IJBPAS, October, 2014, 3(10): 2199-2222

ISSN: 22774998

THE ANTIOXIDANT AND ANTI-TUMOR ACTIVITIES OF THE LEBANESE

ERYNGIUM CRETICUM L.

DIRANI Z1, MAKKI R1, RAMMAL H1, 2*, NASERDDINE S1, HIJAZI A1, KAZAN HF3,
NASSER M1, DAHER A1 AND BADRAN B1
1
Doctoral School of Science and Technology, Research Platform for Environmental Science
(PRASE), Faculty of Sciences, Lebanese University, Lebanon
2
Faculty of Agriculture and Veterinary Science, Lebanese University, Lebanon
3
Laboratory of Experimental Hematology, Institut Jules Bordet, Universit Libre de Bruxelles,
Belgium
*Corresponding Authors E Mail: [email protected]
ABSTRACT
To determine the phytochemical screening of Eryngium creticum L. in order to investigate its in
vitro anti-oxidant, anti-proliferative and cytotoxic activities. The effect of period of growth of the
plant on the chemical composition was also studied. Aqueous and ethanolic extracts from
different parts (leaves, stems, roots, and the whole plant) of the fresh plant E. creticum from the
first and second harvest were performed to fractionate the chemical constituents into
individual fractions or extracts. The extracts were tested for different secondary metabolites
content by classical phytochemical screening tests, antioxidant (DPPH radical scavenging,
superoxide radical scavenging), cytotoxic and cell viability (Neutral red assay on HeLa cells),
and apoptotic activity (DNA fragmentation assay on HeLa cells). Our results showed that the
different parts of first and second harvest of E. creticum contain alkaloid, tannin, coumarin, saponin,
flavonoid, polyphenol and reducing sugars in different concentrations. Moreover, the 4 parts of this plant
have exerted antioxidant activity that may be due to their phenolic content and they have also inhibited
the viability of HeLa cell line in a time-dependent (072 h) and dose-dependent (0250 M) manner.
Finally, we demonstrated that the ethanolic extracts from leaves of the second harvest were the most
potent (at 48 h) with an IC50 value 47.24 g/ml.

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The in vitro anti-oxidant and antiproliferative effects of crude aqueous and ethanolic extracts
from first and second harvest of E. creticum leaves, stems, root and whole plant indicate that it
has sufficient potential to warrant further examination and development as a new anti-cancer
agent.
Keywords: Eryngium creticum, Phytochemical Screening, Antioxidant Activity,
Antiproliferative Activity, Cytotoxicity, Cell Viability
INTRODUCTION
According to the Global Burden of Disease, estimated the overall prevalence for the use of
cancer is a leading cause of death worldwide, herbal products to be 13% to 63% among
accounting for 8 million deaths in 2010 [1]. It cancer patients [6]. In a study on the use of
is a complex disease caused by numerous CAM by breast cancer survivors in Canada,
factors ranging from environmental factors to the investigators found that 67% of the
hereditary genetics. Current treatment of respondents in a randomized survey reported
cancer can be categorized into three groups: using CAM [7]. The study concluded that
surgery, chemotherapy, and radiation CAM use is common among Canadian breast
therapy [2-4]. Surgical removal of tumor and cancer survivors and that many are discussing
cancerous growth can effectively treat 50% of CAM therapy options with their
cancer but cannot eliminate all cancer cells, physicians [7]. Thus, many investigations are
which results in high recurrence rate [3]. now being carried out to discover naturally
Chemotherapy and radiation therapy have the occurring compounds, which can suppress or
potential of removing all cancer cells but both prevent the process of carcinogenesis [8, 9].
lack the specificity to cancer cells since Lebanon is among the countries that are
normal cells are also often targeted by most of highly rich in medicinal plants in the
these treatments, thus harming normal body Mediterranean region. In this study, we are
cells in the process and causing countless interested by a Lebanese food plant,
side-effects. The focal point of cancer Eryngium creticum.
research is still the search for effective and Eryngium creticum, a perennial plant which
specific anti-cancer treatments [5]. belongs to the Umbellifereae family. It is
There is widespread use of Complementary found principally in Lebanon, Palestine,
and Alternative Medicine (CAM) in Jordan, and Syria. It is cultivated for use as
developed countries [5, 6]. A recent study vegetable mainly in salad. E. creticum is

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traditionally used as diuretic, laxative. E. creticum plants were collected during 2013
Submerged roots and seeds in water had been in 2 different periods, the first one in March
drunk to treat the kidney stone and the (the plant is edible and maturing) and the
infections, skin diseases and tumors. It is an second one in May (the plant is mature and
antidote, used in the treatment of the snakebite non-consumable) from South Lebanon.
[10]. E. creticum also showed anti- Preparation of Extracts
inflammatory and anti-microbial activities The stems, leaves and fresh roots, as well as
[11]. It was also used in the treatment of liver the whole plant of E. creticum were well
diseases, poisoning, anemia and infertility washed and cut into small pieces that are then
[12]. This plant has shown an antioxidant placed in the selected solvent (100 g of each
property by inhibiting the lipid peroxidase in of the plant in 500 ml of the selected solvent:
the liver of the rat [13]. distilled water or ethanol). After a period of
Very little data on cytotoxicity and anti-tumor maceration and stirring for 8 hours at room
activities of this plant exist in literature. For temperature and then at 37 C, the macerate
that, our study aimed to determine the obtained was filtered to remove insoluble
phytochemical screening for aqueous and residues. The filtrate was then condensed with
ethanolic extracts of two harvest periods of E. a rotary evaporator to half evaporation and the
creticum leaves, stems, roots and the whole filtrates were then frozen before being
plant, and to evaluate their antioxidant power lyophilized powder to be processed.
using hydrogen peroxide radical and DPPH Phytochemical screening tests
radicals and for the first time the cytotoxicity To study the phytochemical composition of
and the anti-proliferative activity against the aqueous and ethanolic extracts of the
cervical cancer cell line (Hela) by using stems, leaves, roots and the whole plant,
neutral red cytotoxicity assay. The mechanism qualitative detection of secondary metabolites
of anticancer activity of this plant extracts has was performed according to Muanda [14].
however not been reported. Therefore, the
anticancer mechanism of the E. creticum Detection of Alkaloids
extract via apoptosis induction was The detection and determination of alkaloids
investigated in the current study. are practiced by using the reagent of
MATERIALS AND METHODS Dragendorff. The appearance of orange-red
Plant Material precipitate indicates the presence of alkaloids.

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Detection of Tannins Detection of Terpenoids

After filtration of 10 ml of each of the plant 1 ml of acetic anhydride and 2 ml of
parts extract, the filtrate obtained was mixed concentrated sulfuric acid are added to 1 ml
with 2 to 3 ml of a solution of ferric chloride of each filtered part extract. The presence of a
(FeCl3 1%). The occurrence of blue color reddish brown color to the surface of mixture
indicates the presence of tannins. indicates the presence of terpenoids.
Detection of Resins Detection of the Volatile Oil
10 ml of each filtered extract is mixed with 20 10 ml of each extract were filtered through a
ml of hydrochloric acid (HCl 4 filter paper to saturation of the paper by the
%). The presence of the resins is indicated by extract. Then, it is placed under UV light. The
the observation of the turbidity of a mixture. appearance of a pink color gloss on the paper
Detection of Coumarins indicates the presence of the volatile oil.
Test tubes containing 5 ml of each extract are Detection of Flavonoids
covered by filter paper by a saturated In test tubes each containing 5 ml of part
solution of sodium hydroxide (NaOH). These extract, 5 ml of potassium hydroxide (KOH
tubes are then placed in a water bath and are 50%) were added. The observation of a
boiled for 15 minutes. The filter papers are yellow color indicates the presence of
subjected to UV radiation, and the appearance flavonoids.
of a bright yellow color indicates the presence Detection of Carbohydrates
of coumarins. Benedict Test: To 0.5 ml of the filtrate, 0.5 ml
Detection of Saponins of Benedict has been added and then boiled
2 ml of each part extracts were stirred for 2 minutes. The red brick precipitate
vigorously for 5 minutes on Vortex. The indicates the presence of sugar.
presence of saponin is indicated by the Evaluation of the Antioxidant Activity
appearance of more or less significant foam. For each of the prepared samples, an
Detection of Phenols evaluation of the antioxidant activity was
In 5 ml beakers, each filtered part extract is performed according to the method of
mixed with 1 ml of FeCl3 (1%) and 1 ml of Rammal et al., [15] using the free radical 2,2 -
K3(Fe (CN)6) (1%). The appearance of a blue diphenyl- 1 - picrylhydrazyl (DPPH) and the
color indicates the greenish presence of H2O2 test.
phenols. DPPH Radical Scavenging

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Increasing concentrations of each sample (0.1, Dulbecco's Modified Eagle Medium (Sigma
0.2, 0.3, 0.4 and 0.5 mg/ml) have been Chemical Company), containing 10% fetal
prepared. To 1 ml of each dilution of the part bovine serum, penicillin 1% antibiotic
extract prepared, 1 ml of the DPPH reagent solution (penicillin 50 U/ml and streptomycin
was added. The solution was incubated for 30 0.5 mg/ml). Once cell confluence had been
minutes at room temperature in the dark, and reached, Hela cells were transferred, under
then the absorbance was measured at 517 nm. sterile conditions, into 48-multiwell plates
The antioxidant activity is calculated using (10.000 cells/well) for the toxicity tests.
the following equation: Neutral Red Analysis
% Antioxidant activity = [(Abs control - Cell viability was performed using Neutral
Abs sample) / Abs control] x 100 Red assay based on the initial protocol as
The control was prepared by mixing 1 ml of described earlier [16, 17]. Neutral Red, a
DPPH with 1 ml of used solvent (distilled chromogenic dye, is an indicator of lysosomal
water or ethanol) and the blank was composed activity. Live cells demonstrate a
of 1 ml of the selected solvent. chromogenic change with Neutral Red that is
Scavenging with Hydrogen Peroxide detected spectrophotometrically. Briefly,
(H2O2) cells were detached from the tissue culture
A solution of H2O2 (40 mM) was prepared in flask with 2 ml of trypsin solution. The cell
PBS (pH 7.4). Various concentrations of plant pellet was obtained by centrifugation at 1.000
part extracts were added to a solution of H2O2 rpm for 5 minutes. The density of the viable
(0.6 ml, 40 mM), and the absorbance was cells was counted by the trypan blue
measured at 230 nm after 10 minutes against exclusion in a haemocytometer. Cells were
a blank containing PBS without H2O2. The then plated in 96-well microtiter plate, at a
antioxidant activity is calculated using the concentration of 1 104 cells/well and
following equation: incubated in a humidified 37C, 5% CO2
% Antioxidant activity = [(Abs control - incubator that allows the cells to adhere. After
Abs sample) / Abs control] x 100 24 h, the cells were treated with five different
Cells and Cell Culture concentrations of aqueous and ethanolic
Cervical cancer (HeLa) cell line were grown extracts: 50, 100, 150, 200, and 250 g/ml
in 25 cm tissue culture flasks (Nunclon TM, each being tested in three replicates. The
Nunc) at 37C, 5% CO2 in DMEM: plates were incubated for 24, 48 and 72 h at

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37C in a 5% CO2 incubator. The untreated DNA Fragmentation Detection Assay

cells were regarded as a negative control, The DNA fragmentation was used to identify
whilst cells incubated only with ethanol apoptosis by observing the biochemical
(0.5%, v/v) were used as a vehicle control. No changes. Briefly, cancer cells were treated
effect due to the ethanol was observed. with the 200 g/ml of ethanolic root extracts
Arsenic was used as the positive control. At of first and second harvest of E. creticum for
24, 48 and 72 h, the old medium was replaced 48 h. Cells were then collected and washed
with 100 l of fresh medium containing 40 with media. Then cell suspension was
g/ml neutral red and incubated for 3 h. This transferred to microcentrifuge tube (1.5 ml)
is to allow the uptake of the vital dye into the and centrifuged at 1500 rpm for 5 min to
lysosomes of viable and undamaged cells. collect the cell pellet. The DNA in the cell
Then, the media was discarded and cells were pellet was extracted using "QIAGEN kit
washed twice with 100 l of 1X PBS. The (Zymo Research, Belgique), and 5 g of each
intracellular accumulation of neutral red dye DNA sample was analyzed by electrophoresis
was extracted in 200 l of a 50% ethanol-1% on 2% agarose gels containing 0.1 mg/ml
acetic acid lysing solution. ethidium bromide. The migration was done at
The optical density (OD) of the eluted dye 75V. After electrophoresis, DNA fragment
was read at 490 nm using a microplate reader. was analyzed by using UV luminated camera.
The experiments were conducted in Statistical Analysis
triplicates. The percentage of inhibition of Data are presented as mean standard
each of the test samples was calculated deviation (SD) of three independent
according to the following formula using the experiments and statistical significance was
OD values obtained: determined using the Independent Student's t-
Percentage of inhibition (%) = (OD control test and Prism 6.0 software (GraghPad
OD sample)/OD control 100 Software, Inc.). A significant difference was
The average of three replicates was then considered if P <0.05.
obtained. Cytotoxicity of each test agent is RESULTS AND DISCUSSION
expressed as IC50 value. The IC50 for each Phytochemical Screening
extract was extrapolated from the graphs of The results of the phytochemical screening
the percentage inhibition versus concentration represented in Tables 1 and 2 show that the
of test agents [1620]. different parts of E. creticum are rich in

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various secondary metabolites at different influence the distribution of metabolites

concentrations depending on the solvent used. between the two harvests especially in the
Indeed, we note the presence of saponins and second harvest where these metabolites could
coumarins in the aqueous extract from the have been replaced by other compounds
different studied parts of this plant. which may play important roles in various
The ethanolic extract is rich in secondary biological activities. This was not the case
metabolites including: phenols, flavonoids, with the roots, because in fact the roots are in
alkaloids, carbohydrates and resins. Thus, the soil, so the variation of the temperature
there is a difference in the distribution of and water is negligible. The extract of the
metabolites extracted using the same solvent whole plant which represents the mixture of 3
between the leaves, stems and roots of this parts of the plant (leaves + stems + roots)
plant. Leaves are richer in metabolites shows a composition richer in secondary
compared to stems and roots, which assigns metabolites in comparison with the aqueous
them greater biological availability. extract in the first and the second harvest, but
Consequently, E. creticum by its richness in in lower quantities than the other portions.
different secondary metabolites may have This result seems to be normal because the
several medical importances. proportion of each part is divided by 3. This
In addition, comparing the first and second shows that the effect of the combination of
harvest we see a difference in the distribution plant extracts is additive.
of metabolites for the first harvest which Antioxydant Activity
contains more metabolites than the second The activity of chemical compounds draws
one, except roots that are rich in secondary our attention because of their potential role in
metabolites in the second harvest more than the prevention of certain human diseases. A
the first. This can be attributed to the effect of number of studies indicated that flavonoids,
the maturation of the plant, wherein in the polyphenols and triterpenoids have an
first harvest (in March, the plant is maturing antioxidant activity and ability to scavenge
and consumables) it scored more water and free radicals (Frankel et al. 1995). These
less temperature, whilst in the second harvest phytoconstituents can have multiple
(in May, the plant is mature and non- biological effects against tumors, heart
consumable) it had received less water and disease and various diseases due to their
more temperature, and this can in turn trapping free radicals. Hence our study was to

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evaluate the antioxidant power of the extracts Concerning the whole plant, this extract
through two different in vitro methods: DPPH showed an antioxidant activity of 55% and
and H2O2. 59% for the first and the second harvest
DPPH respectively, which represents an activity
Figures 1 (A, C) show the increase in the equal to the average of the effect of three
antioxidant activity of the aqueous extract of separate parts (Figure 1 D).
leaves, stems and roots of E. creticum with The obtained results show a difference in the
increasing concentrations. This increase antioxidant activity between the aqueous and
reached 77% , 89% and 70% at the the ethanolic extract in favor of the ethanolic
concentration of 0.5 mg/ml of leaves, stems which is in agreement with our results
and roots, respectively, for the first harvest, obtained in the phytochemical screening.
while for the second one, it reached 73%, Indeed this extract was richer in flavonoids
59% and 34% at the same concentration of and polyphenols, which have an important
the leaves, stems and roots respectively. The role in the antioxidant activity.
aqueous extract of the whole plant shows an Furthermore, comparing the antioxidant
antioxidant activity of 72% at the same activity between the first and second harvest
concentration for the first harvest and 49% for indicates that it is higher in the first one.
the second. This confirms, as phytochemical To confirm the results obtained by the DPPH
screening, that in both the effect of the method, we used another in vitro method; the
combination of plant extracts is additive, and H2O2.
the antioxidant effect obtained is nearly equal The aqueous extracts at the concentration of
to the average of the effect of three separate 0.5 mg/ml of the leaves, stems, roots and
parts (Figure 1 C). whole plant of E. creticum from the first
The ethanolic extract shows an antioxidant harvest showed an antioxidant activity of
activity of 93%, 82% and 44% for leaves, 92%, 87%, 68% and 74% respectively. This
stems and roots, respectively, at a antioxidant activity was approximately 87%,
concentration of 0.5 mg/ml for the first 76%, 66% and 68% for the leaves, stems,
harvest whilst for the second harvest this roots and whole plant of the second harvest, at
activity was about 56%, 65% and 61% at the the concentration of 0.5 mg/ml (Figure 2 A,
same concentration for the leaves, stems and C).
roots, respectively (Figure 1 B, D).

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On the other hand, the ethanolic extracts of of the HeLa cell line using the Neutral Red
leaves, stems, roots and the whole plant Cytotoxicity/ Viability Assay after the
showed maximum antioxidant activity of treatment of this cancerous cell line for 24, 48
92%, 78%, 71% and 66% respectively for the and 72 hours with increasing concentrations
first harvest, whilst in the second harvest this (50, 100, 150, 200, and 250 g/ml) of these
activity was 75%, 73%, 66% and 70% for extracts. The effect of inhibition by these
these same parts respectively (Figure 2 B, D). extracts from the first and second harvest was
Hydrogen peroxide is an important reactive dose and time-dependent (Figures 3, 4). The
species of oxygen because of its ability to percentage of inhibition was calculated
penetrate biological membranes. But, it can according to the formula mentioned above.
be toxic if it's converted into hydroxyl radical The results of the neutral red assay shows that
in the cell. the different concentrations of aqueous
Scavenging of H2O2 by the plant extracts can extracts of the stems from the first and second
be attributed to the phenolic compounds harvest have no antiproliferative effect at
giving an electron to H2O2, and therefore different studied times (Data not shown)
reduce the H2O. Our results showed that whilst the different concentrations of aqueous
extracts from the plant are capable of extract of the leaves and the whole plant from
scavenging H2O2 depending on the the first harvest and the different
concentrations used. These results showed concentrations of aqueous extract of the roots
that E. creticum has an important antioxidant plant from second harvest showed little
activity and therefore it can be considered a antiproliferative effect (because they do not
good natural source that could be used in the exceed 30% inhibition) at different studied
treatment of diseases associated to the times (Figure 3 A, B and E). The aqueous
oxidative stress. extracts of the second harvest showed an
Cell viability and cytotoxicity effect of antiproliferative activity greater than that of
crude aqueous and ethanolic extract of E. the first for the leaves and for the whole plant
creticum in HeLa cancer cells (Figure 3 C, F). On the other hand, the
An evaluation of the antiproliferative activity aqueous extracts of the roots, from the first
of each of the extracts prepared from leaves, harvest showed an antiproliferative activity
stems, roots and of the whole plant of E. greater than that of the second harvest
creticum was done by measuring the viability (Figure 3 D). At the 150, 200, and 250 g/ml

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dose, leaf extracts from second harvest were effective, and treatment with this dose for 72
effective, and treatment with these doses for hours resulted in about 87 to 97% inhibition
48 and 72 hours resulted in about 90 % in Hela cells viability (Figure 4 A, C, D, E,
inhibition on Hela cells (Figure 3 C). As G and H).
shown in Figure 3 D, treatment with root In order to determine the concentration
extract from first harvest at the 250 g/ml required to achieve a 50% inhibition of cells
dose for 72 h also resulted in significant induced by each aqueous and ethanolic
inhibition (90%) of viability of HeLa cells. extracts, the dose response curve was plotted.
The results indicated that E. creticum aqueous The results of cytotoxic assay were mentioned
extracts from the first and second harvest as IC50 (g/ml) in Table 3 and Table 4. It is
exerted anti-proliferative activity by clear by our results that ethanolic leaves (at
decreasing the viability of HeLa cancer cells. 24 h) and aqueous whole plant extracts (at 48
On the other hand, our results showed that the h) from second harvest have the highest
majority of the ethanolic extract of E. antiproliferative activity on the HeLa cell
creticum from the first and second harvest lines with an IC50 of 47.24 1.23 to 52.33
showed a significant antiproliferative effect at 0.91 g/ml respectively.
different doses and different times (Figure 5). Apoptotic Effects of the Extracts from E.
The different concentrations of the stem creticum in HeLa Cells
ethanolic extract from first harvest showed In order to determine whether the investigated
little antiproliferative effect (because they do plant extracts have pro-apoptotic activities,
not exceed 35% inhibition) at different we examined occurrence of internucleosomal
studied times (Figure 4 B) whilst the DNA fragmentation (180 bp laddering), the
ethanolic extracts of the stem from the second hallmark of apoptosis by agarose gel
harvest showed an antiproliferative activity electrophoresis of genomic DNA isolated
greater than that of the first harvest. As shown from Hela cells treated with plant extracts of
in Figure 6 F, treatment with root extract E. creticum. The morphological changes of
from the second harvest at the 250 g/ml dose the nuclei DNA after being treated with
for 72 h also resulted in significant inhibition ethanolic root extracts from the first and
(70%) of viability of HeLa cells. At the 250 second harvest of E. creticum (200 g/ml) for
g/ml dose, leaf, root and whole plant extracts 48 hours are shown in Figure 6. The results
from first and second harvest were very showed a destruction of genomic DNA and

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appearance of band at approximately 180 bp that the cytotoxicity of E. creticum extracts is

due to apoptosis of the HeLa cells treated based on their prominent proapoptotic effects.
with E. creticum. These analyses confirmed

Table 1: Comparison of the phytochemical composition of crude aqueous extracts from leaves, stems, roots
and the Whole Plant (WP) of E. creticum
First harvest Second harvest

Leaves Stems Roots WP Leaves Stems Roots WP

Alkaloids - - - - + - + -
Tannins + - - + - - - -
Resins - - - - - - - -
Coumarins + ++ + + + ++ ++ ++
Saponins +++ +++ ++ +++ ++ + ++ +++
Phenols - - + +++ - ++ - ++
Terpenoids - - - - - ++ - ++
Volatil oils - - - - - - - -
Flavonoids - - - - +++ + - +
Carbohydrates - - - - - - ++ +
Glucosides - - - - - - - -

Table 2: Comparison for the phytochemical composition of crude ethanolic extracts from leaves, stems, roots
and the Whole Plant (WP) of E. creticum
First harvest Second harvest

Leaves Stems Roots WP Leaves Stems Roots WP

Alkaloids + - ++ +++ - + ++ +
Tannins - + + ++ - - - -
Resins +++ + - ++ - + + -
Coumarins - ++ + + + + ++ +
Saponins ++ ++ + +++ + + - +
Phenols - + - + + + - +
Terpenoids - - + + - - - -
Volatil oils - - - - - - - -
Flavonoids ++ ++ + +++ - + ++ +++
Carbohydrates ++ + ++ + - ++ + +
Glucosides - - - - - - - -

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(1) Aqueous extracts from the first and (2) second harvests. Tabulated values are mean SD of three
replicates

(1) Ethanolic extracts from the first and (2) second harvests. Tabulated values are mean SD of three
replicates

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Figure 1: The in-vitro antioxidant activity of the aqueous (A, C) and ethanolic (B, D) crude extracts of
different parts of E. creticum measured by the DPPH radical scavenging activity

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Figure 2: The in-vitro antioxidant activity of the aqueous (A, B) and ethanolic (C, D) crude extracts of
different parts of E. creticum measured by the H2O2 radical scavenging activity

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Figure 3: Effect of crude aqueous extract of E. creticum plant on viability (% of inhibition) of HeLa cancer
cells as determined by Neutral Red assay. (A-C) HeLa cells were treated with (0-250 g/ml) of aqueous
extract from the first (A) and second harvest (C) of E. creticum leaf for 24-72 h. (B-F) HeLa cells were treated
with (0-250 g/ml) of aqueous extract from the first (B) and second harvest (F) of the whole plant for 24-72 h.
(D-E) HeLa cells were treated with (0-250 g/ml) of aqueous extract from the first for 24-72 h (D) and second
harvest (E) for 72 h of E. creticum roots. Each experiment was done in triplicate. Data are expressed as mean
SD. *p < 0.05 was considered to be statistically significant, **p < 0.01 very significant, and ***p < 0.001
highly significant

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Figure 4: Effect of e than ol i c extract of E. creticum plant on viability of HeLa cancer cells as determined by
Neutral Red assay. (A-D) HeLa cells were treated with (0-250 g/ml) of e thanol i c extract from the first
harvest of E. creticum leaf (A), stem (B), roots (C) and whole plant (D). (E-H) HeLa cells were treated with (0-
250 g/ml) of e than ol i c extract from the second harvest of leaf (E), stem (F), roots (G) and whole plant (H).
Proliferation was measured by Neutral Red assay. Each experiment was done in triplicate. Data are
expressed as mean SD. *p < 0.05 was considered to be statistically significant, **p < 0.01 very significant,
and ***p < 0.001 highly significant

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0 g/ml 50 g/ml
100 g/ml

150 g/ml 200 g/ml

250 g/ml

Figure 5: Microscopic view of HeLa cells treated with ethanolic root extracts from the first harvest of E. creticum
for 72 h. HeLa cells after 72 hours incubation with (0-250 g/ml) of e than ol i c root extracts from the first
harvest of E. creticum. The results presented are from one experiment representative of three carried out, and
were photographed with a microscope ( 40)

Figure 6: The latter stage of apoptosis (DNA laddering) in HeLa cells induced by e than ol i c root
extracts (200 g/ml) from the first (E.R.1) and second harvest (E.R.2) of E. creticum for 48 hours. The new
band generated at approximately 180 bp due to apoptosis is indicated by arrow

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DISCUSSION scorpion venom and antagonize cobra. It was

Many scientific studies show that the plant found that E. creticum extract will prolong the
kingdom provides a rich source of potential life of guinea pig when combined with
cancer chemopreventive and therapeutic. The scorpion venom [24]. The E. creticum was
main agents currently used in clinical practice also tested for their inhibitory activity of
in the treatment of malignant diseases different methicillin-resistant Staphylococcus
originate from plants: vinca alkaloids, aureus [25]. It was demonstrated that the
taxanes, camptothecins and ethanolic extracts of the E. creticum have
epipodophyllotoxins [21]. In the last decades, effective antimutagenic activity. However, the
researchers of anticancer natural products aqueous extracts lacked any inhibitory
chemistry focused their research in a wide efficacy [26]. E. creticum showed antimycotic
variety of natural compounds, especially on and antihyperglycemic activities [27, 28].
phenolic compounds. Numerous The crude aqueous extract of E. creticum leaf
phytochemicals derived from edible plants and stem was found to possess antioxidant
have been reported to affect different activity [29-30]. It was detected that the
intracellular signaling pathways implicated in consumption of 100 g of fresh E. creticum
the initiation, promotion and progression of leaves and stems could provide antioxidants
cancer. The antitumor effects of plant equivalent to (78.50 0.80) mg of vitamin C
compounds have been associated with the and (50.42 0.50) mg of vitamin C,
induction of free radicals, anti-inflammatory respectively. The ethanol extracts either from
activity, induction of cell cycle the aerial parts or roots of Eryngium species
arrest or apoptosis, inhibition of tumor showed apparent antioxidant, antinociceptive
angiogenesis and metastasis [22, 23]. and anti-inflammatory activity [31].
It is reported that root extracts of the different Regarding the link between inflammation and
Eryngium species are used in traditional cancer, chemicals with anti-inflammatory
medicine for diurectic, antidiabetic and properties targeting the small molecules or
antispasmodic purposes as well as in the regulating signaling cascades implicated in
treatment of asthma, liver diseases, low-back inflammation and carcinogenesis are regarded
pain, and cough and in certain poisoning as key cancer chemopreventive drugs.
conditions. An aqueous extract of the roots of Research into the anti-cancer potential of
E. creticum was found to be able to neutralize herbal extracts has been studied as a

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cancer treatment or prevention [22]. On the attributed to the actions of different E.

other hand, data about the potential anticancer creticum constituents on target molecules of
activity of extracts and phytochemicals of the signal transduction pathways that regulate
plants from the genus Eryngium are rare. The cell proliferation and apoptosis. Furthermore,
cytotoxicity and anti-proliferative effect of E. each of the investigated extracts exhibited
creticum human breast adenocarcinoma considerably stronger cytotoxicity to HeLa
MCF-7 cells have been documented. The cells. The prominent antitumor properties of
aqueous and ethyl acetate extracts of both these extracts need to be examined further in
leaves and stems of E. creticum didnt show in vivo studies. It would be interesting to
any cytotoxicity on MCF-7 cell line whilst the investigate their action towards different
methanolic extract of both studied parts had PBMC subpopulations and elucidate the
inhibited the growth of MCF7 by 72% and potential mechanisms through which they
68% respectively at a concentration of 50 M stimulate proliferation.
[29]. However, the molecular mechanisms of Several studies have shown that saponins,
the anti-proliferative effect of E. creticum flavonoids, phenols, and tannins have an
phytochemical extracts have not been studied. important role in the anticancer and in the
The anti-proliferative effect of crude aqueous antioxidant activity. Phenolics, flavonoids,
and ethanolic extracts of E. creticum aerial and tannins from crude aqueous extracts of P.
parts and roots on human cervical carcinoma indica were shown to have anticancer
HeLa cell line have not yet been studied. In potential by the inhibition of ATP-binding
contrast to previous studies, this is the first cassette transports in cancer cells [32].
study to examine the extract effect from the Saponins from Radix astragali were found to
second harvest of plant. suppress colon cancer cell carcinogenic
Our results demonstrate the selective dose- activity by reducing vascular endothelial
dependent cytotoxic actions of the crude growth factor [33]. Flavonoid intake was
aqueous and ethanolic extracts from first and found to be associated with a significant
second harvest of E. creticum leaves, stems, reduction in the risk of gastric cancer in
root and whole plant against human cervical women [34] whilst proanthocyanidins from
carcinoma HeLa cell line. The observed grape seeds was found to inhibit pancreatic
selectivity in the antitumor effects of the cancer cell growth and to induc apoptosis
extracts against HeLa cell line could be [35]. The cycle arrest and pro-apoptotic

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IJBPAS, October, 2014, 3(10)
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effects of coumarin were shown against caspases implicated in the employed apoptotic
bladder cancer cell line [36]. The phenolic pathways.
and flavonoid coumpouds of Citrus CONCLUSION
aurantium bloom were found to have In conclusion, the results of this study suggest
antioxidant properties, anti inflammatory and that aqueous and ethanolic extracts of first
anti cancer activity against human cancer cell and second harvest of E. creticum has a
lines (MCF-7; MDA-MB-231), and human promising anti-oxidant, antiproliferative and
colon adenocarcinoma (HT-29) [37]. The cytotoxic effect on HeLa cervical carcinoma
presence of tannins, flavonoids, phenolics, cells. In addition, it was found for the first
and saponins, among others in the extracts time that the second harvest of E. creticum
used in the present study, may be responsible ethanolic extracts induced tumor apoptosis.
for the antiproliferative activities on the HeLa Further investigations are needed to determine
cell line. In our study, it was observed that the entire anti-cancer molecular mechanism of
ethanolic leaves and aqueous whole plant E. creticum extracts. Also, it is very important
extracts from second harvest might be a to determine the cancer- suppressive effect of
significant source of novel promising the tested extracts in in vivo experiments.
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