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Petroselinum crispum has antioxidant properties, protects against DNA damage and inhibits proliferation and migration of cancer cells

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Background Petroselinum crispum (English parsley) is a common herb of the Apiaceae family that is cultivated throughout the world and is widely used as a seasoning condiment. Studies have shown its potential as a medicinal herb. In this study, P. crispum leaf and stem extracts were evaluated for their antioxidant properties, protection against DNA damage in normal 3T3-L1 cells, and the inhibition of proliferation and migration of the MCF-7 cells.ResultsThe dichloromethane extract of P. crispum exhibited the highest phenolic content (42.31 ± 0.50 mg GAE g−1) and ferric reducing ability (0.360 ± 0.009 mmol g−1) of the various extractions performed. The extract showed DPPH radical scavenging activity with an IC50 value of 3310.0 ± 80.5 µg mL−1. Mouse fibroblasts, 3T3-L1, pre-treated with 400 µg mL−1 of the extract showed 50.9% protection against H2O2-induced DNA damage, suggesting its potential in cancer prevention. The extract (300 µg mL−1) inhibited H2O2-induced MCF-7 cell migration by 41 ± 4%. As cell migration is necessary for metastasis of cancer cells, inhibition of migration is an indication of protection against metastasis.ConclusionP. crispum has health promoting properties with the potential to prevent oxidative stress-related diseases and can be developed into functional food.
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Research Article
Received: 13 November 2014 Accepted article published: 13 January 2015 Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/jsfa.7078
Petroselinum crispum has antioxidant
properties, protects against DNA damage and
inhibits proliferation and migration of cancer
cells
Esther Lai-Har Tang, Jayakumar Rajarajeswaran, ShinYee Fung and
MS Kanthimathi*
Abstract
BACKGROUND: Petroselinumcrispum (English parsley) is a common herb of the Apiaceae family that is cultivated throughout the
world and is widely used as a seasoning condiment. Studies have shown itspotential as a medicinal herb. In this study, P. crispum
leaf and stem extracts were evaluated for their antioxidant properties, protection against DNA damage in normal 3T3-L1 cells,
and the inhibition of proliferation and migration of the MCF-7 cells.
RESULTS: The dichloromethane extract of P.crispum exhibited the highest phenolic content (42.31 ±0.50 mg GAE g1) and ferric
reducing ability (0.360 ±0.009 mmol g1) of the various extractions performed. The extract showed DPPH radical scavenging
activity with an IC50 value of 3310.0 ±80.5 𝛍gmL
1. Mouse fibroblasts (3T3-L1) pre-treated with 400 𝛍gmL
1of the extract
showed 50.9% protection against H2O2-induced DNA damage, suggesting its potential in cancer prevention. The extract (300 𝛍g
mL1) inhibited H2O2-induced MCF-7 cell migration by 41% ±4%. As cell migration is necessary for metastasis of cancer cells,
inhibition of migration is an indication of protection against metastasis.
CONCLUSION: Petroselinum crispum has health-promoting properties with the potential to prevent oxidative stress-related
diseases and can be developed into functional food.
© 2015 Society of Chemical Industry
Keyword s: Petroselinum crispum; antioxidant activity; antiproliferative activity ; DNA protection; MCF-7 cell migration; hydrogen peroxide
INTRODUCTION
Petroselinum crispum (Mill) Nyman ex AW Hill, commonly known
as English parsley, is a culinary and medicinal herb of the Api-
aceae family that grows up to 30 – 100 cm high.1The herb has
been used to flavor the cuisines of South East Asia, China, India,
South America and Mexico.2Although native to Europe and west-
ern Asia, the herb is now cultivated and consumed throughout the
world.3The leaves and stems, either fresh or dried, as well as the
seeds, have been employed in the food, pharmaceutical and cos-
metic industries.4In folk medicine, the aerial part of P. crispum is
used to treat hemorrhoids, the stem for urethral inflammation, and
the root is used to pass kidney stones5and improve brain func-
tion and memory.6Additionally, P. crispum is used as a carmina-
tive, stomachic, emmenagogic, abortifacient and nutritive agent.7
Studies have shown that P. crispum has hypoglycemic, diuretic,
hypolipidemic, antimicrobial, anticoagulant and hepatoprotective
activities.8
The chemical composition and pharmacological properties
of P. crispum have been previously reported in various studies.
The herb contains flavonol glycosides of quercetin, apiol, myris-
ticin and luteolin. Terpenes, phthalides, furanocoumarins, apiin,
carotenoids, ascorbic acid and tocopherol are also present in
P. crispum.9,10 Supplementation of diets with fresh P. crispum
leaves increases the antioxidant capacity of plasma in rats11 and
decreases oxidative stress in humans.12 Zheng et al.13 reported the
inhibition of benzo[a]pyrene-induced tumorigenesis in the lungs
of mice by myristicin, a major volatile aromatic constituent of
parsley leaf oil. The vast health-promoting properties associated
with P. crispum warrant further study. Previous investigations
on P. crispum mostly focused on its antioxidant properties.14
As phenolic compounds and antioxidant activities depend on
variety, location and growth conditions of the plant, data on the
antioxidant activity of P. crispum are still relevant and useful. The
effect of P. crispum leaves and stems on the two most common
cancers in humans breast cancer and colon cancer are unclear
and lacking thus far. The main aim of this work was to investigate
the antioxidant activities and protection against DNA damage by
Correspondence to: MS Kanthimathi, Centre for NaturalProducts and Drug D is-
covery (CENAR) and Department of Molecular Medicine, Faculty of Medicine,
University of Malaya, 50603 Kuala Lumpur, Malaysia.
E-mail: kanthi@ummc.edu.my
Centre for Natural Products and Drug Discovery (CENAR) and Department of
Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala
Lumpur, Malaysia
J Sci Food Agric (2015) www.soci.org © 2015 Society of Chemical Industry
www.soci.org EL-H Tang et al.
extracts of P. crispum leaves and stems and their inhibition of the
proliferation and H2O2-induced migration of the breast cancer cell
line MCF-7. To the best of our knowledge, this is the first study
reporting on the effects of P. crispum on DNA protection and
inhibition of MCF-7 cell migration.
EXPERIMENTAL
Materials and methods
Analytical-grade solvents were purchased from Fisher Scientific
(Loughborough, UK). Dimethyl sulfoxide (DMSO) and H2O2were
purchased from Univar (Ingleburn, NSW, Australia). Chemicals,
polyphenolic standards (gallic acid, quercetin, rutin), proteinase
K and RPMI-1640 were obtained from Sigma-Aldrich (St Louis,
MO, USA). Dulbecco’s Modified Eagle Medium (DMEM) was
purchased from Lonza (Basel, Switzerland) and fetal bovine
serum (FBS) was obtained from iDNA Biotechnology, Singapore.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT), Folin– Ciocalteu phenol reagent, tris(hydroxymethyl)
aminomethane and ethidium bromide were purchased from
Merck (Darmstadt, Germany). Sodium dodecyl sulfate (SDS) was
from Bio-Rad (Hercules, CA, USA). TRIzol®reagent was purchased
from Life Technologies (Carlsbad, CA, USA). RNase A and DNA
ladders were obtained from Thermo Scientific (Carlsbad, CA,
USA). Ultrapure water from a Milli-Q-plus filter system (Millipore,
Billerica, MA, USA) was used throughout the experiments.
Cell lines and cell culture conditions
Two human breast adenocarcinoma cell lines – MCF-7 and
MDA-MB-231 – and the human colorectal adenocarcinoma cell
line HT-29 were used in the antiproliferative study. The MCF-7
cell line expresses the estrogen receptor whereas MDA-MB-231
cells do not. The study on the inhibition of cell migration, trypan
blue dye exclusion assay and DNA fragmentation analysis was per-
formed using MCF-7 cells. Mouse fibroblasts (3 T3-L1) were used in
the comet assay to estimate protection against H2O2-induced DNA
damage. All cells were purchased from the American Type Culture
Collection (Manassas, VA, USA). MCF-7 and HT-29 cancer cells
were routinely cultured in RPMI-1640. MDA-MB-231 and 3T3-L1
were grown in DMEM. The cells were supplemented with 10%
fetal bovine serum (FBS), 100 U mL1penicillin and 100 μgmL
1
streptomycin. Cells were grown at 37 C in a humidified incubator
with 5% CO2.
Plant material
Fresh P. crispum leaves and stems were purchased from the
local market in Kuala Lumpur, Malaysia. The plant was identi-
fied by Dr M Sugumaran, Institute of Biological Sciences, Uni-
versity of Malaya, and the voucher specimen (KLU47745) was
deposited in the University of Malaya herbarium. The leaves and
stems were washed under running tap water and finally rinsed
with distilled water. The plant parts were then freeze dried,
weighed, ground into fine powder and stored at 20 Cuntil
extraction.
Preparation of P. crispum extracts
Powdered leaves and stems of P. crispum were extracted through
sequential extraction using hexane, dichloromethane, ethyl
acetate, methanol and water. Briefly, 120 g powdered leaves and
stems were extracted in 600 mL hexane (1:5 w/v) for 6 h at 40 Con
a hotplate stirrer. Extracts were then filtered through Whatman no.
1 filter paper and the resulting residue was re-extracted twice with
fresh hexane. The remaining residue was subsequently extracted
three times each with dichloromethane, followed by ethyl acetate,
methanol and water. Each filtrate (except for the aqueous extract,
which was concentrated to dryness in a freeze-dryer) was con-
centrated to dryness under reduced pressure at 40 Cusinga
rotary evaporator. The dried extracts were stored at 20 C. For
bioassays, the dried extracts were dissolved in DMSO and diluted
in ultrapure water to make appropriate extract concentrations.
The final concentration of DMSO in reaction mixtures was less
than 1%. All dissolved extracts were kept at 4 C throughout the
experiments.
Determination of total phenolic content
The total phenolic content of P. crispum extracts was determined
using the Folin-Ciocalteau method,15 with some modifications.
Briefly, 500 μL of 1:10 Folin– Ciocalteau phenol reagent was added
to 1 0 μL of sample (dissolved in 10% DMSO), standard or positive
control. The mixture was mixed and allowed to stand for 5min
before the addition of 350 μL of 10% sodium carbonate. The
resulting reaction mixture was incubated in the dark at room
temperature for a further 2 h. Absorbance was then measured at
765 nm using a spectrophotometer. Gallic acid (50– 500 mg L1in
10% DMSO) was used as the standard. Rutin and quercetin were
used as positive controls. Results were expressed in milligrams
of gallic acid equivalents (GAE) per gram of dried extract. All
experiments were carried out in triplicate.
Ferric reducing antioxidant power (FRAP) assay
Ferric reducing activity of P. crispum extracts was estimated
based on the assay by Benzie and Strain16 with slight mod-
ifications. A working reagent was prepared fresh by mixing
10 mL of 300 mmol L1acetate buer with 1mL of 10mmolL
1
2,4,6-tripyridyl-s-triazine (TPTZ) in 40 mmol L1hydrochloric acid
and 1 mL of 20 mmol L1ferric chloride hexahydrate (FeCl3.6H2O).
The freshly prepared FRAP reagent was pre-warmed at 37Cfor
5 min, after which a blank reading was taken at 595 nm using
a plate reader. Subsequently, 3 μL sample (dissolved in 10%
DMSO), standard or positive control and 9 μL water were added to
90 μL of the FRAP reagent. Absorbance readings were measured
instantly upon addition of the FRAP reagent and again at 4 min
after the start of the reaction. The change in absorbance in the
4 min reaction was calculated by comparison with a FeSO4.7H2O
standard cur ve (100 –1000 μmol L1) tested in parallel. Rutin
and quercetin were used as positive controls. Results were
expressed as millimoles of ferric reducing activity of the extracts
per gram of dried extract. All experiments were carried out
in triplicate.
1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging
assay
The radical scavenging activity of P. crispum extracts was deter-
mined by the DPPH radical scavenging assay,17 with some modi-
fications. Petroselinum crispum extract (20 μL) was added to 120 μL
of 0.04 mg mL1DPPH solution in methanol. The extracts tested
ranged from 0 to 5000 μgmL
1(dissolved in 10% DMSO). The solu-
tions were mixed well and incubated in the dark for 30min. The
reduction of DPPH absorption was measured at 515 nm using a
plate reader. Rutin and quercetin were used as positive controls.
All determinations were performed in triplicate. The DPPH radi-
cal scavenging activity was calculated according to the following
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Antioxidant, DNA-protective and anticancer properties of Petroselinum crispum www.soci.org
equation:
Percentage inhibition =(Absorbancecontrol Absorbancesample)
Absorbancecontrol ×100
The results were expressed as half-maximal inhibitory concen-
tration (IC50), i.e. the concentration of the plant extract required to
scavenge 50% of the total DPPH radicals available.
Inhibition of proliferation (MTT assay)
The antiproliferative activity of P. crispum extracts on MCF-7,
MDA-MB-231 and HT-29 cancer cell lines was estimated using
the MTT assay as described by Mosmann.18 Briefly, cells supple-
mented with 5% FBS were seeded (5 ×103cells per well) in 96-well
plates and were allowed to grow at 37 C in a humidified atmo-
sphere with 5% CO2. After 24 h incubation, the cells were treated
with different concentrations of extract (0500 μgmL
1) for a fur-
ther 48 h. Vehicle-control wells with cells only and diluent-control
wells with similar DMSO concentrations as in the treatment were
included. After incubation, 10 μL of 5 mg mL1MTT bromide in
phosphate-buffered saline (PBS) were added to each well. The
plates were reincubated for 4h, after which media and MTT were
removed by aspiration. DMSO (100 μL) was added to each well
to dissolve the formazan crystals. Absorbance was read using a
microtiter plate reader at 595 nm. All measurements were per-
formed in triplicate. The percentage inhibition of cell proliferation
was calculated using the following formula:
Percentage inhibition =(Absorbancecontrol Absorbancetreated)
Absorbancecontrol ×100
Comet assay
The DNA protective effect of P. crispum was estimated using the
comet assay.19 Mouse fibroblasts (3 T3-L1) were cultured in 12-well
culture plates (1 ×105cells per well) for 24 h. The cells were then
pre-treated with dichloromethane extracts of P. crispum, at con-
centrations of 100 –400 μgmL
1for a further 24 h. The control con-
tained DMSO instead of extract. After pre-treatment, cells were
treated with 100 μmol L1of H2O2(final concentration in the well)
for 60 min on ice to induce DNA damage. Cells were then harvested
using a cell scraper, centrifuged and resuspended in 1mL PBS. The
cell suspension (25 μL) was mixed with 75 μL of 0.6% low-melting
agarose and the suspension was spread on a frosted microscopic
slide pre-coated with 250 μL of 0.8% normal melting agarose, cov-
ered with a cover slip, and then allowedto solidify on ice for 10 min.
The cover slips were removedand the slides were immersed in cold
lysis solution containing 1% SDS, 2.5 mol L1NaCl, 100 mmol L1
Na2ethylenediaminetetraacetic acid (Na2EDTA), 1% Triton X-100
and 10% DMSO (with the DMSO added just before use) for 1h
at 4 C in the dark. The slides were arranged in an electrophore-
sis tank filled with pre-chilled electrophoretic buffer (1 mmol L1
Na2EDTA and 300 mmol L1NaOH) and incubated for 20 min. Elec-
trophoresis was conducted in the same buffer at 25 V (300mA) for
20 min. The slides were washed with 0.4 mol L1Tris –HCl (pH 7.5)
and stained with 20 μgmL
1ethidium bromide for viewing under
a fluorescence microscope. The comet tail length was measured
using an ocular micrometer. A total of 50 individual cells were
screened per slide.20 The assay was carried out in triplicate. Results
were expressed in percent DNA protection, calculated using the
following formula:
DNAprotection (%)=(taillengthcontrol taillengthtreatment )
taillengthcontrol ×100
Scratch motility assay
The inhibitory effect of P. crispum on MCF-7 cell migration was
tested using the scratch motility assay. MCF-7 cells (3.5×105cells
per well) were seeded in a 24-well plate and grown for 24h.
The confluent cell monolayer was then scratched vertically with a
pipette tip, washed twice with PBS and incubated with media con-
taining P. crispum dichloromethane extract (0, 200, 300, 400, 500
and 600 μgmL
1)with5%FBS.H
2O2wasaddedintoeachwellat
a final concentration of 1 μmol L1in the cell suspension to stim-
ulate the proliferation and migration of MCF-7 cells. The number
of cells in the denuded area were photographed and counted at 0
and 24 h incubation. The experiment was performed in triplicate.
The percentage inhibition was calculated as described by Sato and
Rifkin.21 Percentage inhibition =100 [(cell no. in denuded area of
sample / cell no. in denuded area of control)×100].
Trypan blue dye exclusion assay
A hemocytometer-based trypan blue dye exclusion cell quanti-
tation and viability assay was used to confirm the antiprolifera-
tive activity of P. crispum. MCF-7 cells were seeded in a six-well
plate (1.5 ×106cells per well) supplemented with 5% FBS and
allowed to grow at 37 C in a humidified atmosphere with 5%
CO2. After 24 h incubation, the cells were treated with differ-
ent concentrations of extract (0800 μgmL
1) for a further 48 h.
Diluent-control wells with similar DMSO concentrations as in the
treatment were included. Following treatment, the cells were col-
lected using 0.25% trypsinEDTA, pelleted and resuspended in
medium. The cells were then stained with an equal volume of
0.2% (w/v) trypan blue solution and the number of viable cells was
counted using a hemocytometer under an inverted microscope.
All measurements were performed in triplicate.
DNA fragmentation analysis
Agarose gel electrophoresis was used to investigate DNA fragmen-
tation in cells treated with P. crispum. MCF-7 cells (2 ×106mL1)
were grown in a 75 cm2culture flask for 24 h. The cells were
then treated with P. crispum dichloromethane extract (500 or
800 μgmL
1) for 24 or 48 h. After treatment, cells were lysed and
the DNA was extracted using TRIzol®reagent according to the
manufacturer’s protocol. Extracted DNA was treated with RNase
A(3mgmL
1)at37
C for 1 h and proteinase K (200 μgmL
1)at
50 C for 2 h. The purified DNA was stored at 20 C until DNA
electrophoresis. The experiment was performed in triplicate. Iso-
lated DNA samples were subjected to electrophoresis in 1.8% (w/v)
agarose gel (in TAE buffer) impregnated with 0.5 μgmL
1ethidium
bromide and run at 90 V for 50 min. The gel was observed under
UV illumination and visualized using a gel documentation system
(UVP, USA).
Statistical analysis
Data are presented as mean ±standard deviation (SD). Statistical
analysis was performed by one-way analysis of variance (ANOVA)
with Tukey’s multiple comparisons and Student’s t-test. A P-value
of <0.05 was considered statistically significant. Pearson’s correla-
tion coefficient was used to assess the correlation between TPC,
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www.soci.org EL-H Tang et al.
Table 1. Phenolic content, ferric reducing antioxidant power and DPPH radical scavenging activity of Petroselinum crispum extracts
P. cri s p u m extract/
positive controls
Total phenolic content
(mg GAE g1)
FRAP value
(mmol g1)
DPPH radical scavenging
activity (μgmL
1)
Hexane 20.17 ±1.35b 0.075 ±0.002b 4485.0 ±78.0b
Dichloromethane 42.31 ±0.50d 0.360 ±0.009d 3310.0 ±80.5a
Ethyl acetate 32.17 ±2.24c 0.139 ±0.006c 4712.0 ±87.0c
Methanol 24.77 ±1.24b 0.027 ±0.004a ND
Aqueous 9.63 ±2.60a 0.014 ±0.003a ND
Positive control
Rutin 649.93 ±13.34 1.789 ±0.214 42.7 ±2.3
Quercetin 1275.62 ±56.03 14.444 ±0.934 22.2 ±0.9
Results are presented as means ±SD (n=3).
IC50 values are presented for the DPPH radical scavenging activity.
Values within the same column with different letters (a–d) are significantly different at P<0.05 from the different extracts. ND, not detected.
FRAP and DPPH radical scavenging activity. Statistical Package
for the Social Sciences (SPSS) version 18.0 (Chicago, IL, USA) and
Microsoft Excel 2007 (Roselle, IL, USA) were used for the statistical
and graphical evaluations.
RESULTS AND DISCUSSION
Total phenolic content
Phenolics are secondary metabolites that are ubiquitously present
in plants. Positive correlations between antioxidant activities
present in medicinal plants with their total phenolic content have
been reported. In our study, the phenolic values of P. crispum
extracts ranged from 9.63 ±2.60 to 42.31 ±0.50 mg GAE g1
(Table 1). The dichloromethane extract displayed the highest
phenolic content (P<0.05) among the extracts. The nature of
the extracting solvent is one of the most important factors in the
extraction of antioxidants, thus explaining the various phenolic
values from different extracts of P. crispum leaves and stems.
A study by Luthria22 investigated the influence of particle size on
phenolic compound extraction of P. crispum with ethanol:water,
50:50 (v/v), using a pressurized liquid extractor. They reported
phenolic values ranging from 18.3 to 22.9 mg GAE g1. In our
study, we showed higher phenolic contentin the dichloromethane
(42.31 ±0.50 mg GAE g1), ethyl acetate (32.17 ±2.24 mg GAE g1)
and methanolic (24.77 ±1.24 mg GAE g1)extractsofP. crispum
compared to that reported by Luthria.22 The dichloromethane
extract in our study also showed higher phenolic content com-
paredtotheP. crispum hydrodistilled ex tract (29.2 ±0.44 mg GAE
g1) reported by Hinneburg et al.23 A possible explanation for
the different phenolic values between these studies and that
reported in our study is that phenolic content in plants differ very
much between cultivars of the same species and are influenced
by genetic factors and environmental conditions.14 In addition,
parameters such as solvent polarity, extraction procedures and
conditions can influence the extraction of phenolic compounds
from P. crispum.24 Our study showed that the dichloromethane sol-
vent resulted in the highest extraction of phenolics from P. crispum
leaves and stems.
Ferric reducing antioxidant power
The FRAP values of P. crispum extracts are presented in Table 1.
Among the extracts tested, the dichloromethane extract of P.
crispum exhibited the highest FRAP value (0.360 ±0.009 mmol g1,
P<0.05).
Table 2. Correlation analysis of total phenolic content with antioxi-
dant activities of Petroselinum crispum extracts
Pearson correlation (r)value
Plant TPC/FRAP TPC/DPPH
P. cri s p u m (leaf and stem) 0.875*0.910*
TPC, total phenolic content; FRAP, ferric reducing antioxidant power;
DPPH, 1,1-diphenyl-2-picryl hydrazyl radical scavenging activity.
*Correlation is significant at the 0.01 level.
Pearson correlation analysis was performed to assess the rela-
tionship between phenolic content and ferric reducing activities
of the leaf and stem extracts. A statistically significant positive
correlation was identified between FRAP and phenolic content
of P. cr i s p um (r=0.875, P<0.01; Table 2). This indicates that phe-
nolic compounds present in P. crispum contributed to their ferric
reducing activities. The reductive ability of the extracts suggests
their ability to donate electrons to reduce ferric tripyridyltriazine
(Fe3+-TPTZ) to the ferrous complex (Fe2+-TPTZ). This implies that
P. crispum extracts may provide antioxidative protection from free
radicals in actual biological systems by donating electrons to rad-
icals and blocking radical chain reactions from causing diseases
related to chronic oxidative stress.25
DPPH radical scavenging activity
The dichloromethane extract of P. crispum showed the lowest IC50
value (3310.0 ±80.5 μgmL
1,P<0.05) compared to the other P.
crispum extracts (Table 1). In a study by Zhang et al.,26 essential
oil from P. crispum showed antioxidant activities in the 𝛽-carotene
bleaching assay (EC50 =5.12 mg mL1) and DPPH scavenging
assay (EC50 =80.21 mg mL1). Hinneburg et al.23 reported that
the P. cr i s p um hydrodistilled extract showed an IC50 value of
12.0 ±0.10 mg mL1in the DPPH scavenging assay. In our study,
the P. crispum extracts showed better DPPH radical scavenging
activity (Table 1) compared to the two studies mentioned above.
A strong and significant positive correlation was seen between
DPPH scavenging activity and phenolic content of P. crispum
(r=0.910, P<0.01; Table 2). This shows that phenolic compounds
of P. crispum could be responsible for the observed DPPH radical
scavenging activity, since these compounds can readily donate
hydrogen atoms to the radical. From the antioxidant study, it
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Antioxidant, DNA-protective and anticancer properties of Petroselinum crispum www.soci.org
Highest percentage inhibition (%)
–10
0
10
20
30
40
50
60
MCF-7
b
bb
bbb
b
aaa
aa
cc
c
MDA-
C
MB-231
Cell line
HT-299
Hexa
Dich
Ethy
Meth
Aque
ane
hloromethane
yl acetate
hanol
eous
e
Figure 1. Anti-proliferative activities of Petroselinum crispum extracts on cancer cell lines, MCF-7, MDA-MB-231 and HT-29. Results are presented as
means ±SD (n=3). Values within the same cell line with different letters (a–c) are significantly different at P<0.05 from the different extracts. Extract
concentration tested: 0 –500 μgmL
1.
is observed that the dichloromethane extract displayed highest
phenolic content and FRAP value while exhibiting best DPPH
radical scavenging activity among the extracts of P. crispum leaves
and stems.
Antiproliferative activity
Extracts of P. crispum (0– 500 μgmL
1) were tested for their effect
on the proliferation of MCF-7, MDA-MB-231 and HT-29 cells,
using the MTT assay. Generally, extracts of P. crispum leaves
and stems exhibited weak cytotoxic activity with percent inhi-
bitions below 50% (Fig. 1). Among the five extracts analyzed,
the dichloromethane extract exhibited the best antiprolifera-
tive activity. At the highest concentration tested (500μgmL
1),
the dichloromethane extract showed a percentage inhibition
of 48.4% ±1.8%, 25.5% ±3.0% and 49.9% ±1.0% on MCF-7,
MDA-MB-231 and HT-29 cells, respectively (Fig. 1). The ethyl
acetate, methanol and aqueous extracts showed less than 20%
inhibition, even at 500 μgmL
1of extract. The different cytotoxic
effects of the various extracts in this study suggest the impor-
tance of using solvents of differing polarity in order to extract
compounds with various polarities that contribute to different
biological activities of the plant extract. Each extract of P. crispum
behaved differently against the cell lines. The distinct effects of
these extracts may be due to the phytodiversity or different mech-
anisms associated with the compounds present in the extracts
and the various susceptibility levels of cell lines to the plant
extracts.27,28
In a study by Yoshikawa et al.,29 the methanolic extract from
the aerial parts of P. crispum (1 and 10 μgmL
1)wasshownto
have potent estrogenic activity and increased MCF-7 cell pro-
liferation. In our study, the methanol extract did not increase
MCF-7 cell proliferation but exhibited a very weak antiprolifer-
ative effect on MCF-7 cells. Our study on P. crispum leaves and
stems showed that the dichloromethane extract displayed best
antioxidant and antiproliferative activities. Hence we selected the
dichloromethane extract for further analysis.
DNA protective activity
Within living cells, reactive oxygen species are constantly being
generated as normal by-products of mitochondrial respiration.
Uncontrolled levels of reactive oxygen species can cause severe
damage to macromolecules, especially DNA, leading to degener-
ative diseases such as cancer.30 H2O2is an oxidizing agent which
produces reactive hydroxyl radicals that can induce strand breaks
associated with DNA damage.20 The comet assay is a quick, sim-
ple and sensitive method for the evaluation of DNA damage,
mainly single-strand and double-strand breaks in individual cells.
The comet tail length is associated with DNA damage. Greater tail
length signifies greater DNA damage.19
Cells pre-treated with the dichloromethane extract of P. crispum
at concentrations of 100 –400 μgmL
1showed a significant
dose-dependent increase in DNA protection (P<0.05) compared
to the control of H2O2treatment alone (Table 3). At 400 μgmL
1of
extract pretreatment, DNA damage was reduced by 50.9% ±6.6%
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www.soci.org EL-H Tang et al.
Table 3. Protection from H2O2-induced DNA damage in 3 T3-L1
fibroblasts pre-treated with Petroselinum crispum extract
P. cri s p u m dichloromethane
extract (μgmL
1) DNA protection (%)
100 19.0 ±6.1*
200 23.1 ±6.9*
300 37.9 ±7.8*
400 50.9 ±6.6*
Results are presented as means ±SD (n=3).
*P<0.05 compared to control (without extract treatment), as tested
by Student’s t-test.
compared to the control, indicating 50.9% DNA protection. The
high phenolic content in the dichloromethane extract of P. crispum
as shown in Table 1 may be responsible for the observed DNA
protective effect. A study on spices (ginger, caraway, cumin,
cardamom, star anise and fennel) has shown a strong positive
correlation between DNA protection and phenols.20 Phenolics in
P. crispum can lower H2O2levels or hydroxyl radicals by increasing
the levels of H2O2-detoxifying enzymes in cells, thus preventing
DNA damage.31 Studies have shown that supplementation of
diets with fresh P. crispum leaves can increase antioxidant capacity
of rat plasma,11 protect against mitochondrial oxidative damage
inthemousebrain
10 and decrease oxidative stress in humans.12
Our study shows that the P. crispum extrac t protected 3 T3-L1
fibroblasts against H2O2-induced DNA damage, suggesting that
appropriate addition of the herb in the daily diet might reduce
the effects of free radical-induced carcinogenesis, hence affording
some protection against cancer.
Inhibition of H2O2-induced MCF-7 cell migration using
the scratch motility assay
Metastasis is the most characteristic aspect of malignant neoplasm
and is the leading cause of the ineffectiveness of chemothera-
peutic drugs and cancer deaths. The scratch motility assay tests
the ability of P. crispum extracts to inhibit migration of can-
cer cells in the denuded area, thus indicating defense against
(A)
(B)
(a) (b) (c)
(d) (e) (f)
20 µm
20 µm 20 µm 20 µm
20 µm 20 µm
P
.
dich
l
extra
crispum
l
oromethane
ct (µg mL
-1
)
200
300
400
500
600
)
Perce
n
ntage inhibi
t
(%)
27 ± 6*
41 ± 4*
30 ± 13
26 ± 8*
18 ± 7
tion
Figure 2. Effect of Petroselinum crispum dichloromethane extract on the inhibition of H2O2-induced MCF-7 cell migration in a denuded area using the
scratch motility assay. (A)Photographs of cell migration (a) in untreated cells and (b) in cells treated with P. c r i sp u m dichloromethane extract at 200 μgmL
1,
(c) 300 μgmL
1, (d) 400 μgmL
1, (e) 500 μgmL
1and (f ) 600 μgmL
1after 24 h. (B) Percentage inhibition presented as means ±SD (n=3). Asterisk
represents *P<0.05 compared to the control (without extract) as tested by Student’s t-test.
wileyonlinelibrary.com/jsfa © 2015 Society of Chemical Industry J Sci Food Agric (2015)
Antioxidant, DNA-protective and anticancer properties of Petroselinum crispum www.soci.org
0.00E+00
5.00E+05
1.00E+06
1.50E+06
2.00E+06
2.50E+06
3.00E+06
3.50E+06
0
20
40
60
80
100
120
0 100 200 400 500 600 700 800
Live cell number
% of control
P. crispum dichloromethane extract (µg mL
1
)
% of control Live cell number
****
**
*
Figure 3. Trypan blue exclusion assay of MCF-7 cells treated with Petroselinum crispum dichloromethane extract. The results are presented as the number
of viable cells counted per well and the percent of viable cells relative to untreated control. The viability of untreated control cells was taken as 100%.
Results are presented as means ±SD (n=3). *P<0.05 compared to the control (without extract) as tested by Student’s t-test.
metastasis.32 H2O2was included in this experiment to induce
the proliferation and migration of MCF-7 cells. The concentra-
tion of H2O2(1 μmol L1) used in this assay has been previously
tested in our laboratory and showed increased cell migration and
proliferation.20
In this study, the scratch motility assay displayed the ability
of P. crispum to suppress H2O2-induced migration of MCF-7 cells
in a denuded area (Fig. 2). Treatment with P. crispum extract at
300 μgmL
1resulted in the highest inhibition of MCF-7 migration
(41% ±4%). At higher concentrations of P. crispum extract, the
inhibitory effect on cell migration decreased; at the highest con-
centration tested, 600 μgmL
1, the inhibition of migration was
the lowest (18% ±7%). The dichloromethane extract of P. crispum
inhibited the migration of MCF-7 cells, but not in a directly pro-
portional manner to the concentration of extract. It is interesting
to note that the inhibition of proliferation induced by the extract
was highest at the highest concentration of 500 μgmL
1. As cell
migration is necessary for metastasis of cancer cells, inhibition
of migration is an indication of protection against metastasis.
Petroselinum crispum prevented migration of MCF-7 cells, thus
showing potential in preventing metastasis. The flavonoids
present in P. crispum, apigenin and luteolin9have been reported
as chemopreventive agents of metastasis due to their ability to
prevent tumor cell motility and invasion.33 Phenolics present
in P. crispum might lower H2O2levels or hydroxyl radicals by
increasing the levels of H2O2-detoxifying enzymes in cells such as
glutathione peroxidase,31 thus preventing cancer cell proliferation
andmigrationinducedbyH
2O2. Antioxidants present in P. crispum
can maintain H2O2levels in cells within physiological levels and
may be associated with the prevention of cancer cell proliferation
and migration.
Trypan blue dye exclusion
Trypan blue is taken up by dead cells that have lost their mem-
brane permeability barrier or dye exclusion capacity, while the
intact plasma membrane of live cells excludes the dye.34 To
assess the antiproliferative effect of P. crispum on MCF-7 cells,
trypan blue exclusion counts were conducted on cells treated
with 0– 800 μgmL
1of dichloromethane extract for 48 h. A sig-
nificant dose-dependent decrease in live cell number (P<0.05)
was observed in cells treated with the dichloromethane extract
of P. crispum compared to the control (Fig. 3). This is shown
by the lower number of live cells counted as the concentra-
tion of dichloromethane extract treatment increased. Using
the trypan blue exclusion assay, the percent of viable cells rel-
ative to untreated control was 55.04%±0.75% at 500 μgmL
1
of dichloromethane extract treatment, whereas at the highest
concentration of extract treatment (800 μgmL
1) cell viability
decreased to 30.10% ±1.48%, indicating the antiproliferative
activity of P. crispum dichloromethane extract on MCF-7 cells.
This antiproliferation profile by trypan blue exclusion assay fur-
ther confirmed the inhibitory effect of P. crispum on MCF-7 cell
proliferation analyzed using the MTT assay (Fig. 1).
DNA fragmentation analysis
DNA fragmentation is a hallmark of apoptosis. In agarose gel elec-
trophoresis, apoptotic cells demonstrate a characteristic DNA ‘lad-
der’ pattern at 200 bp intervals, while necrotic cells are observed
as a ‘smear’ of randomly degraded DNA.35 However, internucleoso-
mal DNA fragmentation is not universal as it may not always occur
during apoptosis.36
In this study, apoptotic DNA fragmentation was analyzed using
agarose gel electrophoresis. DNA isolated from untreated con-
trol cells exhibited one clear band that pointed to the pres-
ence of living cells with intact DNA strand (Fig. 4). A typical
DNA ladder pattern was not evident in MCF-7 cells treated with
dichloromethane extract of P. crispum (500 or 800 μgmL
1)for
24 or 48 h. Instead, a smear pattern of DNA fragmentation was
observed in the extract-treated cells, with more intense smear-
ing seen in cells treated for 48 h, compared to 24 h. The results
could indicate that P. crispum dichloromethane extract kills MCF-7
cells by necrosis in a time-dependent manner, where random DNA
fragmentation occurs through the release of lysosomal DNases to
form a ‘smear’ on agarose gels.37 Conversely, studies have reported
that MCF-7 cells can undergo apoptosis without showing DNA
fragmentation due to lack of caspase-3, which is responsible for
this feature.38,39 Thus, using the results from DNA fragmentation
J Sci Food Agric (2015) © 2015 Society of Chemical Industry wileyonlinelibrary.com/jsfa
www.soci.org EL-H Tang et al.
12345
M2M1
1000 bp
10000 bp
250 bp
500 bp
750 bp
1500 bp
400 bp
100 bp
200 bp
1000 bp
2000 bp
600 bp
800 bp
Figure 4. Electrophoresis of DNA extracted from MCF-7 cells after treatment with Petroselinum crispum dichloromethane extract. M1: 1 kb DNA ladder;
lane 1: DNA from cells of control; lane 2: DNA treated with 500 μgmL
1extract for 24 h; lane 3: DNA treated with 800 μgmL
1extract for 24 h; lane 4: DNA
treated with 500 μgmL
1extract for 48 h; lane 5: DNA treated with 800 μgmL
1extract for 48 h; M2: 100 bp DNA ladder.
alone would not be definitive to accurately ascertain the mode of
cell death (whether by apoptosis or necrosis, or both), in which
P. crispum kills MCF-7 cells.Further work will be needed for more
in-depth investigation into the mechanism of cell death induced
by P. crispum.
CONCLUSIONS
The dichloromethane extract of P. crispum leaves and stems
showed antioxidant activities and also inhibition of proliferation
and cell migration in MCF-7 cells. The extract also protected
against DNA damage induced by H2O2.
Regular addition of P. crispum in the daily diet as food or supple-
ments can help strengthen the antioxidant systems of the body
and reduce the effects of free radical-induced carcinogenesis, can-
cer and subsequent metastasis caused by prolonged and excessive
oxidative stress.
ACKNOWLEDGEMENTS
The study was supported by the University of Malaya Research
University grants RG004/09AFR, PS250/2010B and RG341/11HTM.
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