Cytotoxicity of the Essential Oil of Fennel (Foeniculum vulgare) from Tajikistan

Abstract

The essential oil of fennel (Foeniculum vulgare) is rich in lipophilic secondary metabolites, which can easily cross cell membranes by free diffusion. Several constituents of the oil carry reactive carbonyl groups in their ring structures. Carbonyl groups can react with amino groups of amino acid residues in proteins or in nucleotides of DNA to form Schiff’s bases. Fennel essential oil is rich in anise aldehyde, which should interfere with molecular targets in cells. The aim of the present study was to investigate the chemical composition of the essential oil of fennel growing in Tajikistan. Gas chromatographic-mass spectrometric analysis revealed that the main components of F. vulgare oil were trans-anethole (36.8%); α-ethyl-p-methoxy-benzyl alcohol (9.1%); p-anisaldehyde (7.7%); carvone (4.9%); 1-phenyl-penta-2,4-diyne (4.8%) and fenchyl butanoate (4.2%). The oil exhibited moderate antioxidant activities. The potential cytotoxic activity was studied against HeLa (human cervical cancer), Caco-2 (human colorectal adenocarcinoma), MCF-7 (human breast adenocarcinoma), CCRF-CEM (human T lymphoblast leukaemia) and CEM/ADR5000 (adriamycin resistant leukaemia) cancer cell lines; IC50 values were between 30–210 mg L⁻¹ and thus exhibited low cytotoxicity as compared to cytotoxic reference compounds.

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Article
Cytotoxicity of the Essential Oil of Fennel
(Foeniculum vulgare) from Tajikistan
Farukh Sharopov 1,2 ID , Abdujabbor Valiev 1, Prabodh Satyal 3, Isomiddin Gulmurodov 1,
Salomudin Yusufi 1, William N. Setzer 3and Michael Wink 2,*ID
1Department of Pharmaceutical Technology, Avicenna Tajik State Medical University, Rudaki 139,
Dushanbe 734003, Tajikistan; shfarukh@mail.ru (F.S.); valizoda83@gmail.com (A.V.);
gulmurodov@mail.ru (I.G.); salomudin@mail.ru (S.Y.)
2Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364,
Heidelberg 69120, Germany
3Department of Chemistry, University of Alabama in Huntsville, Huntsville, AL 35899, USA;
prabodhsatyal@gmail.com (P.S.); setzerw@uah.edu (W.N.S.)
*Correspondence: wink@uni-heidelberg.de; Tel.: +49-62-2154-4880
Received: 11 August 2017; Accepted: 16 August 2017; Published: 28 August 2017
Abstract:
The essential oil of fennel (Foeniculum vulgare) is rich in lipophilic secondary metabolites,
which can easily cross cell membranes by free diffusion. Several constituents of the oil carry reactive
carbonyl groups in their ring structures. Carbonyl groups can react with amino groups of amino
acid residues in proteins or in nucleotides of DNA to form Schiff’s bases. Fennel essential oil is rich
in anise aldehyde, which should interfere with molecular targets in cells. The aim of the present
study was to investigate the chemical composition of the essential oil of fennel growing in Tajikistan.
Gas chromatographic-mass spectrometric analysis revealed that the main components of F. vulgare
oil were trans-anethole (36.8%);
α
-ethyl-p-methoxy-benzyl alcohol (9.1%); p-anisaldehyde (7.7%);
carvone (4.9%); 1-phenyl-penta-2,4-diyne (4.8%) and fenchyl butanoate (4.2%). The oil exhibited
moderate antioxidant activities. The potential cytotoxic activity was studied against HeLa (human
cervical cancer), Caco-2 (human colorectal adenocarcinoma), MCF-7 (human breast adenocarcinoma),
CCRF-CEM (human T lymphoblast leukaemia) and CEM/ADR5000 (adriamycin resistant leukaemia)
cancer cell lines; IC
50
values were between 30–210 mg L
−1
and thus exhibited low cytotoxicity as
compared to cytotoxic reference compounds.
Keywords:
Foeniculum vulgare; essential oil; trans-anethole; anise aldehyde; cytotoxicity; cluster analysis
1. Introduction
Fennel (Arpabodiyon, local Tajik name), Foeniculum vulgare Miller, an important member of the
Apiaceae, is widely used for flavouring foods and beverages due to its pleasant spicy aroma [
1
,
2
].
In traditional medicine, the plant and its essential oil have been extensively used as carminative,
digestive, galactogogue and diuretic and to treat respiratory and gastrointestinal disorders [
1
].
It is also used as a constituent in cosmetic and pharmaceutical products [
3
]. The essential oil of
F. vulgare, in particular anethole, exhibits antispasmodic, carminative, anti-inflammatory, estrogenic
and anti-microbial activities [
4
].
In vitro
, fennel oil possesses antioxidant [
5
,
6
], antimicrobial [
7
],
insecticidal [
8
], antithrombotic [
9
] and hepatoprotective activities [
2
]. Furthermore, the essential oil
of fennel exhibits
in vitro
anticancer activity [
10
–
12
]. The
in vitro
cytotoxic, genotoxic, and apoptotic
activities of estragole were suspected to induce hepatic tumors in susceptible strains of mice [10].
Anethole is toxic in high concentrations [
4
]. Because of their lipophilic properties, the secondary
metabolites of essential oils are able to penetrate cytoplasmic membranes by free diffusion. This
Foods 2017,6, 73; doi:10.3390/foods6090073 www.mdpi.com/journal/foods

Foods 2017,6, 73 2 of 11
process can affect membrane fluidity and permeability, transport, ion equilibrium and membrane
potential [13], leading to cell death by apoptosis and necrosis [11].
The essential oil of fennel is rich in secondary metabolites, which carry reactive substituents
(among them carbonyl groups) in their ring structures or side chains. Aldehydes are generally
long-lived and electrophilic compounds, they can react with molecular targets which carry free
amino groups, such as of amino acid residues in proteins or of nucleotides in DNA to form Schiff’s
bases [
14
]. Aldehyde-containing essential oils often exhibit cytotoxicity [
15
,
16
] by reacting with cellular
nucleophiles, including proteins and nucleic acids [13,17].
The chemical composition of the essential oil of F. vulgare from different geographical locations
has been extensively studied [
6
,
18
–
20
]. According to these studies, the major components of fennel oil
are trans-anethole, estragole, fenchone, and limonene depending on the chemotype [
21
–
23
]. The aim
of the present study was to investigate the chemical composition of the essential oil of fennel growing
in Tajikistan (Central Asia) and to explore cytotoxic activity against different human cancer cell
lines. The biological activity and chemical composition of F. vulgare oil from Tajikistan have not been
previously reported.
2. Materials and Methods
2.1. Plant Material
The aerial parts of F. vulgare plants were collected from the Varzob region, Tajikistan on
29 July 2016
. A voucher specimen of the plant material was deposited at the Department of
Pharmaceutical Technology, Avicenna Tajik State Medical University under accession number
TD2016-24. The material was completely dried and hydrodistilled using a Clevenger-type apparatus
for 3 h to give an essential oil yield of 0.5%.
2.2. Gas-Liquid Chromatography-Mass Spectrometry (GLC-MS)
The essential oil from F. vulgare oil was analyzed by GLC-MS using an instrument
(GCMS-QP2010 Ultra, Shimadzu, Tokyo, Japan) operated in the EI mode (
electron energy = 70 eV
),
scan range = 3.0 scans s−1
. The GC column was ZB-5 fused silica capillary with a (5% phenyl)-polymethyl
siloxane stationary phase a film thickness of 0.25 mm. The carrier gas was helium with a column
head pressure 551 kPa and flow rate of 1.37 mL min
−1
. Injector temperature was 250
◦
C and the
ion source temperature was 200
◦
C, increased in temperature rate 2
◦
C min
−1
to 260
◦
C. The GC
oven temperature program was programmed from 50
◦
C initial temperature, increased at a rate of
2◦C min−1
to 260
◦
C. A 5% w/vsolution of the sample in CH
2
Cl
2
was prepared and 0.1
µ
L was
injected in splitting mode (30:1).
Identification of the oil components was based on their retention indices determined by
reference to a homologous series of n-alkanes (Kovats RI), and by comparison of their mass spectral
fragmentation patterns with those reported in the literature [
24
] and stored on the MS library
(NIST 11 (National Institute of Standards and Technology, Gaithersburg, MD, USA), WILEY 10
(
John Wiley & Sons, Inc.
, Hoboken, NJ, USA), FFNSC version 1.2 (Shimadzu Corp., Tokyo, Japan)).
The percentages of each component are reported as raw percentages based on total ion current without
standardization (set 100%).
2.3. Antioxidant Activity
The antioxidant activity of the essential oils was evaluated by 2,2-diphenyl-1-picrylhydrazyl
(DPPH), 2,2
0
-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and ferric reducing antioxidant
power (FRAP) assays. DPPH, ABTS and FRAP assays were performed as described earlier by us [
25
,
26
].

Foods 2017,6, 73 3 of 11
2.4. Cytotoxicity
The potential cytotoxicity of the fennel essential oil against of the five human tumor cell lines
(HeLa, Caco-2, MCF-7, CCRF-CEM and CEM/ADR5000) were determined by the MTT assay. The cells
were seeded at a density of 2
×
10
4
cells/well (HeLa, Caco-2, MCF-7) and 3
×
10
4
cells/well
(CCRF-CEM and CEM/ADR5000). The essential oil was serially diluted in media in the presence of
DMSO at concentrations between 10 mg/L and 5 g/L; 100
µ
L of each concentration was applied to the
wells of a 96-well plates. Cells were incubated with the essential oil for 24 h (HeLa, Caco-2, MCF-7)
and 48 h (CCRF-CEM and CEM/ADR5000) before the medium was removed and replaced with fresh
medium containing 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).
The formazan crystals produced were dissolved in DMSO 4 h later; the absorbance was measured at
570 nm with a Biochrom Asys UVM 340 Microplate Reader (Cambridge, UK).
2.5. Hemolytic Activity
The hemolytic activity was investigated by incubation of serially diluted fennel essential oil
in phosphate-buffered saline with red blood cells (human O+). The hemolytic activity assay was
performed as described earlier [27].
2.6. Hierarchical Cluster Analysis
A total of 68 chemical compositions of F. vulgare essential oils, including the sample from this
study in addition to 66 compositions obtained from the published literature [
5
–
7
,
9
,
23
,
28
–
45
] were
used to carry out the cluster analysis using the XLSTAT software, version 2015.4.01. The essential
oil compositions were treated as operational taxonomic units (OTUs) and the percentages of 34 of
the most abundant essential oil components (trans-anethole, limonene, estragole, fenchone,
α
-pinene,
α
-phellandrene, p-anisaldehyde,
β
-phellandrene,
β
-pinene, exo-fenchyl acetate, p-cymene, myrcene,
(E)-
β
-ocimene, camphor, 10-nonacosanone, piperitenone oxide, sabinene, neophytadiene, cis-anethole,
trans-dihydrocarvone,
γ
-terpinene, carvone, phytol, 1,8-cineole, iso-isopulegol, trans-
β
-terpineol,
endo-fenchyl acetate, camphene, carvacrol, apiole, o-cymene,
δ
-3-carene, linalool, and thymol)
were used to establish the chemical relationships of the F. vulgare essential oil samples using the
agglomerative hierarchical cluster (AHC) method. Pearson correlation was selected as a measure of
similarity, and the unweighted pair-group method with arithmetic average (UPGMA) was used for
definition of the clusters.
2.7. Microscopic Observation
The images of the treated or untreated CCRF cells were obtained and photographed using a by
fluorescence microscopy (BZ-9000, Keyence, Osaka, Japan) in order to investigate morphological changes.
2.8. Data Analysis
The experiments were repeated three times. IC
50
values were calculated using a four parameter
logistic curve (Sigma Plot 11.0 (SYSTAT Software, San Jose, CA, USA)). The data are represented as
means
±
standard deviations. The results of statistical test were determined by using Sigma Plot 11.0
software and also by using the statistical function t-test in Microsoft Excel. A pvalue below 0.05 was
considered to represent statistical significance.
3. Results and Discussion
3.1. Chemical Composition
The essential oil of F. vulgare was analyzed by gas-liquid chromatography—mass spectrometry
(GLC-MS). Thirty components were identified representing 97.7% of total oil composition (Table 1).
Oxygenated terpenoids were the dominant compounds of the essential oil of F. vulgare.

Foods 2017,6, 73 4 of 11
The major components were trans-anethole (
1
) (36.8%), p-anisaldehyde (
2
) (7.7%),
α
-ethyl-p-methoxybenzyl alcohol (
3
) (9.1%), carvone (4.9%), 1-phenylpenta-2,4-diyne (4.7%)
and fenchyl butanoate (4.2%). The main three compounds (trans-anethole, p-anisaldehyde,
α-ethyl-p-methoxybenzyl alcohol) are both ethers, having methoxy functional groups (Scheme 1).
Table 1.
Chemical composition of the essential oil of Foeniculum vulgare according to a GLC-MS analysis.
Compounds % * RT ** RI ***
trans-Anethole 36.8 36.022 1286
α-Ethyl-p-methoxybenzyl alcohol 9.10 54.059 1569
p-Anisaldehyde 7.73 33.825 1254
Carvone 4.87 33.119 1243
1-Phenylpenta-2,4-diyne 4.75 35.875 1283
Fenchyl butanoate 4.23 46.653 1448
Neomenthol 3.62 28.027 1170
(2E)-Dodecenal 3.44 47.807 1467
β-Ethyl-p-methoxybenzyl alcohol 3.27 54.498 1577
trans-Thujone 2.95 24.404 1118
Fenchone 2.75 22.408 1089
Carvacrol 2.15 36.802 1297
Linalyl acetate 1.88 33.503 1249
Unidentified 1.39 42.318 1380
(E)-Chrysanthenyl acetate 1.38 34.015 1256
Thymol 1.03 36.255 1289
Fenchyl isobutanoate 1.03 47.726 1465
(E)-β-Terpineol 1.00 28.642 1179
Linalool 0.75 23.139 1100
cis-Thujone 0.74 23.624 1107
(E)-Dihydrocarvone 0.64 30.422 1204
Unidentified 0.64 48.021 1470
Geranial 0.56 34.747 1267
Myrtenyl acetate 0.54 36.160 1288
exo-Fenchyl acetate 0.48 32.269 1231
Penta-1,3-diynylbenzene 0.46 40.505 1353
Dill ether 0.44 29.148 1186
Methylchavicol (=estragole) 0.42 29.973 1198
Unidentified 0.30 53.885 1567
Caryophyllene oxide 0.25 54.759 1581
Camphor 0.23 26.445 1147
iso-Menthone 0.10 27.050 1156
1-Hexadecene 0.08 48.270 1474
Terpene hydrocarbons: 5.21
Oxygenated terpenoids: 85.78
Others: 6.70
Total identified: 97.67
* Total peak area was set to 100%; ** Retention time; *** Kovats retention index in ZB-5 column.
Foods2017,6,73 4of11
fenchylbutanoate(4.2%).Themainthreecompounds(trans‐anethole,p‐anisaldehyde,
α‐ethyl‐p‐methoxybenzylalcohol)arebothethers,havingmethoxyfunctionalgroups(Scheme1).
Table1.ChemicalcompositionoftheessentialoilofFoeniculumvulgareaccordingtoaGLC‐MS
analysis.
Compounds%*RT**RI***
trans‐Anethole 36.836.0221286
α‐Ethyl‐p‐methoxybenzylalcohol9.1054.0591569
p‐Anisaldehyde 7.7333.8251254
Carvone4.8733.1191243
1‐Phenylpenta‐2,4‐diyne4.7535.8751283
Fenchylbutanoate4.2346.6531448
Neomenthol3.6228.0271170
(2E)‐Dodecenal3.4447.8071467
β‐Ethyl‐p‐methoxybenzylalcohol3.2754.4981577
trans‐Thujone 2.9524.4041118
Fenchone2.7522.4081089
Carvacrol2.1536.8021297
Linalylacetate1.8833.5031249
Unidentified1.3942.3181380
(E)‐Chrysanthenylacetate 1.3834.0151256
Thymol1.0336.2551289
Fenchylisobutanoate1.0347.7261465
(E)‐β‐Terpineol 1.0028.6421179
Linalool0.7523.1391100
cis‐Thujone 0.7423.6241107
(E)‐Dihydrocarvone0.6430.4221204
Unidentified0.6448.0211470
Geranial0.5634.7471267
Myrtenylacetate0.5436.1601288
exo‐Fenchylacetate 0.4832.2691231
Penta‐1,3‐diynylbenzene0.4640.5051353
Dillether0.4429.1481186
Methylchavicol(=estragole)0.4229.9731198
Unidentified0.3053.8851567
Caryophylleneoxide0.2554.7591581
Camphor0.2326.4451147
iso‐Menthone 0.1027.0501156
1‐Hexadecene0.0848.2701474
Terpenehydrocarbons:5.21
Oxygenatedterpenoids:85.78
Others:6.70
Totalidentified:97.67
*Totalpeakareawassetto100%;**Retentiontime;***KovatsretentionindexinZB‐5column.

Scheme 1. Structures of main components of the essential oil Foeniculum vulgare.

Foods 2017,6, 73 5 of 11
In accordance with previously published data,
1
is the main component [
1
,
46
], its content varying
from 5.0 to 85%. However, estragole [
47
,
48
], fenchyl acetate [
7
] and limonene [
6
] have also been
reported as main components of the fennel oil from other origins. Fennel essential oil is known as a
source for anethole [49].
F. vulgare is subdivided into three main chemotypes according to their relative compositions:
(1) estragole chemotype; (2) estragole/anethole chemotype and (3) anethole chemotype [
34
].
The essential oil of F. vulgare from Tajikistan thus belongs to the anethole chemotype, which is widely
distributed [47].
3.2. Cluster Analysis
In order to place the chemical composition of Tajik F. vulgare into context with previous
investigations, a hierarchical cluster analysis was carried out using the essential oil composition
from this study in conjunction with compositions from 66 samples previously reported in the
literature [
5
–
7
,
9
,
23
,
28
–
45
]. The cluster analysis (Figure 1) reveals the major chemotype of F. vulgare
to be an anethole-rich chemotype (CT1), which includes the sample from Tajikistan. There is
also an estragole-rich chemotype (CT2), represented by seven samples, and several chemotypes
represented by only one or two samples each: an estragole/
α
-phellandrene chemotype (CT3),
an anethole/estragole/
α
-pinene chemotype (CT4), an
α
-phellandrene chemotype (CT5), and a
limonene/
β
-pinene/myrcene chemotype (CT6). The anethole-rich cluster can be subdivided
into three chemotypes: an anethole chemotype (CT1a, including the sample from Tajikistan),
an anethole/limonene chemotype (CT1b), and an anethole/camphor chemotype (CT1c) represented
by a single sample from Romania (see Figure 1).
3.3. Antioxidant Activity
The investigation of antioxidant activity of essential oils as lipophilic secondary metabolites
became an interesting aspect of food and pharmaceutical research. Synthetic food additives are
increasingly replaced with plant-based natural ingredients, due to their safety, effectiveness and
consumer acceptance [
50
]. In general, fennel as an edible and medicinal plant represents an interest
through the neutralization of reactive oxygen species in order to prevent the damage of protein, lipid,
and DNA which are supposed to be the main reason for cell aging, oxidative stress-originated diseases
(cardiovascular and neurodegenerative diseases), and cancer.
The essential oil of fennel exhibits low antioxidant activity as compared to the positive control,
caffeic acid. The results of the DPPH, ABTS and FRAP analyses are represented Table 2.
Table 2.
Antioxidant activity of the essential oil Foeniculum vulgare as determined by the ABTS, DPPH,
and FRAP assays *.
Sample DPPH
IC50 (g L−1)
ABTS
IC50 (g L−1)
FRAP
µM Fe(II)/mg of Samples
Foeniculum vulgare 15.6 ±1.1 ** 10.9 ±0.4 ** 194 ±18 **
trans-Anethole 23.4 ±0.1 ** 35.6 ±0.1 ** 104 ±5.2 **
Caffeic acid 0.0017 ±0.0002 *** 0.0011 ±0.0002 *** 2380 ±46 ***
* The data are represented as means
±
standard deviations; ** significant at p< 0.0025; *** significant at p< 0.0001.

Foods 2017,6, 73 6 of 11
Foods2017,6,73 6of11

Figure1.Dendrogramobtainedfromtheagglomerativehierarchicalclusteranalysisof68Foeniculum
vulgareessentialoilcompositions.(CT1)anethole‐richchemotype,(CT1a)anetholechemotype,
(CT1b)anethole/limonenechemotype,(CT1c)anethole/camphorchemotype,(CT2)estragole
chemotype,(CT3)estragole/α‐phellandrenechemotype,(CT4)anethole/estragole/α‐pinene
chemotype,(CT5)α‐phellandrenechemotype,and(CT6)limonene/β‐pinene/myrcenechemotype.

Morocco‐A[39]
Morocco‐B[6]
Iran‐K[36]
Alberta‐E[29]
Brazil‐D[23]
Iran‐F[36]
Brazil‐B[23]
Brazil‐C[23]
Iran‐B[36]
Iran‐D[36]
Iran‐J[36]
Iran‐H[36]
Iran‐I[36]
Italy‐D[37]
Brazil‐A[30]
Italy‐B[37]
France‐D[34]
Italy‐E[37]
Iran‐L[36]
Iran‐A[36]
Iran‐C[36]
Iran‐E[36]
Iran‐G[36]
Romania[41]
Czech‐E[33]
Czech‐F[33]
France‐B[34]
Czech‐A[33]
Czech‐B[33]
Italy‐C[37]
Italy‐F[37]
Egypt‐B[7]
China‐B[32]
Russia‐B[42]
Ukraine‐E[45]
Russia‐A[42]
Ukraine‐A[45]
Ukraine‐D[45]
Ukraine‐B[45]
Ukraine‐C[45]
India[35]
Italy‐G[37]
China‐A[31]
Algeria‐C[28]
Italy‐A[9]
Korea[38]
Algeria‐A[28]
Egypt‐A[5]
Egypt‐E[5]
France‐G[34]
Egypt‐C[7]
France‐C[34]
Tajikistan(this)
Czech‐D[33]
Czech‐H[33]
Czech‐C[33]
Czech‐G[33]
France‐A[34]
France‐E[34]
Egypt‐D[7]
Algeria‐B[28]
Portugal‐A[40]
Turkey‐A[43]
Turkey‐B[43]
Turkey‐C[43]
France‐F[34]
Portugal‐B[40]
0.040.140.240.340.440.540.640.740.840.94
Similarity
CT6
CT5
CT4
CT3
CT2
CT1c
CT1b
CT1
CT1a
Figure 1.
Dendrogram obtained from the agglomerative hierarchical cluster analysis of 68
Foeniculum vulgare essential oil compositions. (CT1) anethole-rich chemotype, (CT1a) anethole
chemotype, (CT1b) anethole/limonene chemotype, (CT1c) anethole/camphor chemotype, (CT2)
estragole chemotype, (CT3) estragole/
α
-phellandrene chemotype, (CT4) anethole/estragole/
α
-pinene
chemotype, (CT5) α-phellandrene chemotype, and (CT6) limonene/β-pinene/myrcene chemotype.
The concentration of 50% inhibition (IC
50
) was the parameter used to compare the DPPH and
ABTS radical scavenging activity. A lower IC
50
(for DPPH and ABTS) and higher FRAP values

Foods 2017,6, 73 7 of 11
indicate higher antioxidant activity. IC
50
values for the antioxidant activity were 15.6 mg mL
−1
(DPPH) and 10.9 mg mL
−1
(ABTS). The IC
50
values of the known antioxidant substance—caffeic
acid—were
0.0017 mg mL−1
for DPPH and 0.0011 mg mL
−1
for ABTS, respectively. Ferric reducing
antioxidant power (FRAP) were equivalent to 193.5
µ
M Fe(II)/mg for oil and 2380
µ
M Fe(II)/mg
for caffeic acid. In agreement with our results, it was reported that the an IC
50
value of DPPH
radical scavenging activity of Foeniculum vulgare essential oil was 15.3 mg mL
−1
[
7
]. According to
the authors [
7
], fennel essential oil reacts with free radicals as a primary antioxidant and, therefore, it
may limit free-radical damage occurring in the human body. In our previous paper, we reported the
antioxidant activity of pure essential oil components, including the main component of the essential oil
of fennel (trans-anethole). It shows weak antioxidant activity. We assume that the phenolic substances
(carvacrol (2.1%) and thymol (1.0%)) are responsible for the observed antioxidant activity. These data
are in agreement with previously reported data [
25
]. However, it is known that the bioactivity of plant
extracts is due to the entire composition of the extract [51,52].
3.4. Cytotoxicity
The cytotoxicity of the oil was tested against HeLa, Caco-2, MCF-7, CCRF-CEM and
CEM/ADR5000 cancer cell lines (Table 3). IC
50
values were 207 mg L
−1
for HeLa, 75 mg L
−1
for
Caco-2, 59 mg L
−1
for MCF-7, 32 mg L
−1
for CCRF-CEM, and 165 mg L
−1
for CEM/ADR5000 cell
lines. As compared to the positive control doxorubicin, the essential oil exhibits low cytotoxicity.
Doxorubicin, an anthracycline antitumor antibiotic is a hydrophilic drug, and shows broad spectrum
anticancer activity [
53
]. The cytotoxicity of F. vulgare oil is most likely due to the lipophilic properties
of essential oil and alkylating properties of the major components trans-anethole and p-anisaldehyde.
Caco-2 and CEM/ADR5000 overexpress the ABC transporter p-gp which can actively pump out any
lipophilic compound that has entered the cell by free diffusion [
54
]). Thus, both cell lines are rather
insensitive towards lipophilic cytotoxic agents. In contrast, the parent cell line CCRF-CEM should be
sensitive. We also suspect that some components of the essential oil are may be substrates for p-gp,
as IC50 values were higher in CEM/ADR5000 cells.
Table 3. Cytotoxicity of the essential oil of Foeniculum vulgare.
Sample HeLa Caco-2 MCF-7 CCRF-CEM CEM/ADR 5000 RBC
IC50, mg L−1*
Foeniculum
vulgare 207 ±13 ** 75 ±4 ** 59 ±5 ** 32 ±1 ** 165 ±15 *** 1100 ±50 **
Doxorubicin 4.5 ±0.6 ** 1.1 ±0.1 ** 1.3 ±0.3 ** 0.25 ±0.2 ** 1.4 ±0.4 ** -
* The data are represented as means ±standard deviations; ** significant at p< 0.0006; *** significant at p< 0.001.
To better understand the mechanism of action of F. vulgare essential oil, we have investigated its
hemolytic effect. The result of hemolytic activity indicates that the oil is able to lyse the cell membrane
albeit with a rather high IC
50
value of 1100 mg L
−1
(Table 3). Moreover, in order to investigate the
effect of essential oil on the cell morphology, the images of untreated and treated CCRF cells with
essential oil were captured by fluorescence microscope. The images are illustrated in Figure 2.
Obtained images indicate that the essential oil can change the morphology of cells. Results of
both hemolysis and microscopic investigation indicate that essential oil also affects the integrity of cell
membranes. This is in agreement with many of the reported data [55].
In addition, trans-anethole, the main component of the essential oil, was examined for its
cytotoxicity in RC-37 cells. Its IC
50
value was 100 mg L
−1
[
56
]. The incubation of hepatocytes
with anethole caused a cell death accompanied by losses of cellular ATP and adenine nucleotide
pools [
57
]. Anethole shows apoptotic activity, as it can damage DNA [
58
]. Thus anethole could be
responsible for the overall cytotoxicity of the essential oil in our study.

Foods 2017,6, 73 8 of 11
Foods2017,6,73 8of11
(a)(b)
Figure2.Theimagesofuntreated(a)andtreated(b)CCRFcellswithessentialoilofFoeniculum
vulgare.
4.Conclusions
Theessentialoiloffennelcontainsseveralbioreactivesecondarymetabolites,suchas
aldehydes.Theoilapparentlyaffectsthestabilityofbiomembranesandinteractswithmolecular
targets,suchasproteinsandDNA,whichcausesalowcytotoxicity.
AuthorContributions:F.S.andM.W.conceivedanddesignedtheexperiments;F.S.,A.V.,P.S.,I.G.,S.I.,and
W.N.S.performedtheexperiments;F.S.,P.S.,W.N.S.,andM.W.analyzedthedata;P.S.,W.N.S.,andM.W.
contributedreagents/materials/analysistools;Allauthorscontributedtowritingandeditingthemanuscript.
ConflictsofInterest:Theauthorsdeclarenoconflictofinterest.
References
1. Mimica‐Dukic,N.;Kujundyic,S.;Sokovic,M.;Couladis,M.Essentialoilcompositionandantifungal
activityofFoeniculumvulgareMill.Obtainedbydifferentdistillationconditions.Phytother.Res.2003,17,
368–371.
2. Rather,M.A.;Dar,B.A.;Sofi,S.N.;Bhat,B.A.;Qurishi,M.A.Foeniculumvulgare:Acomprehensivereview
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6. Ouariachi,E.E.;Lahhit,N.;Bouyanzer,A.;Hammouti,B.;Paolini,J.;Majidi,L.;Desjobert,J.M.;Costa,J.
ChemicalcompositionandantioxidantactivityofessentialoilsandsolventextractsofFoeniculumvulgare
Mill.fromMorocco.J.Chem.Pharm.Res.2014,6,743–748.
7. Shahat,A.A.;Ibrahim,A.Y.;Hendawy,S.F.;Omer,E.A.;Hammouda,F.M.;Abdel‐Rahman,F.H.;Saleh,
M.A.Chemicalcomposition,antimicrobialandantioxidantactivitiesofessentialoilsfromorganically
cultivatedfennelcultivars.Molecules2011,1366–1377.
8. Ghanem,I.;Audeh,A.;Alnaser,A.;Tayoub,G.Chemicalconstituentsandinsecticidalactivityofthe
essentialoilfromfruitsofFoeniculumvulgareMilleronlarvaeofkhaprabeetle(Trogodermagranarium
Everts).HerbaPolonica2013,59,86–96.
9. Tognolini,M.;Ballabeni,V.;Bertoni,S.;Bruni,R.;Impicciatore,M.;Barocelli,E.Protectiveeffectof
Foeniculumvulgareessentialoilandanetholeinanexperimentalmodelofthrombosis.Pharmacol.Res.2007,
56254–260.
10. VillariniM.;Pagiotti,R.;Dominici,L.;Fatigoni,C.;Vannini,S.;Levorato,S.;Moretti,M.Investigationof
thecytotoxic,genotoxic,andapoptosis‐inducingeffectsofestragoleisolatedfromfennel(Foeniculum
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Figure 2.
The images of untreated (
a
) and treated (
b
) CCRF cells with essential oil of Foeniculum vulgare.
4. Conclusions
The essential oil of fennel contains several bioreactive secondary metabolites, such as aldehydes.
The oil apparently affects the stability of biomembranes and interacts with molecular targets, such as
proteins and DNA, which causes a low cytotoxicity.
Author Contributions:
F.S. and M.W. conceived and designed the experiments; F.S., A.V., P.S., I.G., S.I., and W.N.S.
performed the experiments; F.S., P.S., W.N.S., and M.W. analyzed the data; P.S., W.N.S., and M.W. contributed
reagents/materials/analysis tools; All authors contributed to writing and editing the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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