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INDCRO-8539; No. of Pages 7 ARTICLE IN PRESS

Industrial Crops and Products xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Industrial Crops and Products

journal homepage: www.elsevier.com/locate/indcrop

Isolation and identification of an antibacterial compound from

Diplotaxis harra (Forssk.) Boiss
Roudaina Benzekri a,∗ , Lamjed Bouslama a , Adele Papetti b , Mejdi Snoussi c ,
Imen Benslimene a , Majdi Hamami a , Ferid Limam a
a
Laboratory of Bioactive Substances, Center of Biotechnology of Borj Cedria, BP 901, Hammam Lif 2050, Tunisia
b
Nutraceutical & Food Chemical—Toxicological Analysis Laboratory, Department of Drug Sciences, University of Pavia, Viale Taramelli 12, 27100 Pavia, Italy
c
Laboratory of Wastewater Treatment, Water Researches and Technologies Center of Borj Cedria, BP 901, Hammam Lif 2050, Tunisia

a r t i c l e i n f o a b s t r a c t

Article history: The emergence of the antibiotic resistance is an ongoing problem in public health, and therefore the
Received 9 July 2015 search for new natural molecules represents an alternative to synthetic drugs. The aim of this study
Received in revised form 30 October 2015 was to test the antibacterial activity of ten Mediterranean plants (Diplotaxis harra, Ecballium elaterium,
Accepted 23 November 2015
Pergularia tomentosa, Myrtus communis, Solanum villosum, Solanum sodomaeum, Peganum harmala, Lepid-
Available online xxx
ium sativum, Pistacia lentiscus, and Calendula arvensis) against seven pathogenic bacteria (Staphylococcus
aureus, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Klebsiella sp., Salmonella enter-
Keywords:
itidis, and Bacillus cereus) using the disk diffusion method, in order to isolate and identify the active
Antibacterial activity
Diplotaxis harra
compound(s). Dichloromethane extract of D. harra flower showed the best activity against S. aureus
Food-borne pathogen and L. monocytogenes (MIQ = 30 ␮g/disk and 15 ␮g/disk, respectively). This extract was submitted to a
Sulforaphane bio-guided purification using Thin Layer Chromatography (TLC)-bioautography, and an antibacterial frac-
TLC-bioautography tion (MIQ = 2 ␮g/disk) was isolated. The active fraction was characterized by RP-HPLC-DAD-ESI-MSn and
GC–MS GC–MS. Sulforaphane, an isothiocyanate known for its anti-cancer, antioxidant, and anti-inflammatory
HPLC-DAD-ESI-MSn properties, was identified as antibacterial agent in D. harra for the first time. Due to its high antibacterial
activity, sulforaphane could be considered a good candidate for the selection of new natural antibacterial
molecules.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction compounds), natural products constitute the best reservoir of new

The overuse of antibiotics and the emergence of the antimicro-
bial resistance require the search for new alternative compounds
active against pathogenic microorganisms resistant to the avail-
able antibacterial drugs. Herbal medicine plants have traditionally
been used as remedies for human diseases. As they produce a large
variety of secondary metabolites (alkaloids, flavonoids, terpenoids,
steroids, saponins, glycosides, phenolic compounds, and sulphur
Abbreviations: ATCC, American Type Culture Collection; CIP, Collection of Pas-
teur’s Institute; GC–MS, gas chromatography mass spectrometry; HPLC-DAD-ESI-
MS, high-performance liquid chromatography-diode array detector-electrospray
ionization-mass spectrometry; LB, Luria–Bertani; MH, Mueller Hinton Agar; MIQ,
minimal inhibitory quantity; MTT, methyl thiazolyl tetrazolium chloride; MS,
mother solution; TLC, Thin-Layer Chromatography.
∗ Corresponding author. Fax: + 216 79 325 638.
E-mail address: benzekri.roudayna@gmail.com (R. Benzekri).
antibiotics. However, despite the idea that the use of traditional
medicine has no scientific argument and that only few among the
many plants in the World have been investigated, the search for
new compounds coming from medicinal plants remains a hope for
the discovery of new drugs in the treatment of infectious diseases.
In Mediterranean area many plants have been studied for their
biological activities. For example, the seeds of Diplotaxis harra
are rich in glucosinolates acting as plant defense against pests
and are responsible for the bitter and pungent flavor of many
foods such as mustard, radishes, cauliflowers, broccolis (D’Antuono
et al., 2007). The fruit of Ecballium elaterium contains cucurbitacin,
a poisonous substance irritating to skin; its juice is a purgative
used for the treatment of sinusitis (Attard and Sciluna-Spiteri,
2001). The roots of Pergularia tomentosa contain cardenolide gly-
cosides exhibiting cytotoxic activity due to their potent inhibition
of Na+/K + -ATPase (Piacente et al., 2009). The essential oil of Myr-
tus communis contains bio-active substances such as alpha-pinene,

http://dx.doi.org/10.1016/j.indcrop.2015.11.059
0926-6690/© 2015 Elsevier B.V. All rights reserved.

Please cite this article in press as: Benzekri, R., et al., Isolation and identification of an antibacterial compound from Diplotaxis harra
(Forssk.) Boiss. Ind. Crops Prod. (2015), http://dx.doi.org/10.1016/j.indcrop.2015.11.059
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2 R. Benzekri et al. / Industrial Crops and Products xxx (2015) xxx–xxx
cineole and myrtenol, known for their antiseptic and expecto-
rant properties (Bouzabata et al., 2015). Solanum villosum subsp.
villosum exhibit an anti-parasitic effect (against Fasciola hepat-
ica) (Hammami and Ayadi, 2008), and an anti-mosquito larvicidal
activity (against Aedes aegypti, the Dengue and Yellow fever vector)
(Chowdhury et al., 2008), whereas no specific action was men-
tioned for Solanum sodomaeum . The seeds of Peganum harmala
contain psychotropic alkaloids (harmine, harmol, and harmaline)
classified as narcotic substances, and acting as neurotransmitter
mediated by opioid receptors (Farouk et al., 2008). The seeds of Lep-
idium sativum have been shown to reduce asthma symptoms and
improve lung function in asthmatics (Archana and Anita, 2006).
Pistacia lentiscus is efficient against Helicobacter pylori through
resin (mastic gum) secreted by the tree which has the ability to
heal peptic ulcers by killing these bacteria (Marone et al., 2001).
For Calendula arvensis, one report demonstrated that petroleum
ether and chloroform extracts exhibited antibacterial effect against
Escherichia coli and Bacillus subtilis (Abu Hena Mostofa Jamal et al.,
2014).
The objective of the present work was to screen for new natural
antibacterial agents. Ten plants widely spread in the Mediter-
ranean area [D. harra (Forssk.) Boiss. (Brassicaceae), Ecballium
elaterium (L.) A. Rich. (Cucurbitaceae), P. tomentosa L. (Apocy-
naceae), M. communis L. (Myrtaceae), S. villosum Mill. subsp. villosum
(Solanaceae), Solanum sodomeaum Dunal (Solanaceae), Peganum
harmala L. (Nitrariaceae), L. sativum L. (Brassicaceae), Pistacia
lentiscus L. (Anacardiaceae) and C. arvensis L. (Asteraceae)] were
tested for their antibacterial activity against seven bacteria strains
(Staphylococcus aureus, E. coli, Listeria monocytogenes, Pseudomonas
aeruginosa, Klebsiella sp.,Salmonella enteritidis, and Bacillus cereus).
Different parts of each plant were extracted with organic solvents
of increasing polarity (hexane, dichloromethane, ethyl acetate and
methanol) and all extracts (one hundred thirty two) were tested.
The natural antibacterial compound(s) was (were) isolated from
the active extract(s) through bio-guided assays and identified by
chromatographic methods coupled with mass spectrometry.
2. Materials and methods
2.1. Chemicals
All the solvents used for extraction procedures (hexane,
dichloromethane, ethyl acetate, acetone and methanol), and
sodium chloride were obtained from Lab-Scan Analytic Science,
Poland. Formic acid, water, and methanol for MS analyses,
and methyl thiazolyl tetrazolium chloride were purchased from
Sigma–Aldrich, Milan, Italy.
2.2. Plant material
Different aerial parts and roots of ten Mediterranean plants
were collected between March and July 2010 from middle and
south regions of Tunisia. The selected parts for each plant are listed
below. The identification of plant material was confirmed by Prof.
Abderrazak Smaoui (Laboratory of Extremophile Plants—Center of
Biotechnology of Borj Cedria): D. harra (leaf, flower, stem and root),
Ecballium elaterium (fruit, leaf, flower and stem), P. tomentosa (fruit,
leaf, flower and stem), C. arvensis (leaf, flower, stem and root), M.
communis (fruit, leaf and stem), S. villosum Mill. subsp. Villosum (leaf,
stem, mature fruit and immature fruit), S. sodomaeum (fruit, leaf and
stem), Peganum harmala (leaf, stem, seed and flower), L. sativum
(seeds) and Pistacia lentiscus (leaf, stem and fruit).
Please cite this article in press as: Benzekri, R., et al., Isolation and identification of an antibacterial compound from Diplotaxis harra
(Forssk.) Boiss. Ind. Crops Prod. (2015), http://dx.doi.org/10.1016/j.indcrop.2015.11.059
2.3. Plant extracts
The different parts of each plant were freeze dried using Alpha
1-2 LD Freeze Dryer apparatus (Christ, UK), and ground in a blender
Retsch GM200 (Grintomix) until obtaining a fine powder. Five grams
of each dried plant were extracted by maceration using 50 ml of
solvents of increasing polarity, i.e. hexane, dichloromethane, ethyl
acetate, and methanol, under agitation for 24 h at room tempera-
ture. The extracts obtained with each solvent were filtered and then
evaporated to dryness under vacuum in a rotary-evaporation unit
(Vacuum controller V-850, Buchi, Switzerland). Each dried extract
was dissolved in the respective extraction solvent, obtaining a final
concentration of 50 mg/ml (mother solution, MS), and stored at
−20 ◦ C until the assays. The plant extraction procedure was car-
ried out in triplicate. Overall, 132 plant extracts were obtained for
each procedure.
2.4. Bacterial strains
Seven bacterial strains were used through-out this study: S.
aureus ATCC 25,923, E. coli ATCC 25,922, L. monocytogenes ATCC
19,195, Pseudomonas aeruginosa ATCC 27,853, Klebsiella sp. CIP
104,727, Salmonella enteritidis ATCC DMB560, B. cereus ATCC
14,759.
2.5. Antibacterial assay
The evaluation of the antibacterial activity was performed by
the disk diffusion method (Zaidan et al., 2005). Briefly, the bacte-
rial strains were cultured in Luria-Bertani agar (LB, Sigma, Germany)
and incubated at 37 ◦ C overnight. A colony of each bacteria strain
was suspended in 0.9% sodium chloride. The turbidity was adjusted
to 0.5 McFarland to yield approximately 108 CFU/ml. Mueller Hin-
ton Agar (MHA) (Becton Dicknson M. D USA) was prepared according
to the manufacturer’s instruction (35 g of media was mixed with
one liter of distilled water and autoclaved at 121 ◦ C for 15 min) and
dispensed into 90 mm sterile agar plates (Oxoid, UK). Sterile Petri
plates were then inoculated with the test bacterial suspensions
by surface spreading using a cotton swab. Paper disks (Whatman,
diameter = 5 mm; porosity = 6 ␮m) were impregnated with 20 ␮l of
extract mother solution (1000 ␮g/disk) for the screening test, and
with 20 ␮l of solutions at concentration ranging from 25 mg/ml to
0.1 mg/ml for extracts exhibiting inhibition zone at 1000 ␮g/disk.
The impregnated disks were allowed to dry under flow hood (for
3 h) then applied on the agar surface. The inoculated plates were
incubated at 37 ◦ C overnight. Paper disks impregnated only with
extraction solvents were used as negative control. Erythromycin
(15 ␮g/disk), tetracycline (30 ␮g/disk), and oxacillin (5 ␮g/disk)
were used as positive control. Antibacterial activity was deter-
mined as inhibition zone and measured as the minimal inhibitory
quantity (MIQ)/disk. The MIQ is defined as the lowest quantity
(␮g/disk) of extract showing no growth. The experiments were
carried out in duplicate.
2.6. Detection and preparation of the active fraction by
TLC–bioautography
The components of active extract were separated by Thin-Layer
Chromatography (TLC) bioautography. Ten ␮l of mother solution
extract was deposed on pre-coated silica gel 60 F254 aluminum
plate (20 × 20 cm; layer thickness, 0.20 mm; Merck, Germany)
and migrated using hexane/dichloromethane/acetone in the ratio
1:1:1.5-v/v/v as mobile phase. After separation of the different con-
stituents, the plate was allowed to dry at room temperature, and
observed under UV 254 nm. Then, the plate was placed into sterile
Petri plate inoculated with the most sensitive bacterial suspension
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Fig. 1. Total ion chromatogram (TIC) of sulphoraphane (box A), MS spectrum (box B), MS/MS spectrum (box C), and MS3 spectrum (box D).

(108 CFU/ml) against the active extract. Once dried, the plate was
incubated overnight at 37 ◦ C and finally 5 ml of methyl thiazolyl
tetrazolium chloride (MTT) solution (5 mg/ml) were added. The
inhibition zones corresponding to the area of the active fractions
appeared as colored spots on a dark blue background.
For preparative purpose, 500 mg of dried active extract were
fractionated on a silica gel 60 F254 glass plate (20 × 20 cm; Glass
Backed TLC Extra Hard Layer 60 Å; Silicycle, Canada), as previously
described. The bands corresponding to the bacterial growth inhi-
bition zone were scraped off and then dissolved in the migration
solvent (hexane/dichloromethane/acetone) with a modification
of the proportion (1:3:1-v/v/v) under agitation for 20 min. The
dissolved product was filtered and concentrated using a rotary-
evaporation unit. The dried active fraction (35 mg) was dissolved
in 1 ml of 75% ethanol and used for identification of the active com-
pound(s) and for the antibacterial assays after elimination of the
solvent by nitrogen.
ted column oven set at 25.0 ± 0.5 ◦ C, and a diode-array detector
(DAD) set at 254 nm, and a vacuum degasser connected to an
LCQ Advantage Max ion trap mass spectrometer (all from Thermo
Fisher Scientific, Waltham, MA) through an electrospray ionization
(ESI) source. The ion trap operated in data-dependent, full scan
(100–1000 m/z), zoom scan, and MSn mode to obtain fragment ion
m/z with collision energy of 35% and an isolation width of 3 m/z . The
positive parameters of the ion mode ESI source had previously been
optimized at a ionization voltage of 4.5 kV, a capillary temperature
◦
of 250 C, a sheath gas flow rate of 55 arbitrary units (AU), and an
auxiliary gas flow rate of 5 AU. The Thermo Fisher Scientific Excal-
ibur 2.0 software was used for data acquisition and processing. No
marked variations attributable to the nature of the detected frag-
ments or their relative intensities were observed in 3 independent
assays performed to analyze the sample.
2.8. Identification of the active compound(s) by GC–MS

2.7. Identification of the active compound(s) by
HPLC-DAD-ESI-MSn
The separation of the active fraction was carried out using a
Gemini C18 (150.0 × 2.1 mm, i.d., 5 ␮m) with a Hypersil Gold C18
guard column (10.0 × 2.1 mm i.d. 5 ␮m, all from Phenomenex, Tor-
rance, CA). The mobile phase consisted of 0.1% formic acid in
water and methanol 85/15 v/v, at a flow rate of 0.3 ml/min. LC–MS
analyses were performed using a Thermo Finnigan Surveyor Plus
HPLC apparatus equipped with a quaternary pump, a Surveyor Plus
autosampler set at 5 ␮l injection volume, a degasser, a thermostat-
The identification of the isolated active compound was con-
firmed using a gas chromatography HP 5890 (II) interfaced with a
HP 5972 mass spectrometer (5975C inert XL MSD, Agilent Technolo-
gies, Palo Alto, CA, USA) with electron impact ionization (70 eV). The
used capillary column was HP-5 MS (30 m, 0.25 mm, coated with
95% dimethylpolysiloxane, 5% phenyl methyl silicone, 0.25 mm film
thickness; Hewlett-Packard, CA, USA). The column temperature was
programmed to rise from 50 to 240 ◦ C with a rate of 5 ◦ C/min.
The carrier gas was helium with a flow rate of 1.2 ml/min and
the split ratio was 60:1. Scan time and mass range were 1s and
40–300 m/z, respectively. The characterization of the active com-

Please cite this article in press as: Benzekri, R., et al., Isolation and identification of an antibacterial compound from Diplotaxis harra
(Forssk.) Boiss. Ind. Crops Prod. (2015), http://dx.doi.org/10.1016/j.indcrop.2015.11.059
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The used method is the disk diffusion assay. The results correspond to the inhibition diameter (mm) and represent the mean of duplicate values. The deposed quantity is 1 mg/disk for all the extracts mentioned above. Note: (−)
B. cereus ATCC
14759

12
14

22
30
–
–
–
–
–
–
–
–
–
–
–
–
–

–
Fig. 2. Chemical structure of sulforaphane (1-isothiocyanato-4-methylsulfinyl-
butane).
S. enteritidis ATCC

pound was made by comparing retention time, Kovats retention

indice (KI) calculated relative to n-alkanes (Alkane standard solu-
DMB560

tion C8-C20 4070 Fluka) on apolar column HP-5MS. and recorded

mass spectra with those stored in the Wiley/NBS mass spectral
25
16

14
27
–
–
–
–
–
–
–
–
–
–
–
–
–

–
library of the GC–MS data system of the apparatus.

3. Results
E. coli ATCC

The antibacterial activity of the extracts was evaluated by the

determination of the minimal inhibitory quantity (MIQ)/disk. All
25,922

the extracts obtained from C. arvensis, P. tomentosa, Peganum har-

22
12

15
28
–
–
–
–
–
–
–
–
–
–
–
–
–

mala and Solanum Mill. subsp. villosum did not show any activity
against the tested bacteria strains.
Thirteen extracts exhibited moderate activity (1000 ␮g/disk
K. sp. CIP 104727

<MIQ< 125 ␮g/disk) against S. aureus and L. monocytogenes : ethyl

acetate extract of L. sativum seed, hexane extract of Ecballium ela-
terium fruit, methanol and ethyl acetate extracts of M. communis
leaf and fruit, methanol extract of M. communis stem, methanol
11
20
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–

and ethyl acetate extracts of Pistacia lentiscus leaf and stem, ethyl
acetate extract of Pistacia lentiscus fruit, and ethyl acetate extract of
S. sodomaeum stem. Only dichloromethane extracts of D. harra root
and flower revealed significant activity with MIQ values lower than
P. aeruginosa

100 ␮g/disk (MIQ = 60 ␮g/disk for root, and 30 ␮g/disk for flower)
ATCC 27853

against S. aureus, and MIQ = 30 ␮g/disk and 15 ␮g/disk against L.

monocytogenes for root and for flower, respectively. These two
12
23

extracts also showed a moderate activity against B. cereus, E. coli,

–
–
–
–
–
–
–
–
–
–
–
–
–
–
–

and S. enteritidis, but no activity against P. aeruginosa and Klebsiella

sp. (Table 1).
As dichloromethane extract of D. harra flower was the most
L. monocytogenes

active, the attention was focused on this plant part for the isola-
ATCC 19195

no antibacterial activity, H: hexane, DM: dichloromethane, AE: Ethyl acetate, MeOH: methanol.

tion and the identification of the bioactive compound(s). On TLC

plate, a band with retardation factor (Rf ) = 0.33 revealed an inhi-
bition zone when incubated with S. aureus and L. monocytogenes
>50
>50
18
8
12
13
12
11

25
20
20
18
18
10
39
36
33
–

strains using MTT solution. After scraping, this fraction was evalu-
ated for its activity against these two bacteria strains, and the MIQ
values were 2 ␮g/disk for both.
The active fraction was characterized by HPLC-DAD-ESI-MSn .
S. aureus ATCC

The HPLC profile showed only one peak and the MS spectrum reg-
Antibacterial activity of the extracts showing a growth inhibition zone.

istered in positive ionization mode revealed the pseudomolecular

25,923

ion at m/z 178 [M + H]+ ; its fragmentation led to some specific prod-
>50
>50
25

11

12

12

14

38
35
34
10

10

uct ions at m/z 114 corresponding to the base peak and at m/z
–

9
–
8

119. Further m/z 114 fragmentation in MS3 experiment led to m/z

72 as specific product ion (Fig. 1). This fragmentation pattern led
Erythromycin (30 ␮g)
Extract

MeOH

MeOH
MeOH

MeOH

MeOH

to the putative identification of sulforaphane (1-isothiocyanato-4-

Tetracycline (15 ␮g)
DM
DM

methylsulfinyl-butane, SFN) (Fig. 2) by comparing the data here

AE

AE
AE

AE

AE

AE
AE
H

Oxacillin (5 ␮g)

obtained with those reported in literature (Al Janobi et al., 2006;

Wang et al., 2011). This structure was confirmed by GC–MS analy-
Flower

sis and the GC profile showed only sulforaphane peak at 4.752 min
Organ

Leave

Leave
Stem

Stem

Stem
Fruit

Fruit

Fruit
Seed
Root

(Fig. 3), the KI = 1789 and the mass fragments obtained were m/z
(%): 160 (76), 114(10), 85(7), 72(100), 64(15), 55(34), in agree-
ment with previously published data for this compound (Azizi et al.,
2011).
Solanum sodomaeum
Ecballium elaterium

The estimation of the antibacterial effect of sulforaphane was

Lepidium sativum

evaluated by comparing its inhibition diameter with that of stan-

Antibiotics

dard antibiotics (erythromycin, tetracycline and oxacillin), testing

Diplotaxis

Pistacia

all molecules at 10 ␮g/disk (Table 2). Even showing a lower inhi-

Myrtus

lentis-
Table 1

harra
Plant

com-
mu-

bition diameter, the activity of sulforaphane could be considered

cus
nis

interesting if it is taken into account that this compound was

Please cite this article in press as: Benzekri, R., et al., Isolation and identification of an antibacterial compound from Diplotaxis harra
(Forssk.) Boiss. Ind. Crops Prod. (2015), http://dx.doi.org/10.1016/j.indcrop.2015.11.059
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Fig. 3. GC–MS profile of sulforaphane.

extracted from natural product (vs synthetic product for the con-
sidered antibiotics).
4. Discussion
D. harra is a desert plant belonging to the Brassicaceae family.
A phytochemical analysis of the aerial parts showed the presence
of flavonoids, glucosinolates and sterols (Atta et al., 2011). The
flavonoid glycosides of D. harra was reported to have a promis-
ing cytotoxicity with the ability to inhibit tumor cell growth (D.
Mohammed et al., 2011). Concerning its antibacterial activity, Atta
et al. (2011) found that methanolic extracts obtained from the aerial
part were active against E. coli, Pseudomonas aeruginosa, and Kleb-
siella pneumonia, differently from the here exposed results. On the
other hand, Akbar and Al-Yahya (2011) found similar data when the
chloroform extract of the aerial part was tested against S. aureus.
In our study, dichloromethane extracts of D. harra flower and
root exhibited the best antibacterial effect and the compound
responsible for this activity was identified as sulforaphane by
HPLC-DAD-ESI-MSn and confirmed by GC–MS. This product was

Table 2
Antibacterial activity of SFN and three usual antibiotics against the most sensitive tested bacteria strains.

Erythromycin Tetracycline Oxacillin Sulforaphane

Staphylococcus aureus ATCC 25923 32 38 38 15

Listeria monocytogenes ATCC 19195 33 40 39 16

The used method is the disc diffusion assay. The results correspond to the inhibition diameter (mm) and represent the mean of duplicate values. The deposed quantity is
10 ␮g/disk for all the compounds mentioned above.

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previously reported and characterized in some Brassicaceae plants
(Hong et al., 2005; Liang et al., 2005; Wang et al., 2011) as bioac-
tive agent, but never before isolated from D. harra as antibacterial
compound.
Sulforaphane is an isothiocyanate naturally present in widely
consumed vegetables as bioactive metabolite of glucoraphanin, a
characteristic glucosinolate of vegetables belonging to the Brassi-
caceae family (Mithen 2001). Generally, the glucosinolates have
to be metabolized by myrosinase enzyme to their corresponding
isothiocyanate for showing activity (Rouzaud et al., 2003). SFN is
known for possessing some biological activities, such as promising
chemoprotective properties since it showed the ability to induce
cell cycle arrest and apoptosis in HT29 human colon cancer cells and
reduce the incident of several forms of tumors (Gamet-Payrastre
et al., 2000). Moreover, SFN affords a cardiovascular protection via
its antioxidant and anti-inflammatory properties, inducing a reduc-
tion of the oxidative stress, an improvement of the lipid rate, and
a regulation of blood pressure (Meijer et al., 2015). SFN was iso-
lated and identified as antimicrobial agent for the first time in hoary
cress, a plant belonging to the Brassicaceae family (Prochazka and
Komersovaı́, 1959). Devi and Thangam (2012) found that SFN pos-
sess interesting activity against E. coli, V. cholera, B. cereus, S. aureus
and P. aeruginosa; in particular, for S. aureus a 16.5 mm inhibition
zone was generated by 100 ␮g/disk, a concentration ten-fold higher
than that generated a 15 mm inhibition zone in our work. The activ-
ity of SFN was also demonstrated against H. pylori, a potential causal
agent of stomach cancer (Fahey et al., 2002). Despite the high num-
ber of studies on SFN antibacterial activity, only Wu et al. (2012)
studied its action mechanism (on E. coli). These authors demon-
strated that this molecule can affect the energy metabolism and
the cell membrane permeability of the bacteria without destroying
directly the membrane integrity as well it can inhibit the nucleic
acid and protein synthesis.
5. Conclusion
In the present investigation, ten Mediterranean plants were
selected and evaluated for their antibacterial effect against seven
pathogenic bacteria. The most interesting and active extracts were
dichloromethane extract of D. harra flower and roots, showing
very high activity against S. aureus and L. monocytogenes . Bioas-
say guided purification was performed for the most active extract
(flower) using TLC-bioautography followed by preparative TLC.
The active fraction was characterized by HPLC-DAD-ESI-MSn and
GC–MS, and sulforaphane was identified as the bioactive com-
pound. We reported for the first time the presence of SFN in D. harra
. We also confirmed that this molecule, an isothiocyanate belonging
to the Brassicacea family, exhibited significant antibacterial activity,
especially against S. aureus and L. monocytogenes .
Knowing that the overuse of antibiotics is one of the most
important reason of the antimicrobial resistance, the search for
new molecules becomes a priority for the treatment of micro-
bial infections. Thus, sulforaphane could be a promising alternative
compound to common antibiotics. Further investigations regarding
the study of its cytotoxicity and action mechanism are needed to
pick out this molecule as alternative or complement to habitual
drugs.
Conflict of interest
The authors declare that there is no conflict of interests regard-
ing the publication of this article.
Please cite this article in press as: Benzekri, R., et al., Isolation and identification of an antibacterial compound from Diplotaxis harra
(Forssk.) Boiss. Ind. Crops Prod. (2015), http://dx.doi.org/10.1016/j.indcrop.2015.11.059
Acknowledgement
The authors thank Pr. Abderrazak Smaoui (Laboratory of
Extremophile Plants—Center of Biotechnology of Borj Cedria) for
his help in some plant materials collection and their identification.
References
Abu Hena Mostofa Jamal, M., Moniruzzaman, Md., Zulfiqer shaheen, Md.,
Kamruzzaman, M., Rashid, M., Loby Mahmud, Md., Alam Khan, N., Pervin, H.,
Haque Sharif, H., 2014. Analysis of antimicrobial activity of Calendula arevensis
against bacterial pathogens. Int. J. Phytother. Res. 4 (2).
Akbar, S., Al-Yahya, M.A., 2011. Screening of Saudi plants for phytoconstituents,
pharmacological and antimicrobial properties. Aust. J. Med. Herb. 23 (2), 76–87.
Al Janobi, A.A., Mithen, R.F., Gaspera, A.V., Shawc, P.N., Middleton, R.J., Ortori, C.A.,
Barrett, D.A., 2006. Quantitative measurement of Sulforaphane, iberin and
their mercapturic acid pathway metabolites in human plasma and urine using
liquid chromatography-tandem electrospray ionization mass spectrometry. J.
Chromatogr. B 844, 223–234.
Archana, N.P., Anita, A.M., 2006. A study on clinical efficacy of Lepidium sativum
seeds in treatment of bronchial asthma. Iran J. Pharmacol.Ther. 5, 55–59.
Atta, E.M., Hashem, A.I., Ahmed, A.M., Elqosy, S.M., Jaspars, M., El-Sharkaw, E.R.,
2011. Phytochemical studies on Diplotaxis harra growing in Sinai. Eur. J. Chem.
2 (4), 535–538.
Attard, E.G., Sciluna-Spiteri, A., 2001. Ecballium elaterium: an in vitro source of
cucurbitacins. Fitoter 72, 46–53.
Azizi, S.N., Amiri-Beshelib, B., Sharifi-Mehr, S., 2011. The isolation and
determination of sulforaphane from broccoli tissues by reverse phase-high
performance liquid chromatography. J. Chin. Chem. Soc. 58, 906–910.
Bouzabata, A., Cabral, C., Gonçalves, M.J., Cruz, M.T., Bighelli, A., Cavaleiro, C.,
Casanova, J., Tomi, F., Salgueiro, L., 2015. Myrtus communis L: as source of a
bioactive and safe essential oil. Food Chem. Toxicol 75, 166–172.
Chowdhury, N., Ghosh, A., Chandra, G., 2008. Mosquito larvicidal activities of
Solanum villosum berry extract against the dengue vector Stegomyia aegypti,
BMC Complement. Altern. Med. 8, 10.
D’Antuono, L.F., Elementi, S., Neri, R., 2007. Glucosinolates in diplotaxis and eruca
leaves: diversity, taxonomic relations and applied aspects. Phytochemistry 69,
187–199.
Devi, J.R., Thangam, E.B., 2012. Studies on antioxidant and antimicrobial activities
of purified sulforaphane from Brassica oleraceae var rubra. J. Pharm. Res. 5 (7),
3582–3584.
Fahey, J.W., Haristoy, X., Dolan, P.M., Kensler, T.W., Scholtus, I., Stephenson, K.K.,
Talalay, P., Lozniewski, A., 2002. Sulforaphane inhibits extracellular,
intracellular, and antibiotic-resistant strains of Helicobacter pylori and
prevents benzo[a]pyreneinduced stomach tumor. Proc. Natl. Acad. Sci. USA 99,
7610–7615.
Farouk, L., Laroubi, A., Aboufatima, R., Benharref, A., Chait, A., 2008. Evaluation of
the analgesic effect of alkaloid extract of Peganum harmala L.: possible
mechanisms involved. J. Ethnopharmacol. 115 (3), 449–454.
Gamet-Payrastre, L., Li, P., Lumeau, S., Cassar, G., Dupont, M.A., Chevolleau, S., Gasc,
N., Tulliez, J., Terce, F., 2000. Sulforaphane a naturally occurring isothiocyanate,
induces cell cycle arrest and apoptosis in HT29 human colon cancer cells.
Cancer Res. 60, 1426–1433.
Hammami, H., Ayadi, A., 2008. Molluscicidal and antiparasitic activity of Solanum
nigrum villosum against Galba truncatula infected or uninfected with Fasciola
hepatica. Helminthology 82 (3), 235–239.
Hong, F., Freeman, M.L., Liebler, D.C., 2005. Identification of sensor cysteines in
Human Keap1 modified by the cancer chemopreventive agent sulforaphane.
Chem. Res. Toxicol. 18, 1917–1926.
Liang, H., Yuan, Q., Xiao, Q., 2005. Purification of sulforaphane from Brassica
oleracea seed meal using low-pressure column chromatography. J.
Chromatogr. B. 828, 91–96.
Marone, P., Bono, L., Leone, E., Bona, S., Carretto, E., Perversi, L., 2001. Bactericidal
activity of Pistacia lentiscus mastic gum against Helicobacter pylori. J. Chemoth.
13 (6), 611–614.
Meijer, K., Vonk, R.J., Priebe, M.G., Roelofsen, H., 2015. Cell-based screening assay
for anti-inflammatory activity of bioactive compounds. Food Chem. 166,
158–164.
Mithen, R., 2001. Glucosinolates–biochemistry genetics, and biological activity.
Plant Growth Regul. 34, 91–103.
Mohammed, D., ElSharkawy, E.R., Matloub, A., 2011. Cytotoxic flavonoids from
Diplotaxis harra (Forssk.) Boiss. growing in Sinai. J. Med. Plants Res. 5 (20),
5099–5103.
Piacente, S., Milena Masullo, M., De Ne’ve, N., Dewelle, J., Arafa Hamed, A., Kiss, R.,
Mijatovic, T., 2009. Cardenolides from Pergularia tomentosa display cytotoxic
activity resulting from their potent inhibition of Na+/K + -ATPase. J. Nat. Prod
72, 1087–1091.
Prochazka, Z., Komersovaı́, I., 1959. Isolation of sulforaphane from Hoary cress
(Lepidium draba L.). Cesko. Slov Farm 8, 373–376.
Rouzaud, G., Rabot, S., Ratcliffe, B., Duncan, A.J., 2003. Influence of plant and
bacterial myrosinase activity on the metabolic fate of glucosinolates in
gnotobiotic rats. Br. J. Nutr. 90, 395–404.
Wang, H., Lin, W., Shen, G., Khor, T.O., Nomeir, A.A., Kong, A.N., 2011. Development
and validation of an LC–MS–MS method for the simultaneous determination of
G Model
INDCRO-8539; No. of Pages 7 ARTICLE IN PRESS
R. Benzekri et al. / Industrial Crops and Products xxx (2015) xxx–xxx 7

Sulforaphane and +its metabolites in rat plasma and its application in
pharmacokinetic studies. J. Chromatogr. Sci. 49, 801–806.
Wu, H.Z., Fei, H.J., Zhao, Y.L., Liu, X.J., Huang, Y.J., Wu, S.W., 2012. Antibacterial
mechanism of sulforaphane on E. coli. Sichuan Da Xue Xue Bao Yi Xue Ban. 43
(3), 386–390.
Zaidan, M.R.S., Noor Rain, A., Badrul, A.R., Adlin, A., Norazah, A., Zakiah, I., 2005.
In vitro screening of five local medicinal plants for antibacterial activity using
disc diffusion method. Trop. Biomed. 22 (2), 165–170.

Please cite this article in press as: Benzekri, R., et al., Isolation and identification of an antibacterial compound from Diplotaxis harra
(Forssk.) Boiss. Ind. Crops Prod. (2015), http://dx.doi.org/10.1016/j.indcrop.2015.11.059