AACI) was derived from the product of antioxidant (ORAC) activity for each of major anthocyanins present in blackcurrant and grape, and the sum of anthocyanins recovered from purified fruit extracts to determine the extent that the total antioxidant activity derived from different anthocyanin combinations. Blackcurrant contained four predominant anthocyanins, cyanidin3-glucoside (Cy3G), delphinidin3-glucoside (Dp3G), cyanidin3-rutinoside (Cy3R), and delphinidin3- rutinoside (Dp3R). Major anthocyanins found in grape were malvidin3-glucoside (Mv3G), Dp3G, Cy3G, petunidin3-glucoside (Pt3G), and peonidin3-glucoside (Pn3G). A greater (p<0.05) total antioxidant capacity existed for blackcurrant compared to grape when measured by ORAC and ABTS methods. An antioxidant synergy was confirmed for blackcurrant and wind grape thus indicating that this phenomenon is a factor for characterizing total antioxidant activity in both blackcurrant and wine grape.
Article
Characterization and quantification of anthocyanins in selected tea cultivars processed into black (aerated) and green (unaerated) tea products was carried out in this study. The anthocyanins were extracted from tea products processed from a number of newly bred purple leaf coloured Kenyan tea cultivars (Camellia sinensis) using acidified methanol/HCl (99:1 v/v). Extracted anthocyanins were purified by C18 solid phase extraction (SPE) catridges and characterised by HPLC-UV–Visible. They were identified according to their HPLC retention times, elution order and comparison with authentic standards that were available. Total monomeric anthocyanins were determined by the pH-differential method. Although the tea cultivars gave different yields of anthocyanins, the unaerated (green) teas had significantly (p ⩽ 0.05) higher anthocyanin content than the aerated (black) teas. This was attributed to the degradation of anthocyanins by polyphenol oxidase products (catechin O-quinones) formed during the auto-oxidation (fermentation) process of black tea manufacture. Of the six most common natural anthocyanidins, five were identified in the purified extracts from purple leaf coloured tea, in both aerated (black) and unaerated (green) teas namely; delphinidin, cyanidin, pelargonidin, peonidin and malvidin. The most predominant anthocyanidin was malvidin in both tea products. In addition, two anthocyanins namely, cyanidin-3-O-galactoside and cyanidin-3-O-glucoside were also identified. Tea catechins were also identified in the tea products derived from the purple coloured tea cultivars namely, epigallocatechin (EGC), catechin (+C), epicatechin (EC), epigallocatechin gallate (EGCG), and epicatechin gallate (ECG). Correlation between the total catechins versus the total anthocyanins and anthocyanin concentration in unaerated teas revealed significant negative correlations (r = −0.723∗ and r = −0.743∗∗, p ⩽ 0.05 and p ⩽ 0.01, respectively). However, in aerated (black) tea the correlations were insignificant (r = −0.182 and r = −0.241, p > 0.05).
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Neurodegeneration in Parkinson's, Alzheimer's, or other neurodegenerative diseases appears to be multifactorial, where a complex set of toxic reactions, including oxidative stress (OS), inflammation, reduced expression of trophic factors, and accumulation of protein aggregates, lead to the demise of neurons. One of the prominent pathological features is the abnormal accumulation of iron on top of the dying neurons and in the surrounding microglia. The capacity of free iron to enhance and promote the generation of toxic reactive oxygen radicals has been discussed numerous times. The observations that iron induces aggregation of inert alpha-synuclein and beta-amyloid peptides to toxic aggregates have reinforced the critical role of iron in OS-induced pathogenesis of neurodegeneration, supporting the notion that a combination of iron chelation and antioxidant therapy may be one significant approach for neuroprotection. Tea flavonoids (catechins) have been reported to possess divalent metal chelating, antioxidant, and anti-inflammatory activities, to penetrate the brain barrier and to protect neuronal death in a wide array of cellular and animal models of neurological diseases. This review aims to shed light on the multipharmacological neuroprotective activities of green tea catechins with special emphasis on their brain-permeable, nontoxic, transitional metal (iron and copper)-chelatable/radical scavenger properties.