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White Tea (Camellia sinensis Kuntze) Exerts Neuroprotection against

Hydrogen Peroxide-Induced Toxicity in PC12 Cells

Article  in  Plant Foods for Human Nutrition · March 2011

DOI: 10.1007/s11130-010-0203-3 · Source: PubMed

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Plant Foods Hum Nutr
DOI 10.1007/s11130-010-0203-3
ORIGINAL PAPER
White Tea (Camellia sinensis Kuntze) Exerts
Neuroprotection against Hydrogen Peroxide-Induced
Toxicity in PC12 Cells
Víctor López & Maria Isabel Calvo
# Springer Science+Business Media, LLC 2011
Abstract Tea is a popular beverage whose consumption is
associated with prevention of certain disorders. The
objective of the study was to investigate the potential
neuroprotective effect of white tea extract (WTE) on
hydrogen peroxide induced toxicity in PC12 cells. Cells
were treated with various doses of WTE (10–250 μg/ml)
before exposition to 250 μM hydrogen peroxide and cell
survival was determined through the MTT and LDH assays.
Oxidative stress was quantified in the cells after treatments
as intracellular reactive oxygen species (ROS) production
and the antioxidant activity of the extract was assessed in a
cell free system in terms of free radical scavenging capacity.
Results showed that WTE has a significant protective effect
in the PC12 cell line against hydrogen peroxide as cell
survival was significantly superior in WTE-treated cells
compared to hydrogen peroxide-treated cells. A reduction
on intracellular oxidative stress as well as radical scavenging
properties were produced by WTE. Results suggest that WTE
protects PC12 cells against H2O2-induced toxicity, and that
an antioxidant mechanism through ROS scavenging may be
in part responsible for cells neuroprotection.
Keywords Tea . Camellia sinensis . PC12 cells .
Antioxidant . Neuroprotection . Medicinal plants
V. López (*)
Faculty of Health Sciences, San Jorge University,
Autovía A-23 Zaragoza-Huesca km. 510,
50830 Villanueva de Gállego, Zaragoza, Spain
e-mail: ilopez@usj.es
M. I. Calvo
Deparment of Pharmacy and Pharmaceutical Technology,
School of Pharmacy, University of Navarra,
Irunlarrea sn,
31080 Pamplona, Spain
Introduction
Tea is the most consumed beverage worldwide after water.
It is prepared as an infusion with the leaves of Camellia
sinensis Kuntze (Theaceae), a shrub native to China and
India. There are different types of tea depending on
botanical varieties, geographical origin and processing.
Concerning the grade of fermentation, oolong and black
tea are partially or totally fermented, respectively, whereas
green and white tea are not. White tea is prepared with
young leaves and buds before fully opened, plucked and
dried with minimal processing [1]. Tea is drunk for pleasure
and its beneficial effects are also known since ancient times.
Apart from the energizing effect due to methylxanthines such
as caffeine, tea consumption is nowadays associated with a
decrease of cardiovascular disorders, metabolic diseases such
as obesity, and chemoprevention [2]. Many of these
pharmacological effects are related to the antioxidant activity
of tea polyphenols [3].
Oxidative processes in central nervous system (CNS)
may induce neurodegenerative disorders and neuronal
death. Food antioxidants may help the antioxidant capacity
of the organism to prevent from diseases associated with
reactive oxygen species (ROS) and other oxidative damage
inducing agents. In this sense, white tea has been tested for
protective and antioxidant effects using hydrogen peroxide
as toxic agent in PC12 cells.
Materials and Methods
White Tea Extract (WTE) Preparation
WTE was prepared with 50 g of dry white tea leaves
acquired from Tealand (Vitoria, Spain) that were extracted

with 500 ml of 50% methanol after maceration at 4 °C for
24 h. Solvent was removed in a rotatory evaporator, the
extract was then lyophilized and the obtained powder was
kept in glass vials at −40 °C. Stock solutions of WTE were
prepared in Dulbecco’s phosphate buffered saline (DPBS)
under sterile conditions before the experiments.
Cell Culture
Rat pheochromocytoma (PC12) cells were obtained from
the American Type Culture Collection. Cells were grown
in DMEM supplemented with 10% heat inactivated
horse serum, 5% heat-inactivated fetal bovine serum,
penicillin (10 U/ml), streptomycin (10 μg/ml) and
0.2 mM sodium pyruvate. Cultures were incubated in
the presence of 5% CO2 at 37 °C and 100% relative
humidified atmosphere.
Cell Viability
The cytotoxicity detection kit was purchased from Roche
(Indianapolis, USA). Dulbecco’s modified eagle’s medium
(DMEM), penicillin-streptomycin, fetal bovine serum
(FBS), horse serum (HS), sodium pyruvate and Dulbecco’s
phosphate buffered saline (PBS) were obtained from
Gibco (Barcelona, Spain). 3-(4,5-dimethylthiazol-2 yl)-
2,5-diphenyltetrazolium bromide (MTT), hydrogen per-
oxide and 2′,7′-dichlorofluorescein diacetate (DCFH-DA)
were purchased from Sigma-Aldrich (Spain). PC12 cells
viability was studied under exposure to WTE using the
MTT assay [4]. Briefly, cells were seeded in 96-well
microplates at a density of 20×103 cells/well and grown
for 48 h at 37 °C. Cells were treated with various
concentrations of WTE (10–1,000 μg/ml) for 24 h and a
MTT solution was added and incubated for 1 h at 37 °C.
Cell survival (%) was measured as reduction of MTT into
formazan at 550 nm.
Protective Effects on PC12 Cells by the MTT and LDH Assays
The protective effect of WTE against toxicity induced by
H2O2 in PC12 was carried out as previously published by
López et al. [5] using MTT and lactate dehydrogenase
(LDH) assays. Cells were seeded as described above and
treated with WTE (50, 100, 250 μg/ml) for 24 h. PC12 cells
were then exposed to DPBS containing 250 μM H2O2 for
30 min. The MTT assay was performed 24 h after hydrogen
peroxide exposure. Cells were seeded at a density of 2,000
cells/well and treated with WTE (50, 100, 250 μg/ml) for
24 h. PC12 cells were then exposed to DPBS containing
250 μM H2O2 for 30 min. The LDH assay was performed
24 h after hydrogen peroxide exposure through measure-
ment of released LDH into the incubation using a
Plant Foods Hum Nutr
commercial kit from Roche® following the manufacturer’s
instruction.
Measurement of Intracellular Reactive Oxygen Species
(ROS) Formation
Intracellular reactive oxygen species (ROS) formation
was evaluated with the purpose of investigating the
potential antioxidant mechanism. Cells were seeded in
96-well microplates at 20,000 cells/well and grown for
48 h. Cells were incubated with PBS containing 10 μM
2′,7′-dichlorofluorescein diacetate (DCFH/DA) for
30 min. DCFH-DA solution was removed and cells were
treated with the extracts at different doses and 250 μM
H2O2. Intracellular oxidative stress was determined mea-
suring the fluorescence intensity at 485 nm excitation and
530 nm emission for 2 h.
Free Radical Scavenging Properties by the DPPH
Photometric Assay
The 2,2-diphenyl-1- picrylhydrazyl (DPPH) test was used to
evaluate the radical scavenging capacity (RSC) of WTE in cell
free systems according to a published procedure [6] using
ascorbic acid as positive control substance. Different concen-
trations were tested and absorbance was measured at 517 nm
after 30 min of reaction with a stock solution of DPPH.
Phytochemical Screening
WTE was analyzed in a flavonoid system [6], 100 μg of
extract was applied to Merck silica-gel 60F254 and eluted
in ethyl acetate:formic acid:glacial acetic acid:water
(100:11:11:26). The plate was sprayed with natural
products- PEG (viewed under 366 nm) to detect flavo-
noids, using a mixture solution of rutin-chlorogenic acid-
100
Cell survival (% MTT reduction)
80
**
60
40 *** ***
20
0
Control 10 50 100 250 500 1000
WTE (µg/ml)
Fig. 1 Effect of WTE on PC12 cells after exposure for 24 h. ** p<
0.01; *** p<0.001 versus control (non-treated cells)
Plant Foods Hum Nutr

a 180
100
Cell survival (% MTT reduction)

*** *** *** 160

80

% (ROS)
140
60
120
40
100
20
80
0 0 15 30 45 60 90 120
Control 10 50 100 250 Exposure time (min)
H2O2 H2O2 + 100 µg/ml WTE
WTE (µg/ml)
H2O2 + 10 µg/ml WTE H2O2 + 250 µg/ml WTE

H2O2 (250 µM) H2O2 + 50 µg/ml WTE

b 80
Fig. 3 Time-dose dependent effect of WTE on intracellular reactive
oxygen species (ROS) production induced by hydrogen peroxide. A
Cell death (% LDH release)

significant decrease on intracellular ROS was detected in cells treated

with 100 and 250 μg/ml WTE compared to H2O2 group (cells treated
60 with 250 μM H2O2)

** Results and Discussion

40

Tea is a widely consumed beverage in daily human diet

20
*** *** with proved beneficial effects [1], currently being the major
source of dietary flavonoids in U.S. adults [7]. In addition,
non-fermented teas, such as green and white, are considered
0 good sources of natural antioxidants. Whereas green tea
Control 10 50 100 250
composition and bioactivity has been in-depth investigated
WTE (µg/ml) [8–13], there are few studies about white tea extracts.
Antioxidant and lipolytic activities of white tea have been
H2O2 (250 µM) reported [14, 15]; however, the neuroprotective effect of
this variety has never been established in neuronal-like
Fig. 2 Neuroprotective effect of WTE on hydrogen peroxide
induced toxicity in PC12 cells by the MTT (a) and LDH (b) assays. cells. In the present study, we have evaluated the protective
** p<0.01; *** p<0.001 versus hydrogen peroxide-treated cells.
Control group refers to non-treated cells 100

80
% DPPH inhibition

hyperoside as reference. WTE was tested for alkaloids

60
using Draggendorff’s reagent.

40
Statistical Analysis
Ascorbic acid
Data are mean ± SD of at least three independent experi- 20
ments. Statistical significance was evaluated through the WTE
most appropriate test using GraphPad Prism v.5.0. One-way 0
analysis of variance (ANOVA) followed by Dunnett’s 0 5 10 15 20
multiple comparison post-test was used for MTT and Dose (µg/ml)
LDH assays whereas ROS data were analyzed through Fig. 4 Free radical scavenging profile of WTE expressed as % of DPPH
two-way ANOVA. IC50 values from DPPH experiment inhibition compared to ascorbic acid. IC50 values for WTE and ascorbic
were compared using Student t-test. acid were 1.0±0.1 and 7.4±0.4, respectively, *** p<0.001

effect of white tea against oxidative stress in rat pheochro-
mocytoma (PC12) cells because the PC12 cell line retains
features of dopaminergic neurons [16].
PC12 cells were firstly treated with different concen-
trations of WTE for 24 h to test the extract cytotoxicity. The
effect of the extract on the cells is shown in Fig. 1. Doses
over 250 μg/ml were considered toxic in the PC12 cultures
as cell survival was significantly reduced until approxi-
mately 30% (p<0.01). We next evaluated the protective
effect of WTE on H2O2-induced toxicity in PC12 cells.
Cells were treated for 24 h with different concentrations of
extract (10–250 μg/ml) before exposure to 250 μM H2O2
and cell survival was determined through MTT and LDH
assays. Concerning these assays, treatments with 50, 100
and 250 μg/ml of WTE protected PC12 against hydrogen
peroxide toxicity (Fig. 2). Significant differences were
detected comparing cells exposed to H2O2 versus cells
treated with WTE before exposition to the toxic agent
(p<0.01). In order to elucidate mechanisms by which WTE
provides protective effects, intracellular oxidative stress and
free radical scavenging properties were measured. Thus, cells
were treated with increasing concentrations of WTE and
250 μM H2O2, quantifying levels of intracellular ROS for
2 h. Results demonstrate that PC12 cells treated with
WTE in the presence of hydrogen peroxide reduced
intracellular ROS formation in a time-concentration
dependent manner (Fig. 3). 250 μg/ml WTE was the
most active concentration, decreasing ROS formation in
59% after 2 h treatment (p<0.001). Regarding antioxidant
properties in the cell free system, WTE (IC50-value of
1.0 μg/ml) was able to scavenge DPPH radicals more
efficiently than ascorbic acid (IC50-value of 7.4 μg/ml)
(Fig. 4).
Oxidative stress in CNS is known to induce neuronal
death and to be involved in certain disorders such as
Alzheimer’s and Parkinson’s diseases. Consequently, the
consumption of food plants with neuroprotective effects in
our diet may represent a strategy to prevent from a wide
range of diseases. Although tea composition is complex, it
has been reported to contain metylxanthines (caffeine),
polyphenols (flavonoids), amino acids (theanine) and
vitamins (ascorbic acid and B-complex) [17]. In our
phytochemical screening, WTE revealed flavonoids (yellow
spots viewed at 366 nm), though Draggendorff’s test was
negative for caffeine. Isolated constituents from tea have
previously been demonstrated to exert protective effects in
neuronal cells; for instance, studies reveal neuroprotective
effects of epigallocatechin-3-gallate [18] and the amino acid
theanine [19]. In this sense, results suggest that protection
may be due to a synergistic effect among various
compounds.
More in-depth research is needed to define health
benefits and clinical effects of white tea. However, these
Plant Foods Hum Nutr
findings demonstrate the protective effect of a highly
consumed tea variety against oxidative stress in PC12 cells,
whose neuroprotection is mediated, at least in part, through
free radical scavenging properties.
Acknowledgements University of Navarra Foundation and Association
of Friends of the University of Navarra are thanked for financial support.
Dr. M.P. Gómez-Serranillos (Complutense University of Madrid) is
thanked for providing PC12 cells.
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