Investigating the Antiproliferative and Antioxidant Properties of Pancratium maritimum L. (Amaryllidaceae) Stems, Flowers, Bulbs, and Fruits Extracts

Abstract

Pancratium maritimum stems, flowers, bulbs, and fruits extracts were investigated for their antiproliferative and antioxidant properties. Total phenols and total flavonoids were also determined. The in vitro antiproliferative activity was tested against seven cancer cell lines such as C32, HeLa, MDA-MB-231, PC3, A549, MCF-7, and LNCaP by using SRB assay. Interesting results were obtained with stems ethanol extract (ET1) against C32 cells (IC 50 of 27.1 μ g/mL) and fruits aqueous extract (AQ) against MCF-7 cell line (IC 50 of 36.5 μ g/mL). To define the antioxidant activity, four tests such as DPPH, ABTS FRAP, and β -carotene bleaching tests were used . The most promising ABTS scavenging capacity was observed with fruits ethanol extract (ET1) that showed an IC 50 value of 6.9 μ g/mL. According to the correlation results, the phenols and flavonoids content could provide a fundamental contribution to the antioxidant and antiproliferative activity of P. maritimum extracts.

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Research Article
Investigating the Antiproliferative and Antioxidant
Properties of Pancratium maritimum L. (Amaryllidaceae)
Stems, Flowers, Bulbs, and Fruits Extracts
Mariarosaria Leporini,1Giorgia Catinella,2Maurizio Bruno ,2Tiziana Falco,1
Rosa Tundis,1and Monica R. Loizzo 1
1Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Rende (CS), Italy
2DipartimentodiScienzeeTecnologieBiologicheChimicheeFarmaceutiche(STEBICEF),UniversityofPalermo,Palermo,Italy
Correspondence should be addressed to Maurizio Bruno; maurizio.bruno@unipa.it
and Monica R. Loizzo; monica rosa.loizzo@unical.it
Received 29 June 2018; Revised 22 August 2018; Accepted 19 September 2018; Published 30 September 2018
Academic Editor: Adolfo Andrade Cetto
Copyright ©  Mariarosaria Leporini et al. is 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.
Pancratium maritimum stems, owers, bulbs, and fruits extracts were investigated for their antiproliferative and antioxidant
properties. Total phenols and total avonoids were also determined. e in vitro antiproliferative activity was tested against seven
cancer cell lines such as C, HeLa, MDA-MB-, PC, A, MCF-, and LNCaP by using SRB assay. Interesting results were
obtained with stems ethanol extract (ET) against C cells (IC50 of . 𝜇g/mL) and fruits aqueous extract (AQ) against MCF- cell
line (IC50 of . 𝜇g/mL). To dene the antioxidant activity, four tests such as DPPH, ABTS FRAP, and 𝛽-carotene bleaching tests
were used.e most promising ABTS scavenging capacity was observed with fruits ethanol extract (ET) that showed an IC50 value
of . 𝜇g/mL. According to the correlation results, the phenols and avonoids content could provide a fundamental contribution
to the antioxidant and antiproliferative activity of P. maritimum extracts.
1. Introduction
e genus Pancratium (Amaryllidaceae family) comprises
about  species. P. maritimum L. or marine narcissus, is
a plant species typical of sandy coasts, widely disseminated
from the Mediterranean to the Black Sea, including the part
of the Atlantic coasts []. Although widely distributed, P.
maritimum populations have declined signicantly due to
urbanization, tourism development, alteration and destruc-
tion of dune systems, and overharvesting []. P. maritimum
is used in the traditional medicine of several Mediterranean
countries for its antimicrobial, antimalarial, purgative, antivi-
ral, immune-stimulant, antalgic, anticancer, antifungal, and
antioxidant properties [–].
Several studies focused on alkaloids as the main bioactive
constituents [, ] while few reports investigated the nonal-
kaloidal composition of P. maritimum.
Cancer and chemoprevention represent a major challenge
for health professionals worldwide [, ]. e pharma-
cological strategy, although eective in some cases, causes
numerous toxic eects due to the action that these drugs also
have towards healthy tissue cells that possess a high fraction
of proliferating cells, such as bone marrow cells and epithelial
cells []. e research activity in the eld of oncology is
therefore increasingly directed towards the selection of new
molecules with greater activity and less toxicity towards
healthy tissues. erefore, the development of a safe, nontoxic
plant protection product is justied []. Oxidative stress is
closely related to all aspects of cancer, from carcinogenesis
to the tumor-bearing state, from treatment to prevention
[]. Reducing oxidative stress is related to the anticancer
eect. Nowak et al. [] report the importance of chemo-
prevention with natural compounds to reverse, suppress,
or prevent the development of invasive cancer. Moreover,
Hindawi
Evidence-Based Complementary and Alternative Medicine
Volume 2018, Article ID 9301247, 7 pages
https://doi.org/10.1155/2018/9301247

 Evidence-Based Complementary and Alternative Medicine
natural antioxidants can eliminate free radicals such as singlet
oxygen or peroxides by donating hydrogen and chelating
metal ion. ese activities decrease DNA damage, reduce
lipid peroxidation, and inhibit cell proliferation that isclosely
related to cancer development [, ]. Hence, the studies
on natural products characterized by both antioxidant and
antiproliferative activities have gained increasingly greater
importance. Following our previous studies, the aim of this
work is to assess the in vitro antiproliferative activity against
seven human cancer cell lines and antioxidant properties,
in relation to the phenols and avonoids content of P.
maritimum owers,bulbs,stems,andfruits.
2. Materials and Methods
2.1. Chemicals and Reagents. All chemicals and reagents used
in this study were purchased from Sigma-Aldrich Chemical
Co. Ltd (Milan, Italy) and VWR International (Milan, Italy)
and, unless specied otherwise, were analytical grade or
higher.
2.2. Plant Materials. Flowers, stems, bulbs, and fruits of
Pancratium maritimum were collected in September  in
Lascari (Palermo, Italy) (∘󸀠󸀠󸀠 N, ∘󸀠󸀠󸀠 E,  m s/l)
on a sandy soil. Voucher specimens (No. MB /) were
identied by Dr. E. Schimmenti and deposited in the Depart-
ment STEBICEF, University of Palermo, Palermo, Italy.
2.3. Extraction Procedure. Fresh owers, stems, bulbs, and
fruits of P. maritimum were blended and extracted with two
dierent methodologies: (a) sequential extraction with
petroleum ether (ETP), ethanol (ET), and water (H2O, %
of H2SO4)(AQ)and(b)macerationwithethanol(ET)(
x  mL). Petroleum ether and ethanol were evaporated at
low pressure, ∘C, using a Rotavapor Buchi R- (Buchi,
Milan, Italy), whereas the water extracts were freeze-dried
with Scanlaf Coolsafe -.
2.4. Total Phenols Content. Total phenols content was eval-
uated by using the Folin-Ciocalteau method as previously
reported []. A solution of Folin-Ciocalteau reagent and
% sodium carbonate was mixed with sample. e mixture
was incubated at room temperature for  h. e absorbance
was measured at 𝜆=  nm using a UV-Vis Jenway 
spectrophotometer. e total phenols content was expressed
as mg chlorogenic acid equivalents/g of extract.
2.5. Total Flavonoids Content. Total avonoids content was
determined following the method previously described [].
e extract was mixed with % aluminum chloride solution
and le to incubate at room temperature for  min. e
absorbance was measured at 𝜆=  nm using a UV-Vis
Jenway  spectrophotometer. e total avonoids content
was expressed as mg quercetin equivalents/g of extract.
2.6. Radical Scavenging Activity
2.6.1. DPPH Test. ,-Diphenil--picrylhydrazyl (DPPH)
radical scavenging activity was evaluated following the
method previously described []. Dierent concentrations
of the extract were mixed with DPPH (. mM) and le to
incubate at room temperature for  min. e absorbance
was measured at 𝜆=  nm using a UV-Vis Jenway 
spectrophotometer. e DPPH radicals scavenging activity
was calculated as follows: DPPH scavenging activity = [(A0−
A1/A0)×], where A is the absorbance of the blank and
A is the absorbance in the presence of the extract. Ascorbic
acidwasusedaspositivecontrol.
2.6.2. ABTS Assay. ABTS assay was done following the
methodology previously described []. A solution of ABTS
radical cation (ABTS+) and potassium persulphate was pre-
pared. Aer  h the solution was diluted with ethanol until
an absorbance of . ±. measure at 𝜆=nmusing
a UV-Vis Jenway  spectrophotometer. e extract and
diluted ABTS+solution were mixed and aer  min and the
absorbance has been read again. e ABTS scavenging ability
was calculated as follows: ABTS scavenging activity (%) =
[(A0−A)/A0]× where A0is the absorbance of the control
reaction and A is the absorbance in the presence of extract.
Ascorbic acid was used as positive control.
2.6.3. 𝛽-Carotene Bleaching Test. e 𝛽-carotene bleach-
ing test was performed following the procedure previously
described []. A solution of 𝛽-carotene, linoleic acid, and
 % Tween was prepared. Aer evaporation of the solvent
by using a rotary evaporator the  mL of water was added. e
emulsion was transferred into dierent tubes containing .
mL of extract at dierent concentrations. e absorbance was
measured at 𝜆=  nm using a UV-Vis Jenway  spec-
trophotometer. Propyl gallate was used as positive control.
2.6.4. Relative Antioxidant Capacity Index (RACI) Calcula-
tion. e statistical application RACI was used to evaluate
the antioxidant capacity of extracts []. e standard scores
were obtained from data from dierent chemical methods
without unrestricted units and no variance between the
methods.
2.6.5. Global Antioxidant Score (GAS). e T-scores were
used to calculate the value of Global Antioxidant Score
(GAS). T-score is calculated by the following equation: T
−score = (X −min)/(max −min), where min and max,
respectively, represent the smallest and largest values of
variable X among the investigated extract [].
2.7. Antiproliferative Activity
2.7.1. Cell Culture. Seven cancer cell lines, namely, human
Caucasian breast carcinoma (MCF-, ECACC N∘:),
Human cervix epitheloid carcinoma (HeLa, ECACC N∘:
), human Caucasian breast adenocarcinoma (MDA-
MB-, ECACC N∘:), amelanotic melanoma (C,
ATC C N∘:CRL-), lung carcinoma A (ECACC No.
), human Caucasian prostate carcinoma (LNCaP,
ECACC N∘:), and human Caucasian prostate ade-
nocarcinoma (PC, ECACC N∘: ), were used in
our experiments. All media, buers, trypsin, and dyes were

Evidence-Based Complementary and Alternative Medicine 
lter-sterilized prior to use and warmed to ∘C. e MDA-
MB-, C, and LNCaP cells were cultured in RPMI 
medium, while MCF-, HeLa, A, and PC cells were
cultured in DMEM. Both media were supplemented with
% fetal bovine serum, % L-glutamine, and % penicillin/
streptomycin. e cell lines were maintained at ∘Cina
% CO atmosphere with % humidity. e cultures were
passed once a week by trypsinization using a : dilution of
standard Trypsin-EDTA solution. Cells counts and viability
were performed using a standard trypan blue cell counting
technique.
2.7.2. Sulforhodamine B Assay. e antiproliferative activity
was performed by using the protein-staining sulforhodamine
B (SRB) assay as previously described []. Cells were tryp-
sinized, counted, and placed in -well plates at optimal
plating density of each cell line determined over a range -
 ×4to ensure exponential growth throughout the exper-
imental period and to ensure a linear relationship between
absorbance at  nm and cell number analyzed by the SRB
assay and incubated to allow for cell attachment. Aer  h
the cells were treated with serial dilutions of the samples. Each
sample was initially dissolved in DMSO and further diluted
in medium to produce dierent concentrations. One hundred
microliters/wells of each dilution were added to the plates
in six replicates to obtain the nal concentrations ranging
from  to  𝜇g/mL for the sample. e nal mixture used
for treating the cells contained not more than .% of the
solvent (DMSO), the same as in the solvent-control wells.
Aer  h of exposure  𝜇L of ice-cold % trichloroacetic
acid (TCA) was added to each well, le for  h at ∘C, and
washed with distilled water. e TCA-xed cells were stained
for  min with  𝜇L of .% (w/v) SRB in % acetic acid.
Plates were washed with % HOAc and air-dried overnight.
For reading plate, the bound dye was solubilised with  𝜇L
of  mM tris base (tris[hydroxymethyl]aminomethane). e
absorbance of each well was read on a Molecular Devices
SpectraMax Plus Plate Reader (Molecular Devices, Celbio,
Milan, Italy) at  nm. Cell survival was measured as the
percentage absorbance compared to the untreated control.
Vinblastine sulfate salt, doxorubicin, and taxol were used as
positive control.
2.8. Statistical Analysis. All experiments were carried out in
triplicate. Data are expressed as mean ±standard deviation
(SD). e concentration giving % inhibition (IC50)was
calculated by nonlinear regression with the use of Prism
GraphPad Prism version . for Windows (GraphPad So-
ware, San Diego, CA, USA). e concentration-response
curve was obtained by plotting the percentage inhibition
versus concentration. Dierences within and between groups
were evaluated by one-way analysis of variance test (ANOVA)
followed by multicomparison Dunnett’s test compared with
the positive control.
3. Results and Discussion
3.1. Extraction Yield, Total Phenols, and Flavonoids Content.
P. maritimum owers, fruits, stems, and bulbs were extracted
T : Total phenols and total avonoids content of P. maritimum
extracts.
Sample Total Phenols ContentaTotal Flavonoids Contentb
Flowers
ETP . ±. . ±.
ET . ±. . ±.
AQ . ±. . ±.
ET . ±. . ±.
Fruits
ETP . ±. . ±.
ET . ±. . ±.
AQ . ±. . ±.
ET . ±. . ±.
Stems
ETP . ±. . ±.
ET . ±. . ±.
AQ . ±. . ±.
ET . ±. . ±.
Bulbs
ETP . ±. . ±.
ET . ±. . ±.
AQ . ±. . ±.
ET . ±. . ±.
1Data are expressed as mean ±SD (n= ). ETP: p etroleum ether extract; ET:
sequential extraction with ethanol; AQ: sequential extraction with water;
ET: maceration with ethanol; amg of chlorogenic acid equivalents/g of
extract. bmg of quercetin equivalents/g of extract.
by using two methods. Firstly, plant materials were sequen-
tially with petroleum ether (ETP), ethanol (ET), and water
(AQ). Extracts with the following yields (%) were obtained:
owers (ETP, .%) (ET, .%) (AQ, .%); stems (ETP,
.%) (ET, .%) (AQ, .%); bulbs (ETP, .%) (ET, .%)
(AQ, .%); fruits (ETP, .%) (ET, .%) (AQ, .%).
e second methodology consisted in the extraction of
fresh and blended plant materials with ethanol (ET) to
give, aer solvent evaporation, the following yields %: owers
(.%); stems (.%); bulbs (.%); fruits (.%). All samples
were stored at ∘C for further investigations.
e importance to determine the content of phenols in
plant extracts is related to the antioxidant capacity of these
bioactive compounds that are able to act as reducing agents,
free radical scavengers, metal chelators, or deactivators of
singlet oxygen and/or display simultaneously more than one
of these functions [].
Table  showed the total phenols and total avonoids
content of dierent P. maritimum extracts. Flowers ethanol
extract showed the highest total phenols content with value
of . mg of chlorogenic acid equivalents/g of extract.
Similar results are observed also with fruits and stems ethanol
seq. extracts. Fruits ethanol seq. extract showed, also, the
highest value of total avonoids content with value of .
mg of quercetin equivalents/g of extract. Recently, Johnson et
al. [] reported the total avonoids content of P. t r i  o r u m
extracts with values ranging from . to . mg

 Evidence-Based Complementary and Alternative Medicine
T : Antiproliferative capacity [IC50 (𝜇g/mL)] of P. maritimum extracts.
P. maritimum MCF- HeLa MDA-MB- C A LNCaP PC
Flowers
ETP . ±.∗∗∗ . ±.∗∗∗ NA . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗
ET . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗
AQ . ±.∗∗∗ . ±.∗∗∗ NA . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗
ET . ±.∗∗∗ . ±.∗∗∗ NA . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗
Fruits
ETP . ±.∗∗∗ . ±.∗∗∗ NA . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗
ET . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗
AQ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗
ET . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗
Stems
ETP . ±.∗∗∗ . ±.∗∗∗ NA . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗
ET . ±.∗∗∗ . ±.∗∗∗ NA . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗
AQ . ±.∗∗∗ NA . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗
ET . ±.∗∗∗ NA NA . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗
Bulbs
ETP . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗
ET NA . ±.∗∗∗ NA . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗
AQ . ±.∗∗∗ . ±.∗∗∗ NA . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗
ET . ±.∗∗∗ NA  . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗
Positive control
Vinblastine . ±. . ±. . ±.
Doxorubicin . ±. . ±. . ±. . ±.
Taxo l  .  ±. . ±. . ±. . ±.
Data are expressed as mean ±standard deviation (SD) (n= ). ETP: petroleum ether extract; ET: sequential extraction with ethanol; AQ: sequential extraction
with water; ET: maceration with ethanol; MCF-: human Caucasian breast carcinoma HeLa: Human cervix epithelioid carcinoma; MDA-MB-: human
Caucasian breast adenocarcinoma; C: amelanotic melanoma; A: lung carcinoma; LNCaP: human Caucasian prostatecarcinoma; PC: human Caucasian
prostate adenocarcinoma. MCF-, HeLa, and MDA-MB-: one-way ANOVA ∗∗∗p<.  followed by multicomparison Dunnett’s test: ∗∗∗p<. compared
with doxorubicin. C, A, and LNCaP: one-way ANOVA ∗∗∗p<. followed by multicomparison Dunnett’s test: ∗∗∗ p<. compared with vinblastine.
of GAE/g of extract for chloroform and methanol extracts,
respectively.
Previously, Taie et al. [] evaluated the total phenol and
avonoid content of P. maritimum root, bulb, leaves, owers,
and seeds and found the highest value in leaves (. mg
gallic/g extract and . mg quercetin/g extract, respectively).
3.2. Antiproliferative Activity. P. maritimum extracts were
tested to evaluate their antiproliferative activity on dierent
cancer cell lines including human Caucasian breast carci-
noma (MCF-), human cervix epithelioid carcinoma (HeLa),
human Caucasian breast adenocarcinoma (MDA-MB-),
amelanotic melanoma (C), lung carcinoma (A), human
Caucasian prostate carcinoma (LNCaP), and human Cau-
casian prostate adenocarcinoma (PC). Data are reported
in Table . All extracts showed antiproliferative eects in a
concentration-dependent manner. e stems ethanol extract
(ET) was the most active against C cells with an IC50 value
of . 𝜇g/mL, followed by the petroleum ether extract (ETP)
of bulbs (IC50 value of . 𝜇g/mL). Both these results are
of interest if compared to the positive control vinblastine
with an IC50 value of . 𝜇g/mL. e other IC50 values are
in the range .-. 𝜇g/mL. e stems ethanol extract
(ET) showed an activity higher than that of vinblastine (IC50
value . vs . 𝜇g/mL of positive control) also against lung
carcinoma cells.
Except for the ETP extract, the most promising results
against MCF- cell line were obtained with fruits extracts
with IC50 values in the range .-. 𝜇g/mL. Promising
values were obtained with bulbs petroleum ether extract
and aqueous extract that inhibited HeLa and LNCaP cells
growth with IC50 values of . and . 𝜇g/mL, respectively.
Recently, Tayoub et al. [] evaluated the eects of Iranian
P. maritimum bulbs, leaves, owers, and roots on human
breast cancer cells MDA-MB-. For this purpose plant
material was extracted by maceration with ethanol. e
antiproliferative activity was assessed using BD biosciences
cell viability kit with exposure time of , , , and 
hours of exposure. As in our experiments all extract inhibited
cancer cell in a dose-dependent manner however a more
pronounced cell growth inhibitory activity was observed also
in dependence of the time. Generally, bulbs showed more
antiproliferative activities than leaf extract. Bulbs ethanol
extract showed the most promising activity aer  h of
exposure with IC50 value of . mg/mL. e cytotoxic
activity is mediated by cell cycle cell arrest at S and G/M

Evidence-Based Complementary and Alternative Medicine 
T : Antioxidant activity of P. maritimum extracts.
P. maritimum DPPH test
(IC50 𝜇g/mL)
ABTS test
(IC50 𝜇g/mL)
𝛽-carotene bleaching
test
(IC50 𝜇g/mL)
𝛽-carotene bleaching
test
(IC50 𝜇g/mL)
FRAP test
𝜇M Fe (II)/g RACI GAS
 min  min
Flowers
ETP . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.  ∗∗∗ . ±.∗∗∗ . .
ET . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ -. .
AQ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ .  ±.∗∗∗ NA -. .
ET . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . .
Fruits
ETP % . ±.∗∗∗ .% .% NA -. .
ET . ±
.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.  ∗∗∗ . ±.∗∗∗ . .
AQ .% . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . .
ET . ±
.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ -. .
Stems
ETP . ±
.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . .
ET . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . .
AQ .% . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ NA -. .
ET . ±
.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ -. .
Bulbs
ETP . ±
.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ NA . .
ET % . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ NA -. .
AQ . ±
.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ . ±.∗∗∗ -. .
ET .% . ±.∗∗∗ . ±.∗∗∗ . ±.  ∗∗∗ NA . .
Positive control
Ascorbic acid . ±. . ±.
Propyl gallate . ±. . ±.
BHT . ±.
Data are expressed as mean ±standard deviation (SD) (n= ). ETP: petroleum ether extract; ET: sequential extraction with ethanol; AQ: sequential extraction
with water; ET: maceration with ethanol; RACI: Relative Antioxidant Capacity Index; GAS: Global Antioxidant Score. DPPH Radical Scavenging Activity
Assay: one-way ANOVA ∗∗∗p<. followed by multicomparison Dunnett’s test: ∗∗∗p<. compared with ascorbic acid. ABTS test: one-way ANOVA
∗∗∗p<. followed by a multicomparison Dunnett’s test: ∗∗∗p<. compared with ascorbic acid. 𝛽-carotene bleaching test at  minutes of incubation:
one-way ANOVA ∗∗∗p<. followed by multicomparison Dunnett’s test: ∗∗∗p<. compared with propyl gallate. 𝛽-carotene bleaching test at 
minutes of incubation: one-way ANOVA ∗∗∗p<.  followed by multicomparison Dunnett’s test: ∗∗∗ p<. compared with propyl gallate. Ferr ic Reducing
Antioxidant Power (FRAP): one-way ANOVA ∗∗∗p<. followed by multicomparison Dunnett’s test ∗∗p<. compared with BHT.
phases. e expression of cyclin B, Bcl-, and Ki was also
aected by plant extracts.
Based on the indications of the National Cancer Institute,
plant extracts with an IC50 value less of  𝜇g/ml are to
be considered as promising anticancer agents that needed
further investigation []. In this preliminary study, the focus
of our interest was on P. maritimum crude extracts. Further
studies will be done in order to identify phytochemicals
responsible of the activity and their mechanism of action.
3.3. Antioxidant Activity. Despite the presence of the several
antioxidant defence systems to neutralize oxidative stress,
oxidative damage may occur to cell structure and may induce
somatic mutations and neoplastic transformation. Indeed,
cancer initiation and progression hasbeen linked to oxidative
stress by inducing DNA damage, increasing DNA mutations,
and cell proliferation []. Counteracting oxidative stress
with potent antioxidant agents is a very active eld of
research. Herein, the antioxidant activity of P. maritimum
extracts was examined using dierent in vitro methods.
All samples showed concentration-dependent antioxidants
eects. Data are reported in Table .
e most promising scavenging capacity was observed
with fruits ethanol extract (ET) that inhibited ABTS

 Evidence-Based Complementary and Alternative Medicine
radicals with an IC50 value of . 𝜇g/mL, followed by owers
petroleum ether extract that showed an IC50 value of .
𝜇g/mL in DPPH assay. A signicant protection of lipid
peroxidation was observed with owers aqueous extract that
showed IC50 values of . and . 𝜇g/mL,aerand
 minutes of incubation, respectively. A moderate ferric
reducing activity for all tested samples was observed.
Previously, Nikolova et al. [] found an IC50 value greater
than  𝜇g/mL for the P. maritimum methanol bulbs extract.
A promising DPPH and ABTS radical scavenging activity
was observed also with Egyptian P. maritimum owers
and leaves methanol extracts []. In particular owers and
leaves extracts recorded the highest DPPH radical scavenging
potential with percentage of . and .%, respectively.
Moreover, owers signicantly inhibited ABTS+⋅ with per-
centage of .%. e leaves antioxidant potential was con-
rmed, also in Tunisian P. maritimum []. Leaves extract
showed stronger ORAC and DPPH inhibition compared to
bulbs extract. e comparison of diethyl ether and ethyl
acetate fractions of the aqueous extract of P. f o e t i d u m leaves
conrmed that the DPPH radical scavenging potential is
related to the total phenols content []. e key role of
phenols content in antioxidant capacity, with particular ref-
erence to the free radical scavenging activity, was previously
evidenced by Elmastas et al. [].
In our study, a positive correlation was found with total
phenols content and DPPH, 𝛽-carotene aer  and 
minutes of incubation, and FRAP test. In addition, a positive
correlation between total avonoids content and DPPH and
FRAP test was observed. e Relative Antioxidant Capacity
Index (RACI) and the Global Antioxidant Score (GAS) are
calculated and values are comprised in the range .- and
.-., respectively.
4. Conclusions
In this study, we investigated the total phenols and avonoids
content of P. maritimum stems, owers, bulbs, and fruits
extracts and their antiproliferative and antioxidant prop-
erties. e antiproliferative eect of the ethanol extract of
stems against C and A- cells may be related to their
antioxidant activity. Moreover, the ethanol extract of fruits,
with the higher content of avonoids, presents the highest
radical scavenging activity in ABTS test. In conclusion, the
results revealed that P. maritimum extracts can provide a good
source of antioxidant compounds and showed signicant
antiproliferative eects.
Abbreviations
ABTS: ,󸀠-Azinobis
(-ethylbenzothiazoline--sulfonic acid)
diammonium salt
DMSO: Dimethyl sulfoxide
DPPH: ,-Diphenyl--picrylhydrazyl
FRAP: Ferric Reducing Ability Power
GAS: Global Antioxidant Score
IC50: Concentration giving % inhibition
RACI: Relative Antioxidant Capacity Index
ROS: Reactive Oxygen Species
SD: Standard deviation
SRB: Sulforhodamine B
TCA: Trichloroacetic acid.
Data Availability
e data used to support the ndings of this study are
available from the corresponding author upon request.
Conflicts of Interest
e authors declare no conicts of interest.
Authors’ Contributions
Maurizio Bruno and Rosa Tundis conceived and designed
the experiments; Mariarosaria Leporini and Tiziana Falco
performed the experiments; Giorgia Catinella analyzed the
data; Mariarosaria Leporini wrote the paper; Monica R.
Loizzo supervised the project.
Acknowledgments
is work was supported by grant from MIUR-ITALY
PRIN “Top-Down and Bottom-Up Approach in the
Development of New Bioactive Chemical Entities Inspired on
Natural Products Scaolds” (Project no. MSCKCE ).
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