Hindawi Publishing Corporation

Evidence-Based Complementary and Alternative Medicine

Volume 2011, Article ID 156765, 11 pages
doi:10.1155/2011/156765

Research Article
Antiapoptotic and Antioxidant Properties of
Orthosiphon stamineus Benth (Cat’s Whiskers):
Intervention in the Bcl-2-Mediated
Apoptotic Pathway

Siddig Ibrahim Abdelwahab,1 Syam Mohan,2 Manal Mohamed Elhassan,2

Nabil Al-Mekhlafi,2 Abdelbasit Adam Mariod,3 Ahmad Bustamam Abdul,2
Mahmood Ameen Abdulla,4 and Khalid M. Alkharfy5
1 Department of Pharmacy, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia
2 Instituteof Biosciences, Universiti Putra Malaysia, 43400 Serdang, Malaysia
3 Food Science and Technology Department, College of Agricultural Studies, Sudan University for Sciences and Technology,

P.O. Box 71, Khartoum North, Sudan

4 Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia
5 Department of Clinical Pharmacy, College of Pharmacy, King Saud University, Riyadh 11451, Saudi arabia

Correspondence should be addressed to Siddig Ibrahim Abdelwahab, siddigroa@yahoo.com

Received 14 July 2010; Revised 13 October 2010; Accepted 30 November 2010

Copyright © 2011 Siddig Ibrahim Abdelwahab et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.

Antiapoptotic and antioxidant activities of aqueous-methanolic extract (CAME) of Orthosiphonstamineus Benth(OS), and its
hexane (HF), chloroform (CF), n-butanol (NBF), ethyl acetate (EAF) and water (WF) fractions were investigated. Antioxidant
properties were evaluated using the assays of Folin-Ciocalteu, aluminiumtrichloride, β-carotene bleaching and DPPH. The role of
OS against hydrogen peroxide induced apoptosis on MDA-M231 epithelial cells was examined using MTT assay, phase contrast
microscope, colorimetric assay of caspase-3, western blot and quantitative real-time PCR. Results showed that EAF showed the
highest total phenolic content followed by CAME, NBF, WF, CF and HF, respectively. Flavonoid content was in the order of the
CF > EAF > HF > CAME > NBF > WF. The IC50 values on DPPH assay for different extract/fractions were 126.2 ± 23, 31.25 ±
1.2, 15.25 ± 2.3, 13.56 ± 1.9, 23.0 ± 3.2, and 16.66 ± 1.5 μg/ml for HF, CF, EAF, NBF, WF and CAME, respectively. OSreduced the
oxidation of β-carotene by hydroperoxides. Cell death was dose-dependently inhibited by pretreatment with OS. Caspase-3 and
distinct morphological features suggest the anti-apoptotic activities of OS. This plant not only increased the expression of Bcl-2,
but also decreased Bax expression, and ultimately reduced H2 O2 -induced apoptosis. The current results showed that phenolics
may provide health and nutritional benefits.

1. Introduction Orthosiphon stamineus Benth (family: Lamiaceae) or

In the past, herbs often represented the original sources of
most drugs, but nowadays, alternative medicines are used
widely in all over the world. With increasing recognition of
herbal medicine as an alternative form of health care, screen-
ing of medicinal plants for biologically active extracts and/or
compounds has become an important source of drugs [1–3],
many of them with unique pharmacological and chemical
properties. Herbal-derived remedies need a powerful and
deep assessment of their pharmacological qualities [4].
Misai Kucing (Malay for “Cat’s Whiskers”) is commonly used
as Java Tea. Java Tea is a medicinal plant, native in South
East Asia (Malaysia, Indonesia, and Thailand) and some
part of Tropical Australia. It is a herbaceous shrub which
grows to a height of 1.5 meter. This plant is best used for
treating the ailments or problems of kidney and bladder due
to its mild diuretic action. It is used as a remedy for kidney
stone and nephritis. It is believed to have antiallergenic,
antihypertensiveand anti-inflammatory properties. OS is
also used for treating gout, diabetes, hypertension, and
2 Evidence-Based Complementary and Alternative Medicine
rheumatism. It is also reported to possess antifungal, and
it exhibits considerable antibacterial activity. Java tea/misai
kucing is appearing in many products where safe diuretic
action is required such as detoxification, water retention,
hypertension, weight loss, and for kidney stones. The
products appear in form of tablets, capsules, tea sachets,
drinks, raw herbs, dried leaves, or extracts [5–13].
O. stamineus contains several chemically active con-
stituents such as polyphenols, terpenoids, oleanolic acid,
and sterols [14]. The polyphenols are the most dominant
constituents in the leaf, which has been reported to be effec-
tive in reducing oxidative stress by inhibiting the formation
of lipid peroxidation products in biological systems [15].
Recently, the diuretic activity of methoxy flavonoids isolated
from OS was reported [16]. Phenolic compounds such as
lipophilic flavones, caffeic acid derivatives (rosmarinic acid
and 2,3-dicaffeoyltartaric acid), eupatorine, cichoric acid,
sinensetin and methoxy flavones were found in O. stamineus
[17, 18]. Three highly oxygenated 2,3-secoisopimarane-type
diterpenes, named secoorthosiphols A, B, and C, have been
isolated from the aerial parts of this plant [19].
The therapeutic benefit of medicinal plants is usually
contributed to their antioxidant properties [20]. Phenolic
compounds possess diverse biological activities such as
anti-inflammatory, anticarcinogenic, and antiatherosclerotic
activities. These activities of phenolic compounds might be
related to their antioxidant activities [21]. Other studies
showed that there were significant correlations between
phenolic compounds and antioxidant properties of medic-
inal plants. Therefore, the current study was conducted
to investigate the antioxidant and antipoptotic potency of
aqueous-methanol extract/fractions of O. stamineus.
2. Materials and Methods
2.1. Plant Materials. Leaves of OS (Lamiaceae) were collected
in July 2009 from the Institute of Bioscience, Universiti Putra
Malaysia, Selangor, Malaysia. Authentication was done at the
same institute where the voucher specimen under the plant
name was deposited. The freshly collected leaves were washed
and weighed. After that the parts were cut in small and dried
at 50–60◦ C for 5 days. The dried leaves were weighed and
then grounded to powder form. The powder form of plants
were weighed and kept in an airtight plastic bag at room
temperature.
2.2. Aqueous-Methanolic Extraction and Liquid-Liquid Frac-
tionation of Orthosiphon stamineus. Extraction was done
according to the method recommended by Mariod et al. [22].
Thirty grams of the dried ground leaves of OS were extracted
successively with 60% methanol (3 × 300 mL) to obtain crude
methanolic extract (CAME) at room temperature for two
days each, then combined, and concentrated by removing
solvent by rotary evaporator (Buchi, Flawil, Switzerland).
The obtained CAME (8.27 g) was fractionated using hexane,
chloroform, ethyl acetate, n-butanol, and water individually,
where the residue from each fractionation step was used
to obtain the subsequent fraction as shown in Figure 1.
Each extraction process involved homogenisation of CAME
and its fractions in the respective solvent at 13,000 rpm for
15 min followed by sonication at constant temperature of
30◦ C for 1 h. The CAME and its fractions (hexane fraction
HF, ethyl acetate fraction EAF, chloroform fraction CF, n-
butanol NBF, water fraction WF) were filtered through filter
paper Whatman no. 1. Then solvents were removed by using
rotary evaporator (Buchi, Flawil, Switzerland). The yield of
each extract and its fractions was measured before kept in
refrigerator for further analysis.
2.3. Total Phenolic Content (TPC). TPCs of HF, EAF, CF,
NBF, WF, and CAME were determined using Folin-Ciocalteu
method [23]. Briefly, stock solutions of extract and fraction
of OS were prepared in a concentration of 10 mg/mL in
methanol. Five microliters of each solution were transferred
to 96-well microplate (TPP, USA). To this, 80 μL of Folin-
Ciocalteu reagent (1 : 10) were added and mixed thor-
oughly. After 5 min, 160 μL of sodium bicarbonate solution
(NaHCO3 7.5%) were added, and the mixture was allowed
to stand for 30 min with intermittent shaking. Absorbance
was measured at 765 nm using microplate reader (Molecular
Devices, Sunnyvale, USA). The TPC was expressed as gallic
acid equivalent (GAE) in mg/g extract, obtained from the
standard curve of gallic acid. The gallic acid standard curve
was established by plotting concentration (mg/mL) versus
absorbance (nm) (y = 0.001x + 0.045; R2 = 0.9975), where
y is absorbance and x is concentration in GAE (n = 3).
2.4. Total Flavonoid Content. Total flavonoid contents
(TFCs) were determined by the AlCl3 method [24] using
rutin as a standard. The test samples were dissolved in DMSO
(10 mg/mL). The sample solution (1 mL) was mixed with
one millilitre of AlCl3 (2.0%). After 10 min of incubation
at ambient temperature, the supernatant was transferred
to 96-well plate. The absorbance of the supernatant was
measured immediately at 435 nm using microplate reader
(Molecular Devices, Sunnyvale, USA). TFC was expressed as
rutin equivalents (REs) in milligrams per gram sample. For
the rutin, the curve was established by plotting concentration
(μg/mL) versus absorbance (nm) (y = 5.6752x − 0.0312;
R2 = 0.99). Here y = absorbance, and x = concentration
(n = 3).
2.5. Free Radicals Scavenging of DPPH. Radical scavenging
activity of extract and fractions of OS against DPPH (2, 2-
diphenyl-2-picrylhydrazyl hydrate, Sigma-Aldrich Chemie,
Steinheim, Germany) was determined spectrophotometri-
cally. When DPPH (Violet colour) reacts with an antioxidant
compound, which can donate hydrogen, it is reduced and
produces a yellow colour. The changes in colour were
measured at 517 nm wavelength [25]. Radical scavenging
activity of extract/fractions of OS was measured by slightly
modified method of Chan et al. [25], as described below.
Stock solutions were dissolved in methanol (10 mg/mL).
The working solution was prepared using methanol in a
concentration of 2000 μg/mL. The solution of DPPH in
methanol (2.5 mg/mL) was freshly prepared. Five μL of
Evidence-Based Complementary and Alternative Medicine
O. stamineus leaves
Identification
Dried powder
Aqueous methanolic extract
Extract dissolved in deionised water
Hexane fraction
Chloroform fraction
Ethyl acetate fraction
n-butanol fraction
Figure 1: Extraction and fractionation of O. stamineus.
this solution were mixed with 100 μL of serial dilutions of
samples (15.625–2000 μg/mL) in 96-well plate. The samples
were kept in the dark for 30 min at ambient temperature,
and then the change in absorption was measured (Molecular
Devices Plate Reader, Sunnyvale, USA). Absorption of blank
sample containing the same amount of methanol and DPPH
solution was prepared and measured daily. The experiment
was carried out in triplicate. Radical scavenging activity was
calculated by the following formula:
 
A B − AA
% Inhibition = × 100,
AB
where AB is the absorption of blank sample; AA is the
absorption of tested samples. The inhibitory concentration
50% was determined as well as the kinetics of DPPH
scavenging reaction. Ascorbic acid was also tested against
DPPH as positive control.
2.6. Beta-Carotene Bleaching Assay (BCB). The antioxidant
activity (AOA) of the different extracts/fractions of OS was
evaluated using the β-carotene-linoleic acid assay following
the method of Amarowicz et al. [26]. An aliquot of
5.0 mg from β-carotene was dissolved in 50 mL chloroform.
Linoleic acid 200 μL and 600 mg Tween-20 were mixed
with 1 mL of the chloroform solution. The chloroform was
evaporated under vacuum at 45◦ C for 20 min, then 50 mL
oxygenated water was added, and the mixture was vigorously
shaken to obtain β-carotene-linoleic acid emulsion. Two
hundred μL of this emulsion were distributed in a 96-
well microplate. Methanolic solutions (10 μL, 2 mg/mL) of
3
Drying and grinding
60% methanol, maceration (3X),
filtration, evaporation
Liquid-liquid
- fractionation,
hexane
Residues
Liquid-liquid fractionation,
chloroform
Residues
Liquid-liquid fractionation,
ethyl acetate
Residues
Liquid-liquid fractionation,
n-butanol
Residues
the sample extracts and positive control BHA (butylated
hydroxyanisole) were added into the plate. An equal amount
of methanol was used as negative control. Absorbance was
read at 470 nm after incubation for every 20 min until
120 min at 50◦ C using a microplate reader (Molecular
Devices Plate Reader, Sunnyvale, USA). Tests were performed
in triplicate.
2.7. Antipoptotic Effect of O. stamineus against H2 O2
Induced Cell Injury
(1)
2.7.1. Cell Culture and Drug Treatment. RPMI-1640 medium
was used to culture MDA-M231 cells. Cultures were supple-
mented with 10% fetal calf serum, penicillin (100 U/mL),
and streptomycin (100 μg/mL). Stock cultures of exponen-
tially growing cells were trypsinized and plated (2 × 105
cells/well) into 96-well plate and incubated at 37◦ C for 48 h
prior to use then washed twice with PRMI-1640 medium
and recultured therein. HF, EAF, CF, NBF, WF, CAME, and
quercetin (positive control) dissolved in methanol, and later
diluted with media, was added to the wells and incubated
for 10 min before the addition of hydrogen peroxide (final
concentration 400 μmol/L) [27]. After 2 h of incubation, cell
viability in every well was assayed.
2.7.2. Assessment of Cell Viability. Cell viability could be
quantified using MTT, which yields a purple formazan
product in living cells, but not in dead cells or their lytic
debris. MTT was dissolved in aseptic PBS to a concentration
of 5 mg/mL as a stock solution. MTT was added at the
end of incubation to a final concentration of 0.5 mg/mL
4 Evidence-Based Complementary and Alternative Medicine
and then incubated at 37◦ C for 4 h, and the resultant
formazan product was solubilised using dimethyl sulphoxide
and detected using UV-spectrometer at 570 nm.
2.7.3. Microscopic Observation of Cellular Morphology Using
Phase-Contrast Inverted Microscope. This analysis examines
morphologically whether cell death may be implicated
in mediating H2 O2 among MDA-MB231 cells. The EAF
was chosen in this analysis, since this fraction showed
the most potential results in MTT assay. Morphological
appearance of treated cells (EAF + Hydrogen Peroxide) was
compared with the control cells (Hydrogen Peroxide) by
using the inverted light microscope. Apoptosis was identified
using morphological criteria, including shrinkage of the
cytoplasm, membrane blebbing, and nuclear condensation,
and/or fragmentation.
2.7.4. Colorimetric Assays of Caspase-3. The colorimetric
protease assay of caspase-3 provides a simple and convenient
means for quantifying the enzyme activity that recognize the
amino acid sequence, DEVD (a synthetic tetrapeptide, (Asp-
Glue-Val-Asp), which is the upstream amino acid sequence
of the caspase-3 cleavage site), coupled with p-nitroanilide,
which is released upon substrate cleavage. This assay was
performed using ApoTarget Kit (BioSource International,
Inc., Calif, USA). Cells (2 × 106 ) were treated with hydrogen
peroxide with or without EAF and incubated for 72 hours
while untreated cells acted as control. The cells were lysed by
the addition of 50 μL of chilled cell lysis buffer and incubated
on ice for 10 minutes. The resulting cell lysate was cen-
trifuged for 1 minute at 10,000 xg, and the supernatant was
collected. Fifty microliters of 2X reaction buffer (containing
10 mM DTT) were added to each sample. Then 5 μL of
DEVD-pNA (caspase-3 substrate) was added and incubated
in the dark at 37◦ C for 1 hour. At the end of the incubation
period, the samples were read at 405 nm in a microplate
reader (TECAN, Sunrise, Mannedorf, Switzerland). Data was
presented as optical density (n = 3).
2.7.5. Western Blotting. This assay was used to investigate
the expression of apoptosis-associated protein, Bcl-2. MDA-
M231Cells were lysed using CSK buffer (10 mM pipes,
pH 6.8, 100 mM NaCl, 1 mM MgCl2 , 1 mM EGTA, 1 mM
dithiothreitol, and 1 mM phenylmethanesulfonyl fluoride)
containing 0.1% Triton X-100, 1 mM ATP, and proteinase
inhibitors (Pharminogen, USA). Cell lysates were centrifuged
at 20,000 x g for 30 min, and the protein concentration in
the supernatant was determined with Coomassie method.
Equal amounts of protein were separated by SDS-PAGE and
transferred to a PVDF membrane (Bio-Rad). The membrane
was incubated at 37◦ C for 70 min with primary antibodies in
a blocking solution. After being washed with Tris buffered
saline, the membrane was incubated with conjugated sec-
ondary antibodies (Biosyntech, USA). The immunoreacted
proteins were detected using a chemiluminescence system
(Bio-Rad, USA).
2.7.6. Real-Time Polymerase Chain Reaction. Total RNA was
extracted using RNeasy Mini Kit following the manufac-
turer’s instructions (Qiagen, Germantown, Maryland, USA).
RNA concentrations were quantified using a spectropho-
tometer (Smart Spec, Bio-Rad). RNA quality and integrity
were determined via the A260/A280 ratio and agarose
gels electrophoresis, respectively. cDNA was synthesized
with Revert Aid H Minus M-muLV reverse transcriptase
(Biometra, Goettingen, Germany). Real-time RT-PCR was
performed using an ABI 7700 Prism Sequence Detection
System and TaqMan primer probes (Applied Biosystems,
Foster City, CA). The total reaction volume was 20 μL : 2 μL
cDNA, 10 μL SYBR Premix ExTaq, 0.4 μL of each primer
(10 μM), and 7.2 μL ultrapure water. Cycle parameters were
as follows: activation at 95◦ C for 30 s, 40 cycles of denatura-
tion at 95◦ for 5 s, and then annealing and extension at 60◦ C
for 30 s. Glyceraldehyde-3-phosphate dehydrogenase gene
(GAPDH) was used as an internal control for each sample.
PCR products were detected using gel electrophoresis.
The primer sequences for Bax were
Forward: 5 -TGCTTCAGGGTTTCATCCA-3
Reverse: 5 -GACACTCGCTCAGCTTCTTG-3
The primer sequences for Bcl-2 were
Forward: 5 -GGGAGAACAGGGTACGATAA-3
Reverse: 5 -GCTGGGAGGAGAAGATGC-3
The primer sequences for GAPDH were
Forward: 5 -GGATTTGGTCGTATTGGG-3
Reverse: 5 -TCGCTCCTGGAAGATGG-3 .
2.8. Statistical Analysis. Experimental values are means ±
SD of the number of experiments indicated in the legends.
Significance was assessed by using the one-way ANOVA
followed by multiple comparison test (P < .05 as significant).
Pearson correlation coefficient was used to assess the correla-
tion between phenolic content and antioxidant activities.
3. Results and Discussion
3.1. Efficiency of Aqueous Methanolic Extraction and Fraction-
ation. Methanol has been recommended for the extraction
of phenolic compounds from plant tissues. It is an appropri-
ate solvent due to its ability to inhibit polyphenol oxidase,
which could amend antioxidant activity. High methanol
extraction efficiency has been reported for leaves and flowers
of some plant species [28]. Leaf powder of OS when extracted
previously using aqueous methanolic solvent has shown high
phenolic content and good free radical activity. The amount
of rosmarinic acid, the principal polyphenol in OS leaf, was
confirmed earlier to be higher in aqueous methanol extract,
and the amount of this acid was significantly different from
the other solvent extracts [9, 12]. The current study provide,
for the first time, a standardized method to obtain rich
antioxidant fractions from OS. In this study, amount of
extractable compounds (TECs) was calculated as milligram
extract in each gram of powdered leaves. As shown in Table 1,
the highest TEC was revealed by WF (178.34 mg/g), followed
Evidence-Based Complementary and Alternative Medicine
Table 1: Total extractable compounds (TEC), total flavonoids content (TFC), total phenolic compounds (TPC), and DPPH IC50 (mg/mL)
of different extract/fractions obtained from O. stamineus (n = 3).
Extract/fractions of
TEC (mg/g)
O. stamineus
HF 4.02
CF 7.75
EAF 12.62
NBF 72.93
WF 178.34
CAME 275.66
Values with different superscript letters within the same column are statistically different (P < .05).
by NBF (72.93 mg/g), EAF (12.62 mg/g), CF (7.75 mg/g),
and HF (4.02 mg/g), respectively. The TECs were obviously
varying between the fractions of OS leaves and increase
gradually with the increasing of the solvents’ polarity.
3.2. Total Phenolic and Flavonoids Contents. Since plant
phenolics represent one of the major groups of compounds
behaves as primary antioxidants or free radical scavengers
[29], it was reasonable to determine their total amount in
the extract/fractions of OS using Folin-Ciocalteau method.
The TPC was expressed as gallic acid equivalent in mg/g,
GAE. The content of the total phenolic compounds of CAME
and its different fractions (HF, EAF, CF, NBF, and WF) is
shown in Table 1. Results in this table showed that EAF
showed the highest TPC followed by CAME, NBF, WF, CF,
and HF, respectively. TPC of ethyl acetate fraction (EAF) is
significantly (P < .05) higher in comparison to the different
extract/fractions obtained from OS. Ethyl acetate is often
used as an extraction solvent with a significant selectivity in
the extraction of low-molecular-weight phenolic compounds
and high-molecular-weight polyphenols [30]. On the other
hand, Conde et al. have reported that ethyl acetate allowed
the highest phenolic content and the selective removal of
nonphenolic compounds [31].
Flavonoids are the most common and widely distributed
group of plant phenolic compounds, which usually are very
effective antioxidants [32]. In this study, total flavonoids
content (TFC) of OS was determined (Table 1). The TFC
was expressed as rutin equivalent in mg/g, RE. The results
showed that the flavonoids content was in the order of the
CF >EAF> HF > CAME > NBF > WF with the values of
58.11 ± 9.5, 28.83 ± 9.1, 24.52 ± 2.5, 17.56 ± 5.5, 14.94 ±
1.9, and 13.20 ± 4.2 mg RE/g extract, respectively. From
our results, the chloroform fraction (CF) gave the highest
TFC. There was a significant difference (P < .05) of the
TFC in CF, HF, and EAF compared with the amounts in
NBF, WF, and CAME. Among the various solvents used for
the fractionation, chloroform and hexane have the lowest
polarity; therefore, the amounts of lipophilic flavonoids
were higher, as expected. This indicates that the lipophilic
flavonoids are major phenolic compounds present in OS.
5
Total phenolic content Total flavonoids DPPH IC50
(GAE mg/g content (RE mg/g (μg/mL)
extract/fraction) extract/fraction)
35.507 ± 1.420a 24.52 ± 2.5b 126.2 ± 23
139.813 ± 12.164bd 58.11 ± 9.5c 31.25 ± 1.2
c b
222.927 ± 7.580 28.83 ± 4.1 15.25 ± 2.3
bd a
183.250 ± 14.110 14.94 ± 1.9 13.56 ± 1.9
160.327 ± 15.552bd 13.20 ± 4.2a 23.0 ± 3.2
bd a
189.073 ± 49.15 17.56 ± 5.5 16.66 ± 1.5
It can be observed that the content of phenolics in
the extracts/fractions of OS correlates with their anti-
radical activity (e.g., correlation coefficient between data
of DPPH assay and total phenolic compounds is 0.832,
P < .001), confirming that phenolic compounds are likely
to contribute to the radical scavenging activity of these
plant extract/fractions. Detailed examination of phenolic
composition in plant extract/fractions of OS is required
for the comprehensive assessment of individual compounds
exhibiting antioxidant activity. A previous study had found
that phenolic compounds contributed significantly to the
antioxidant capacity of the medicinal herbs as there was
a direct relationship between antioxidant activity and total
phenolic content [33].
3.3. DPPH Scavenging Activity Test. The DPPH assay has
been widely used to test the free radical scavenging ability
of plant materials and pure compounds [22]. The DPPH is a
stable free radical (purple coloured), which is reduced to α,α-
diphenyl-β-picrylhydrazine (yellow coloured) when reacting
with an antioxidant agent. Antioxidants interrupt free radical
chain oxidation by donating hydrogen from hydroxyl groups
to form a stable end-product, which does not commence or
proliferate further oxidation of lipids in human body [28].
The data obtained from this assay is commonly reported
as IC50 , which is the concentration of antioxidant required
for 50% scavenging of DPPH radicals in the specified time
period.
The DPPH scavenging activities of extract/fractions of
OS are shown in Table 1. The IC50 values for different
extract/fractions were 126.2 ± 23, 31.25 ± 1.2, 15.25 ± 2.3,
13.56 ± 1.9, 23.0 ± 3.2, and 16.66 ± 1.5 μg/mL for HF, CF, EAF,
NBF, WF, and CAME, respectively. As depicted in Table 1, a
correlation was observed between the TPC and IC50 when
the TPC level was high; the IC50 was low which indicates
high antioxidant activity. This is due to the high TPC present
in the leaves of OS that act as free radical scavengers. It also
observed that there is a positive linear relationship (P < .05)
between DPHH inhibition and the polyphenolic content of
extract/fractions of OS, whereby polar extract/fractions have
shown the higher DPPH scavenging activities. The findings
6 Evidence-Based Complementary and Alternative Medicine
1.4
Absorbance at 470 nm
1.2
1 molecular-weight phenolic compounds and high-molecular-
0.8
0.6
0.4
0.2
0 grandifolia [35].
0 20 40 60 80 100
Time (min)
HF WF
EAF CAME
CF Control
NBF BHT
Figure 2: Effect of extract/fractions of O. stamineus on oxidation
of β-carotene/linoleic acid at 50◦ C. BHA: butylated hydroxyanisole,
CAME: crude aqueous-methanolic extract, HF: hexane fraction,
CF: chloroform fraction, NBF: n-butanol fraction, EAF: ethyl
acetate fraction, and WF: water fraction.
of the current study are in an agreement with those of
Akowuah et al. [9] who indicated that the extracts of OS are
free radical inhibitors and primary antioxidants that react
with free radicals and the polar extracts have the highest
free radical scavenging activity. However, the current study
investigated for the first time the antioxidant activities of OS
fractions.
3.4. β-Carotene Bleaching (BCB) Assay. In the BCB assay,
the oxidation of linoleic acid produces peroxyl free radicals
due to the abstraction of hydrogen atom from diallylic
methylene groups of linoleic acid [34]. The free radical then
will oxidise the highly unsaturated β-carotene. The presence
of antioxidants in the sample will reduce the oxidation
of β-carotene by hydroperoxides. Hydroperoxides formed
in this system will be decomposed by the antioxidants
from the extracts. Thus, the degradation rate of β-carotene
depends on the antioxidant activity of the extracts. Effect
of cat whiskers (OS) extracts/fractions (CAME, HF, CF, WF,
NBF, and EAF) on oxidation of β-carotene is shown in
Figure 2. It was clear that the presence of antioxidants in
OS reduced the oxidation of β-carotene by hydroperoxides.
There were significant differences (P < .05) between
the different extracts/fractions, control, and BHA effect as
shown in Figure 2. NBF, EAF, WF, and CAME fractions
were better in their effect on reducing the oxidation of β-
carotene than CF and HF fractions. The degradation rate
of β-carotene clearly depends on the antioxidant activity
of OS. There was a correlation between the degradation
rate and the bleaching of β-carotener, where the extract
with the lowest β-carotene degradation rate exhibited the
highest antioxidant activity (Figure 2). The high antioxidant
activities of n-butanol fraction and ethyl acetate fraction
tested using β-carotene model may be correlated with their
high phenolic content. These findings are in good agreement
with those of Matthaus [29] who found that extraction
with polar solvents gave the highest amounts of phenolic
compounds. Ethyl acetate is often used as an extraction
solvent with a significant selectivity in the extraction of low-
weight polyphenols [30]. Recently ethyl acetate extract has
been observed to possess the highest total phenolic content,
DPPH scavenging ability, and antioxidant activity (in β-
carotene bleaching assay) among the extracts of Pereskia
120
3.5. Effect of OS on Hydrogen Peroxide Induced Apoptosis
3.5.1. Cell Viability. To examine the effect of OS on
oxidant-induced apoptosis, epithelial cells (MDA-M231)
were pretreated with or without EAF and exposed to
H2 O2 (400 μmol/L). Incubation of cells with H2 O2 leads to
the decrease of formazan production compared with the
untreated one, which can be seen from the decrease of
optical density at 570 nm, and it means that cell viability
in H2 O2 -treated wells was much lesser than the untreated
one. However, when cell is incubated with extract/fractions
of O. stamineus (HF, CF, EAF, NBF, WF, and CAME) prior to
H2 O2 exposure, cell viability was higher than that of control
(Table 2). Among the tested samples, EAF and NBF showed
significant (P < .01) protection against hydrogen peroxide
induced apoptosis. Nevertheless, all extract/fractions of the
plant under this investigation have shown significant (P <
.05) in vitro free radical inhibition. This result is in agreement
with the previous study by Zhang and Yang [21] who
reported that flavonoids extracted from the radix of Scutel-
laria baicalensis exhibited inhibitory activity towards H2 O2 -
induced cell injury. This confirms that protecting cells from
antioxidants such as H2 O2 is a vital bioprocess. However,
hydrogen peroxide is made by quite a few enzymes in the
body. In particular, some enzymes breaking down certain
amino acids and fatty acids (D-amino acid oxidase and acyl-
CoA oxidase) make significant amounts of hydrogen perox-
ide. Since hydrogen peroxide can be damaging to normal
tissue, these enzymes are kept inside specialized organelles
inside cells called peroxisomes [36]. Since EAFs were the
most cytoprotective fraction of OS against H2 O2 induced cell
injury, all downstream experiments were conducted to assess
cell death between control and treated cells.
3.5.2. Phase-Contrast Microscope. Recent studies have sug-
gested that hydrogen peroxide, a reactive compound formed
endogenously in the breakdown of superoxide, may intercede
the initiation of apoptosis in various cell types in response to
outer stimuli. Apoptosis, a type of programmed cell death,
is an active process and a way of eliminating a cell from
an organism [37]. Morphological assessment of hydrogen
peroxide treated-MDA-M231 using inverted microscope
demonstrated that suspected apoptosis had occurred due
to exhibition of morphological features changes (Figure 3).
Morphological changes associated with apoptotic cell death
induced by H2 O2 were characterized by the presence of
shrunken cells with surface blebbing and nuclear conden-
sation. However, these cellular events were clearly prevented
when the cells were preincubated with EAF. Confirmation of
Evidence-Based Complementary and Alternative Medicine 7

(a) (b) (c)

(d) (e)

Figure 3: Inhibition of hydrogen peroxide-(H2 O2 -) triggered apoptosis using ethyl acetate fraction (EAF) of O. stamineus. Epithelial cells
were pretreated with or without EAF and later exposed to H2 O2 . Cells were viewed under phase contrast microscopy. (a) MDA-MB-231
control cells; (b–d) cells pre-treated with different concentrations of EAF and incubated with hydrogen peroxide; (e) cells treated with
hydrogen peroxide.

1.2
Optical density (405 nm)

1
0.8
0.6
0.4
0.2
0
Control cells
EAF (31.25 μg/mL) +

EAF (62.5 μg/mL) +

EAF (125 μg/mL) +

Hydrogen peroxide
hydrogen peroxide
hydrogen peroxide
hydrogen peroxide

Figure 4: The colorimetric assay of caspase-3 in MDA-M231 cells treated and untreated with H2 O2 and EAF for 72 hours. Cells were cultured
in RPMI 1640 (75 mL flask) media maintained at 37◦ C and 5% CO2 . ANOVA showed a significant difference (P < .05) between treated and
untreated cells in the activity of caspase-3.
EAF (31.25 μg/mL) +
EAF (62.5 μg/mL) +

hydrogen peroxide
hydrogen peroxide

Hydrogen peroxide
Control

Bcl-2

β-actin

Figure 5: Effect of EAF on hydrogen peroxide- (H2 O2 -) triggered apoptosis. Cells were treated with hydrogen peroxide with or without EAF,
incubated for 72 hours while untreated cells acted as control. After incubation, cells were harvested and used western blotting analysis.
8 Evidence-Based Complementary and Alternative Medicine

15 ∗ 4
Relative quantity

Relative quantity
3.5
of Bcl-2 mRNA

of Bax mRNA
12 3
∗
9 ∗ 2.5 ∗
2 ∗
6 1.5 ∗
3 1
0.5
0 0
Hydrogen peroxide

EAF (31.25 μg/mL) +

EAF (62.5 μg/mL) +

EAF (125 μg/mL) +

Control

Hydrogen peroxide

EAF (31.25 μg/mL) +

EAF (62.5 μg/mL) +

EAF (125 μg/mL) +

Control
hydrogen peroxide

hydrogen peroxide
hydrogen peroxide

hydrogen peroxide
hydrogen peroxide

hydrogen peroxide
Bcl-2 Bax
(a) (b)

Figure 6: Bcl-2 and Bax mRNA expression in MDA-M231. Cells were treated with EAF and then incubated with H2 O2 . After incubation, cells
were harvested and used real-time RTPCR analysis. (a) Bcl-2; (b) Bax. Normalization relative to GAPDH was performed. Results presented
in bar graph are the means ± SD of three independent experiments.

Oxidative injury

Cell membrane

Orthosiphon stamineus Benth

Blc2/Bax
Caspase-3

Apoptosis

Figure 7: Hypothetical scheme of the cytoprotective properties of Orthosiphonstamineus against free radical induced cell death.

apoptosis in the current study was performed using western this enzyme is higher in H2 O2 -reated MDA-M231 cells as
blot, caspase-3 assay, and gene expression. compared to untreated cells. EAF reduced remarkably (P <
.05) the level of caspase-3 in a dose-dependent manner. We
suggest that EAFs prevent apoptosis in epithelial cells by
3.5.3. Colorimetric Assay of Caspase-3. Exposure of epithelial inhibiting caspase-3 during oxidant injury.
cells to hydrogen peroxide resulted in apoptosis and activa-
tion of its associated proteins such as capsase-3. However,
H2 O2 -induced apoptosis was associated with activation of 3.5.4. Effect of EAF Mitochondrial Apoptosis. To examine
caspase-3 [38, 39]. Since EAFs were the most cytoprotective whether the antipoptotic effect of EAF is due to inter-
fraction against H2 O2 induced cell injury, an in vitro vention with Bcl2/Bax regulated apoptosis, proteins and
colorimetric assay of caspase-3 was conducted to assess mRNAs of epithelial cells were studied using western blot
cell death between control and treated cells. As shown in and qRT-PCR, respectively. Bcl-2 protein (Figure 5) and
Figure 4, H2 O2 significantly (ANOVA, P < .05) stimulated mRNA (Figure 6(a)) levels were significantly decreased in
caspase-3, the hallmark enzyme of cell death. The level of cells exposed to H2 O2 compared to levels in control cells
Evidence-Based Complementary and Alternative Medicine 9

Table 2: Effects of extract/fractions of O. stamineus on cell viability.

Treatment∗ Concentrations of extract/fractions of O. stamineus

500 μg/mL 250 μg/mL 125 μg/mL 62.5 μg/mL
HF + Hydrogen Peroxide 25.90 ± 0.95∗ 22.37 ± 1.56∗ 20.61 ± 0.94∗ 23.10 ± 3.18∗
CF + Hydrogen Peroxide 70.81 ± 10.24∗∗ 43.03 ± 2.65∗∗ 24.81 ± 0.09∗∗ 20.66 ± 1.56∗
EAF + Hydrogen Peroxide 100.00 ± 27.75∗∗ 100.00 ± 16.51∗∗ 84.82 ± 3.98∗∗ 56.43 ± 5.98∗∗
NBF + Hydrogen Peroxide 100.00 ± 8.69∗∗ 66.03 ± 0.01∗∗
37.92 ± 0.99∗∗ 29.90 ± 4.00∗∗
WF + Hydrogen Peroxide 65.62 ± 10.70∗∗ 35.20 ± 13.12∗
23.88 ± 1.51∗ 23.41 ± 5.49∗
CAME + Hydrogen Peroxide 91.31 ± 5.32∗∗ 52.64 ± 7.56∗∗
37.53 ± 1.65∗ 26.27 ± 1.86∗
∗∗
Quercetin + Hydrogen Peroxide 95.45 ± 3.72
Hydrogen Peroxide control 15.86 ± 0.23
Control cells 100∗∗
∗
Cell viability after 2 h exposure in 400 μmol/L H2 O2 was assayed by MTT. Cells were preincubated with different tested samples (62.5, 125, 250, and
500 μg/mL) for 10 min before the addition of 400 μmol/L H2 O2 . After 2 h incubation, MTT was added to a final concentration of 0.5 mg/mL and then
incubated at 37◦ C for another 2 hrs; the resultant formazan product was extracted for 4 h with DMSO and detected by UV-spectrometer at 570 nm. Values
are expressed as means ± SD (N = 3). ∗ P < .05, ∗∗ P < .01 versus hydrogen peroxide control.

(Figure 6(a)). In contrast, Bcl-2 levels were significantly
increased from control by various concentration of EAF
in a dose-dependent manner (Figure 6(a)). Bax mRNA
levels were significantly higher in the H2 O2 group. EAF
group has higher Bax expression compared to control group
(Figure 6(b)).
Many genes are involved in the regulation of apoptosis.
Bcl-2 is an antipoptotic regulator, while Bax is a proapoptotic
regulator. The balance between Bcl-2 and Bax expression
plays an important role in sustaining cell morphology and
function. Several studies have shown that Bcl-2 overexpres-
sion can disrupt regulation of the proapoptotic proteins
Bax [38, 39]. Moreover, an increase in Bcl-2 expression
prevents cytochrome c release from the mitochondria,
thereby inhibiting activation of caspases, such as caspase-9
and caspase-3, and preventing apoptosis [39]. Other studies
have demonstrated that Bcl-2 can act as a mitochondrial
membrane channel protein [40]. Thus, cells are active when
Bcl-2 is overexpressed and die if Bax is hyperexpressed [41].
In the present study, we found that expression of Bcl-2 was
downregulated by H2 O2 . These alterations of Bcl-2 protein
expression were restored by EAF. Our findings suggest that
Bcl-2 is involved in mediating the antipoptotic effects of EAF
in H2 O2 -exposed MDA-M231.
4. Conclusion
Despite the wide spread use of medicinal uses of OS
as a remedy for kidney stone, nephritis, gout, diabetes,
hypertension, and rheumatism, evidence from in vitro previ-
ousbiological studies is not sufficient [11]. The current paper
provides strong evidence about the antioxidant properties
of Orthosiphon stamineus and its fractions as well as their
use as cytoprotective against free radical induced cell death.
Antipoptotic effect of this plant is suggested to be through
the intervention in Bcl-2-mediated apoptotic pathways. The
results obtained support the efficacy of natural phenolics
from plant origin in offering protection against oxidative
injury and highlight the fact that phenolic-rich foods may
provide health and nutritional benefits. On the other hand,
epidemiological studies have shown a relationship between
high dietary intakes of phenolic-rich foods and reduced risk
of cardiovascular disease and cancer whereby, the production
and development of many diseases are closely related to
oxidative damages caused by free radicals. So it becomes one
of investigating hotspots that safe and effective antioxidants
in plants are searched.
Conflict of Interests
Authors state that there is no conflict of interests.
Acknowledgment
Authors are very grateful to the staff of Institute of Biosci-
ence, Universiti Putra Malaysia for their technical assistance.
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