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Research Article
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In vitro Anti-oxidant and Anti-cancer Activity of Methanolic Extract from Sanchezia speciosa Leaves |
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Mohammadjavad Paydar,
Yi Li Wong,
Bushra Abdulkarim Moharam,
Won Fen Wong
and
Chung Yeng Looi
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ABSTRACT
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Sanchezia speciosa, is a bushy shrub from Acanthaceae
family which commonly grows in tropical areas of South and Central America.
In this study, we employed MTT assay to test the cytotoxicity of that methanolic
fraction of S. speciosa leaves on MCF-7 human breast cancer, SK-MEL-5
human malignant melanoma and human umbilical vein endothelial cells, HUVEC cells.
The extract showed highest activity on MCF-7 and moderate cytotoxicity towards
SK-MEL-5. In contrast, the extract demonstrated lowest cell growth inhibition
activity on HUVEC cells, indicating better selectivity compare to standard drug,
doxorubicin. In addition, we also performed ORAC assay to determine the radical
scavenging capacity of methanolic extract of S. speciosa leaves. The
extract exhibited nearly similar anti-oxidant activity as quercetin, suggesting
S. speciosa leaves as a potential source of natural anti-oxidant. To
the best of our knowledge, this is the first report on anti-oxidant and cytotoxic
activity of S. speciosa.
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Received: December 14, 2012;
Accepted: January 26, 2013;
Published: April 18, 2013
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INTRODUCTION
Breast cancer is a heterogeneous disease that has become a global public health
problem in the recent years. The incidence of breast cancer has undergone an
upward trend during the recent decades at an annual rate of 3.1% (Forouzanfar
et al., 2011). The number of new cases has increased from 1.38 million
women in 2008 (Ferlay et al., 2010) to more than
1.6 million in 2010 (Forouzanfar et al., 2011).
Breast cancer has been reported as the most usual cause of cancer deaths in
adult women in 2008 (16% of all cancer deaths) (WHO, 2008).
In 2007, 3.242 female breast cancer cases were reported in Malaysia, accounted
for 18.1% of reported cancer cases and 32.1% of female cases (Ariffin
and Nor Saleha, 2011). Breast cancer cell lines are applied by many investigators
to screen and characterize new therapeutics and among these cells, MCF-7 is
the most extensively used model of estrogen positive breast cancer cell line.
MCF-7 cell line was originally established using a pleural effusion from a metastatic
breast cancer patient, at the Michigan Cancer Foundation in 1973 (Soule
et al., 1973) and then these cells were distributed globally to be
used in various investigations.
Anti-oxidants are compounds that prevent damage to cell structures caused by
chemical reactions involving free radicals. Many synthetic anti-oxidants such
as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tertiary
butyl hydroquinone (TBHQ) have been produced since the beginning of their application
in food. However, majority of investigators tend to replace them with natural
anti-oxidants, due to the serious side effects of these synthetic anti-oxidants
(Van Esch, 1986; Grice, 1988;
Miller et al., 2000). Prior to the era of modern
medicine, medicinal plants and herbs have been extensively used for centuries
as the main source of remedies against most ailments. There is a general desire
among modern medicine researchers to utilize pharmaceutical plants as sources
of novel drugs.
Sanchezia speciosa, commonly known as shrubby whitevein, is a semiwoody
evergreen shrub with smooth bright green or purple stems, large variegated leaves
and colorful flowers. Scientific synonym for this plant is S. nobilis.
It is a member of Acanthaceae plant family, occurring in the lowlands of tropical
South and Central America, mostly in Peru and Equador (Clay
and Hubbard, 1977). S. speciosa is usually cultivated as an ornamental
plant in shrub borders and hedges throughout the tropics and in botanical gardens
of temperate areas due to its large, colorful bracts, foliage and flowers and
sometimes even colorful leaves (Leonard and Smith, 1964).
In this study, we evaluated the anti-cancer effect of methanolic extract from
S. speciosa on MCF-7, SK-MEL-5 and HUVEC cells. In addition, we used
ORAC (oxygen radical antioxidant capacity) assay to evaluate the anti-oxidant
potential of S. speciosa.
MATERIALS AND METHODS
Plant material: The leaves of S. speciosa were obtained from
Seri Subah Agrofarm, Negeri Sembilan, Malaysia and verified by their botanist.
The plant leaves specimen was labelled as SS1 and kept at Department of Pharmacology,
Faculty of Medicine, University of Malaya.
Cell culture: The human breast cancer cell line, MCF-7 was purchased
from cell lines service (300273; Eppelheim, Germany), Human Umbilical Vein Endothelial
Cells (HUVEC) cells was obtained from ScienCell Research Laboratories (Carlsbad,
CA) and human malignant melanoma cells, SK-MEL-5 was acquired from American
Type Culture Collection (ATCC, Manassas, VA). MCF-7 and SK-MEL-5 cells were
grown in Dulbeccos Modified Eagle Medium (DMEM, Life Technologies, Inc,
Rockville, MD) supplemented with 10% heat-inactivated fetal bovine serum (FBS,
Sigma-Aldrich, St. Louis, MO), 1% penicillin and streptomycin. HUVEC cells were
grown in Endothelial Cell Medium (ECM, ScienCell Research Laboratories, Carlsbad,
CA) supplemented with 5% heat-inactivated Fetal Bovine Serum (FBS, ScienCell
Research Laboratories, Carlsbad, CA), 1% (ECGS, ScienCell Research Laboratories,
Carlsbad, CA), 1% penicillin and streptomycin (P/S solution, ScienCell Research
Laboratories, Carlsbad, CA). Cells were cultured in tissue culture flasks (Corning,
USA) and were kept in incubator at 37°C in a humidified atmosphere with
5% CO2. For experimental purposes, cells in exponential growth phase
(approximately 70-80% confluency) were used.
Extraction: The fresh leaves of S. speciosa were cleaned, dried
and powdered coarsely. The powder (100 g) was macerated with MeOH (3x300 mL)
for 72 h. The resultant extract was filtered and dried under vacuum to yield
247 mg of the extract. Then the dried fractions were kept at -20°C until
further use.
MTT cell viability assay: The influence of S. speciosa methanolic
leaves extract of was determined by MTT assay (Mosmann, 1983).
MCF-7, SK-MEL-5 and HUVEC cells were treated for different lengths of time including
24, 48 and 72 h. On the first day, 1.0x104 cells were seeded into
a 96-well plate for 24 h incubation assay while 7.0x103 and 5.0x103
cells were seeded for 48 and 72 h incubation assay, respectively. It was followed
by overnight incubation of the seeded cells at 37°C in 5% CO2.
On the next day, the cells were treated with a two-fold dilution series of six
concentrations of methanolic extract from S. speciosa leaves and then
they were incubated at 37°C in 5% CO2, based on the certain time
period of each plate (24, 48, 72 h). MTT solution (4, 5-dimethylthiazol-2-yl-2,5-diphenyltetrazoliumbromide)
was added at 2 mg mL-1 and after 2 h of incubation at 37°C in
5% CO2, DMSO was added to dissolve the formazan christals. The plates
were then read in Chameleon multitechnology microplate reader (Hidex, Turku,
Finland) at 570 nm absorbance. The cell viability percentage after exposure
to S. speciosa leaves extract for 24, 48 and 72 h was calculated by previously
described method (Mosmann, 1983; Looi
et al., 2011). The ratio of the absorbance of treated cells to the
absorbance of DMSO-treated control cells was determined as cell viability (percentage).
The concentration of S. speciosa leaves extract which is required to
reduce the absorbance of treated cells to 50% of the DMSO-treated control cells
was defined as IC50.
ORAC-antioxidant activity assay
Chemicals: Fluorescein sodium salt, AAPH (2, 2'-Azobis (2-methylpropionamidine)
dihydrochloride), quercetin dehydrate and trolox ((±)-6-Hydroxy-2, 5,
7, 8-tetramethylchromane-2-carboxylic acid) were purchased from Sigma-Aldrich.
Oxygen Radical Antioxidant Capacity (ORAC) assay was done based on procedures
previously described with slight modifications (Zulueta
et al., 2009; Arya et al., 2013).
Compounds were diluted to final concentration of 100 μg mL-1,
with total reaction volume of 200 μL. The assay was performed in a 96-well
black microplate, with 25 μL of samples, standard (trolox), blank (solvent/PBS)
or positive control (quercetin). Subsequently, 150 μL of working fluorescein
solution was added to each well of assay plate. The plate was incubated at 37°C
for at least 5 min. Twenty five microlitter of AAPH working solution was then
added to the wells, making up total volume of 200 μL. Fluorescence was
recorded with excitation wavelength of 485 nm and emission wavelength of 538
nm. Data were collected every 2 min for duration of 2 h and were analyzed by
calculating the differences of area under fluorescence decay curve (AUC) of
samples and blank. The values were expressed as Trolox Equivalent (TE).
Statistical analysis: Experimental values were expressed as the Means±Standard
Deviation (SD) of the number of experiments indicated in the legends. GraphPad
Prism 5 software was employed for the analysis of variance (ANOVA). Statistical
significance was defined when p<0.05.
RESULTS AND DISCUSSION
The cytotoxic effect of S. speciosa methanolic extract on cell viability
was determined by MTT assay on MCF-7, SK-MEL-5 and HUVEC cells. The MTT assay
was done in different time points to have a better view of the cell growth pattern
after treatment with S. speciosa methanolic extract. We treated the cells
with different concentrations of doxorubicin, a cancer chemotherapy drug as
positive control. No significant cell inhibitory effect was observed in DMSO
(solvent)-treated samples. As shown in Table 1, the plant
extract induced the highest cell growth inhibition activity on MCF-7 cells.
However, its activity on SK-MEL-5 was moderate and demonstrated lowest cytotoxic
effect on HUVEC normal endothelial cells. In contrast, doxorubicin exhibited
no selectivity against cancer cell-lines or normal endothelial HUVEC cells.
In this study, we showed that S. speciosa methanolic extract inhibited
MCF-7 cell growth in a time and concentration dependant manner (Fig.
1). HUVEC cells were used to determine the level of selectivity of the extract
on normal and cancer cells. As shown in Table 1, the IC50
value of the extract for HUVEC cells in each time point was at least 3 fold
higher than MCF-7 cells. Higher selectivity is crucial to avoid side effects
and ensure the other non-target cells are not/less affected in order to gain
optimal therapeutic effect. As many scientists have turned to natural product
for anti-cancer drug discovery research, methanolic extract of S. speciosa
leaves may contained anti-cancer compounds worthy of further development (e.g.,
bio-assay guided isolation).
ORAC-antioxidant activity assay: ORAC assay has been widely used for
measuring anti-oxidant capacity as it is the only assay which involves the use
of peroxyl radical as pro-oxidant and quantify activity via., Area Under Curve
(AUC) technique (Prior and Cao, 1999). In our experiment,
quercetin was used as the standard for comparison of antioxidant activity. The
assay performed showed that methanol extract of S. speciosa leaves exhibited
comparable anti-oxidant capacity with the standard quercetin (Table
2).
Many diseases such as cancer and atherosclerosis had been reported to be complicated
by excessive production of free radicals and Reactive Oxygen Species (ROS) (Machlin
and Bendich, 1987; Aruoma, 1998). Thus, natural
products which contain anti-oxidant property is useful to alleviate oxidative
stress induced by ROS and possess therapeutic value in treating these chronic
diseases mentioned above. To serve this aim, we performed ORAC assay to determine
the radical scavenging capacity of methanolic fraction of S. speciosa leaves.
The principle of ORAC assay is on the capacity of compounds tested to compete
with the probe in scavenging generated radicals, thus inhibiting the decay of
fluorescent probe (Takashima et al., 2012).
From our results, methanol extract of S. speciosa leaves possess nearly
similar anti-oxidant activity as the standard (quercetin), suggesting S.
speciosa leaves as a potential source natural anti-oxidant. Anti-oxidants
developed from natural product are safer compare to synthetic anti-oxidant which
may have many unwanted side effects.
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Fig. 1: |
Dose-dependent inhibition of MCF-7 cells proliferation treated
with methanolic extract of S. speciosa leaves for different lengths
of time (24, 48 and 72 h) |
Table 1: |
Cytotoxic activity of S. speciosa methanolic fraction
on MCF-7, SK-MEL-5 and HUVEC cells in terms of IC50 |
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Table 2: |
Anti-oxidant capacity of compounds by ORAC method |
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CONCLUSION
The methanol fraction of S. speciosa leaves exhibited significant anti-oxidant
activity, comparable to the standard drug, quercetin. The extract also indicated
highest cytotoxicity against human breast cancer cells, MCF-7, in comparison
with human umbilical vein endothelial, HUVEC and SK-MEL-5 human malignant melanoma
cells. To the best of our knowledge, this is the first report on anti-oxidant
and cytotoxic activity of S. speciosa extracts.
ACKNOWLEDGMENT
This study was supported by University Malaya research grant (RG434-12HTM).
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