Antimicrobial volatile glucosinolate autolysis products from Hornungia petraea (L.) Rchb. (Brassicaceae)

Article abstract of DOI:10.1016/j.phytol.2012.02.017

Plant samples of Hornungia petraea were analyzed for glucosinolate (GLS) autolysis metabolites for the first time. GC-MS analysis of the autolysate and the synthesis of a series (12 compounds) of possible glucosinolate breakdown products revealed/corroborated the presence of glucoaubrietin, glucolimnanthin, glucolepigramin and glucotropaeolin in this species as the most likely "mustard oil" precursors. GLS degradation products identified in the autolysate of H. petraea, benzyl isothiocyanate, 3- and 4-methoxybenzyl isothiocyanate, along with several other structurally related compounds were evaluated for antimicrobial activity in order to possibly pinpoint the role of the latter secondary metabolites in the plant tissues. The assays showed a very high antibacterial activity of the tested isothiocyanates against Sarcina lutea and an antifungal effect against Aspergillus fumigatus and Candida albicans with MIC values in the order of 1 μg/ml value.

Full text of DOI:10.1016/j.phytol.2012.02.017

Phytochemistry Letters 5 (2012) 351–357
Contents lists available at SciVerse ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Antimicrobial volatile glucosinolate autolysis products from
Hornungia petraea (L.) Rchb. (Brassicaceae)
a,
a,b
c
*
´
´
´
´
Niko S. Radulovic , Milan S. Dekic , Zorica Z. Stojanovic-Radic
a
ˇ
ˇ
ˇ
Department of Chemistry, Faculty of Science and Mathematics, University of Nis, Visegradska 33, 18000 Nis, Serbia
b
c
ˇ
Department of Chemical and Technological Sciences, State University of Novi Pazar, Vuka Karadzic´a bb, 36300 Novi Pazar, Serbia
ˇ
ˇ
ˇ
Department of Biology and Ecology, Faculty of Science and Mathematics, University of Nis, Visegradska 33, 18000 Nis, Serbia
A R T I C L E I N F O
A B S T R A C T
Article history:
Plant samples of Hornungia petraea were analyzed for glucosinolate (GLS) autolysis metabolites for the
first time. GC–MS analysis of the autolysate and the synthesis of a series (12 compounds) of possible
glucosinolate breakdown products revealed/corroborated the presence of glucoaubrietin, glucolim-
nanthin, glucolepigramin and glucotropaeolin in this species as the most likely ‘‘mustard oil’’ precursors.
GLS degradation products identified in the autolysate of H. petraea, benzyl isothiocyanate, 3- and 4-
methoxybenzyl isothiocyanate, along with several other structurally related compounds were evaluated
for antimicrobial activity in order to possibly pinpoint the role of the latter secondary metabolites in the
plant tissues. The assays showed a very high antibacterial activity of the tested isothiocyanates against
Sarcina lutea and an antifungal effect against Aspergillus fumigatus and Candida albicans with MIC values in
Received 25 December 2011
Received in revised form 28 February 2012
Accepted 29 February 2012
Available online 14 March 2012
Keywords:
Brassicaceae
Hornungia petraea
Glucosinolate
the order of 1
mg/ml value.
Isothiocyanate
Antimicrobial activity
ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
1. Introduction
years isothiocyanates have also been identified as potent cancer-
prevention agents (Fahey et al., 2001; Halkier and Gershenzon,
Glucosinolates (GLSs), known as mustard oil glucosides, are a
class of nitrogen and sulfur-containing natural products distribut-
ed in 16 dicotyledonous families of the order Capparales (Al-
Shehbaz and Al-Shammary, 1987; Verkerk et al., 2009), but also in
the genus Drypetes (family Euphorbiaceae) (Rodman et al., 1996).
Mostly, glucosinolate-containing genera are clustered within the
Brassicaceae, Capparaceae and Caricaceae families (Fahey et al.,
2001). Representatives of the Brassicaceae family are of particular
importance as vegetables, seasonings and relishes, and sources of
vegetable oil. GLSs are recognized as the active constituents
responsible for many of the physiological activities proposed for
the Brassica vegetables (Holst and Williamson, 2004; Verkerk et al.,
2009). Up to date, approximately 200 GLSs were described (Clarke,
2010; Fahey et al., 2001; Halkier and Gershenzon, 2006). Upon
plant damage, thioglucoside glucohydrolase (E.C.3.2.3.1), or also
called myrosinase, hydrolyzes the relatively non-reactive GLS to
give an array of biologically active compounds, i.e. isothiocyanates,
thiocyanates, nitriles, etc. GLS metabolites have been recognized as
antimicrobial agents for many decades (Fahey et al., 2001) and this
activity has been proposed to be a part of the crucifers defense
2006; Holland et al., 1995; Zhang and Talalay, 1994). Epidemio-
logical studies have shown that they show a protective effect
against cancer – particularly in the bladder, colon and lung (Song
and Thornalley, 2007). In addition to their diverse biological and
physiological properties, GLSs and their breakdown products are
receiving considerable attention of scientists due to their known
great chemotaxonomical significance.
The genus Hornungia Rchb. (Brassicaceae) comprises approxi-
mately 10 species, distributed in Europe, North Africa and west
Asia; one species of this genus grows in Australia, south and North
Africa. In Serbia, the genus is represented by only one species –
Hornungia petraea (L.) Rchb. 1837 (syn. Hutchinsia petraea (L.) R. Br.
ˇ
ˇ
1812; Lepidium petraeum L. 1753; Gonesina or grancika, in Serbian)
´
´
(Jovanovic-Dunjic, 1972). In general, a literature survey revealed
that the genus Hornungia has been poorly investigated. In general,
only two previous references on the phytochemistry of Hornungia
and Hutchinsia taxa can be found regarding the analysis of
glucosinolates and their breakdown products in Hutchinsia alpina
(Bennett et al., 2004; Kjaer et al., 1953).
Although there are many articles in the literature dealing with
the identification of GLSs, it is often very difficult to identify any
consistent results. The difficulty mainly lies in the lack of reliable,
standard methods of GLS analysis, their instability and limited
analytical data required for the identification. Analysis of volatile
GLS autolysis products performed by gas chromatography/mass
´
against pathogen attack (Radulovic et al., 2011). In the past few
* Corresponding author. Tel.: +381 18223430; fax: +381 18533014.
E-mail address: nikoradulovic@yahoo.com (N.S. Radulovic´).
1874-3900/$ – see front matter ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.phytol.2012.02.017

352
N.S. Radulovic´ et al. / Phytochemistry Letters 5 (2012) 351–357
Table 1
Percentage composition of the glucosinolate autolysis volatiles from Hornungia petraea.
RI calc.
Component
Compound class
Percentagea
Method of identification
hp1
hp2
965
Benzaldehyde
OTH
OTH
FAD
NIT
0.1
0.1
0.2
1.2
8.5
2.4
0.1
1.5
25.1
–
–
RI, MS, Co-GC
RI, MS, Co-GC
RI, MS, Co-GC
RI, MS, Co-GC
RI, MS, Co-GC
RI, MS, Co-GC
RI, MS, Co-GC
RI, MS
978
Phenol
–
1106
1135
1361
1373
1404
1480
1599
1623
1703
1841
1900
1962
2000
2100
2141
2200
2300
2400
2500
2600
2700
2800
2834
2900
3000
3100
3103
Nonanal
–
2-Phenylacetonitrile (9)
Benzyl isothiocyanate (1)
2-(3-Methoxyphenyl)acetonitrile (11)
Vanillin
0.2
4.8
1.6
–
ITC
NIT
OTH
NIT
2-(3-Hydroxyphenyl)acetonitrile (14)
3-Methoxybenzyl isothiocyanate (3)
4-Methoxybenzyl isothiocyanate (4)
3-Hydroxybenzyl isothiocyanate (13)
Neophytadiene (isomer II)
Nonadecane
–
ITC
34.4
tr
RI, MS, Co-GC
RI, MS, Co-GC
RI, MS
ITC
ITC
15.9
0.9
tr
22.1
–
TER/OTH
FAD
FAD
FAD
FAD
FAD
FAD
FAD
FAD
FAD
FAD
FAD
FAD
TER
FAD
FAD
FAD
FAD
RI, MS
–
RI, MS, Co-GC
RI, MS, Co-GC
RI, MS, Co-GC
RI, MS, Co-GC
RI, MS, Co-GC
RI, MS, Co-GC
RI, MS, Co-GC
RI, MS, Co-GC
RI, MS, Co-GC
RI, MS, Co-GC
RI, MS, Co-GC
RI, MS, Co-GC
RI, MS, Co-GC
RI, MS, Co-GC
RI, MS, Co-GC
RI, MS, Co-GC
RI, MS, Co-GC
Hexadecanoic acid
Eicosane
1.1
0.2
0.3
2.6
0.4
0.5
0.3
0.6
0.5
1.7
0.9
2.1
12.2
2.1
8.5
3.6
0.3
–
Heneicosane
0.1
0.2
0.1
0.1
–
(Z,Z,Z)-9,12,15-Octadecatrienoic acid
Docosane
Tricosane
Tetracosane
Pentacosane
1.4
–
Hexacosane
Heptacosane
1.2
–
Octacosane
Squalene (all E)
0.9
10.8
3.0
4.2
3.3
Nonacosane
Triacontane
Hentriacontane
a-Tocopherol
Total
93.6
88.5
Grouped components
Percentagea
hp1
hp2
Isothiocyanates (ITC)
Nitriles (NIT)
49.5
5.1
61.3
1.6
Terpenoids (TER)
7.0
0.9
Fatty acids and fatty acid derived compounds (FAD)
Others (OTH)b
28.1
3.9
21.4
3.3
RI calc. – experimentally determined linear retention indices on an HP-5MS column; hp1 and hp2 – samples of H. petraea collected from two different locations (for more
details see Section 3); tr – trace amounts (<0.05%); RI – retention indices matching with literature data (Adams, 2007); MS – mass spectra matching; Co-GC – co-injection
with a pure reference compound; (–) not detected.
Entries in bold letters are compounds believed to be GLS autolysis products.
a
Values are means of triplicate analyses.
b
Unclassified constituents, compounds of possible anthropogenic origin.
spectrometry (GC–MS) is a simple, rapid and, in most cases,
convenient method for the qualitative analysis of glucosinolate-
containing plant species.
GC and GC–MS analyses of the autolysates enabled the positive
identification (with GC co-injection of the standards) of 26
compounds and 3 additional tentatively identified according to
MS and RI analysis matching in both samples, accounting for
88.5–93.6% of the extracted volatiles based on the areas under
the GC chromatograms (Table 1). Seven compounds were
detected in the two H. petraea samples (hp1 and hp2) as being
possible GLS metabolites: 3- (3) and 4-methoxybenzyl isothio-
cyanate (4) (25.1 and 34.4%, and trace amounts, respectively), 3-
hydroxybenzyl isothiocyanate (13) (15.9 and 22.1%), benzyl
isothiocyanate (1) (4.8 and 8.5%), 2-(3-methoxyphenyl)acetoni-
trile (11) (1.6 and 2.4%), 2-(3-hydroxyphenyl)acetonitrile (14)
(1.5%) and 2-phenylacetonitrile (9) (0.2 and 1.2%).
Considering the fact that isomers, in general and especially
those possessing an aromatic core, yield upon EI very similar
mass spectra (in our case the positional and functional group
isomerism of the detected GLS metabolites), and scarce
literature GC retention index data of the compounds in question,
the detected GLS metabolites and their conceivable isomers
were synthesized and GC co-injected with the autolysates in
order to acquire an unambiguous corroboration of their
identities (Fig. 1).
In connection with our previous studies on the glucosinolate
´
autolysis products (Radulovic et al., 2008, 2011), we are interested
in the investigations of the chemical composition of volatile
autolysis products of Serbian Cruciferae, since these have received
almost no attention in this respect. Bearing in mind the scarce
literature data on the chemistry of Hornungia species, in this study
we focused our attention on the glucosinolate autolysis products of
H. petraea. Another goal of this study was to provide a set of
spectral and chromatographic data that would make the detection
and identification of a number of GLS breakdown products easier.
This was accomplished through the synthesis of a series of
regioisomeric compounds of possible GLS origin and their full
spectral characterization and accumulation of GC chromatographic
retention data (RI).
2. Results and discussion
Plant samples of H. petraea, collected at two different
locations, were analyzed for glucosinolate autolysis metabolites.

N.S. Radulovic´ et al. / Phytochemistry Letters 5 (2012) 351–357
353
Fig. 3. Structures of the glucosinolates glucotropaeolin, glucolimnanthin,
glucolepigramin and glucoaubrietin, believed to be the precursors of the
identified ‘‘mustard oils’’ in H. petraea autolysate.
revealed the presence of
b-phenethyl isothiocyanate and of a
second, unknown isothiocyanate (Kjaer et al., 1953). Recently,
benzyl-, 3-hydroxybenzyl-, 4-hydroxybenzyl-, 2-methoxybenzyl-
and 3-methoxybenzyl glucosinolates were found in the same
species (Bennett et al., 2004). The same authors investigated GLS
profiles of a number of GLS-containing species, and noticed a clear
subdivision of the crucifers based on their GLS content into four
groups: (i) short- to medium-chain-length aliphatic; (ii) long-
chain-length aliphatic; (iii) simple aromatic and (iv) highly
substituted aromatic ones. According to this classification, these
two Hutchinsia species could be recognized as crucifers with the
predominance of simple aromatic GLSs. From the aforementioned
observations, an obvious similarity of the GLS profile of the two
Hutchinsia species and of their phylogenetically close Hornungia
taxon exists. This provides support for the suggestion of Appel and
Al-Shehbaz (1997) to reduce the genus Hutchinsia (=Pritzelago) to
synonymy of Hornungia, i.e. to treat H. alpina as a species of the
genus Hornungia.
Fig. 1. Structures of the synthesized/identified benzylic isothiocyanates and
thiocyanates as well as of the phenylacetonitriles.
Due to the limited literature data, and the difficulties
encountered in the synthesis of the chemical instable 3-hydro-
xybenzyl isothiocyanate (13) and 2-(3-methoxyphenyl)acetoni-
trile (14), the presence and identification of these compounds in H.
petraea autolysate were concluded based on MS data, while the
distinction of the regio isomers were made by a comparison of
their retention times with those reported by Olsen and Sorensen
(1980) (only available GC data for these compounds) (Fig. 2). A very
nice fit (correlation coefficient R = 0.99878) was observed for the
compounds in common to this work and the present one.
Identification of the above-mentioned compounds (1, 3–4, 9,
11, 13–14; Fig. 1) in the autolysates suggests the occurrence of
benzyl (glucotropaeolin), 3-methoxybenzyl (glucolimnanthin), 3-
hydroxybenzyl-(glucolepigramin) and 4-methoxybenzyl glucosi-
nolate (glucoaubrietin) in this species as the possible ‘‘mustard oil’’
precursors (Fig. 3).
The synthesized compounds 1–5, 7–9 were evaluated for
antimicrobial activity in microdilution assays against eight patho-
genic bacterial and two fungal strains. The microorganisms listed in
Table 2 were selected according to two criteria: GLS breakdown
products were previously foundto possessan effect on the growthof
such and related strains of human pathogenic bacteria and fungi
Previous studies on the glucosinolates in H. alpina, a species
closely related to H. petraea, the only studied from this genus,
´
(Radulovic et al., 2011) and some of these may be present in the soil
associated with the plant species and hence could represent direct
plant pathogens (e.g. Aspergillus fumigatus, O’Gorman et al., 2008).
The tested compounds were showed to be active against all of tested
microorganisms, with the activity ranging from 1 to 1250
mg/ml for
inhibitory and 1 to 5000 g/ml for microbicidal activity (Table 2).
m
Amongthetestedcompounds,themostactiveweretheonesbearing
an isothiocyanate group – benzyl isothiocyanate (1) against A.
fumigatus (MIC = 9
ybenzyl isothiocyanate (3) against Sarcina lutea (MIC = MBC = 5
ml and MIC = MBC = 2 g/ml, respectively) and 4-methoxybenzyl
isothiocyanate (4) against C. albicans (MIC = 1 g/ml; MBC = 2 g/
ml) and A. fumigatus (MIC = MBC = 1 g/ml). This significant and
mg/ml; MBC = 10
mg/ml), 2- (2) and 3-methox-
mg/
m
m
m
m
non-selective both antibacterial and antifungal activities of the
tested isothiocyanates, connected with their mode of release by
mechanical stress, suggests that isothiocyanates (and their precur-
sor glucosinolates) play a significant role in the defense of the plants
against various phytopathogens.
Fig. 2. A plot showing the correlation between GC retention times (Rt, min) of 2-
phenylacetonitrile (9), benzyl isothiocyanate (1), 2-phenethyl isothiocyanate, 2-(3-
hydroxyphenyl)acetonitrile (14) and 3-hydroxybenzyl isothiocyanate (13),
respectively, from the present study and those reported by Olsen and Sorensen
(1980) demonstrating the matching of the retention data utilized in the
3. Experimental procedures
identification procedure with
a correlation coefficient of R = 0.99878. The
3.1. General experimental procedures
following compounds were plotted, Rt in our study, Rt previously reported: 9,
8.44, 12.1; 1, 14.25, 15.3; 2-phenethyl isothiocyanate (synthesized compound in
our lab, but not the part of this study), 16.73, 16.7; 14, 17.08, 17.3 and 13, 22.30,
19.9.
All chemicals were commercially available and used as received
(Aldrich, USA; Merck, Germany; Fluka, Germany), except that the

354
N.S. Radulovic´ et al. / Phytochemistry Letters 5 (2012) 351–357
Table 2
Antimicrobial activity of the synthesized benzyl isothiocyanates 1–4 and thiocyanates 5, 7–8 as well as of the phenylacetonitrile 9 – minimal inhibitory (MIC) and minimal
bactericidal/fungicidal concentrations (MBC/MFC).
Bacterial strain
Compound (mg/ml)
Tetracycline
(mg/ml)
1
2
3
4
5
7
8
9
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MBC
S. aureus (isolate 1)
S. aureus (isolate 2)
S. enteritidis
K. pneumoniae
S. lutea
0.07
0.01
0.01
0.03
0.01
0.07
0.03
0.01
0.07
0.03
0.01
0.03
0.01
0.15
0.15
0.03
0.15
0.03
0.07
0.03
0.005
1.25
0.60
0.15
0.15
0.03
0.30
0.07
0.005
5.00
1.25
0.60
0.15
0.03
0.03
0.03
0.002
0.30
0.60
0.15
0.15
0.03
0.07
0.07
0.002
0.30
1.25
0.60
0.15
0.01
0.03
0.01
0.03
0.30
1.25
0.62
0.60
0.30
0.07
0.01
0.07
0.60
1.25
1.25
0.60
0.30
0.07
0.07
0.15
1.25
1.25
0.07
1.25
0.60
0.07
0.15
0.30
1.25
1.25
0.15
0.15
0.07
0.07
0.03
0.07
0.30
0.15
0.01
0.30
0.15
0.07
0.15
0.07
0.30
0.30
0.07
0.60
0.30
0.03
0.07
0.15
0.60
0.60
0.07
0.60
0.30
0.07
0.07
0.15
0.60
1.25
0.60
0.60
0.30
0.03
0.07
0.15
0.60
0.60
0.07
0.60
0.30
0.07
0.07
0.15
0.60
1.25
0.60
0.025
0.025
0.025
0.012
0.002
0.025
0.025
0.006
E. coli
Shigella sp.
B. cereus
Fungal strain
Compound (mg/ml)
Nystatin
(mg/ml)
1
2
3
4
5
7
8
9
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MFC
C. albicans
0.03
0.60
0.01
0.15
0.07
1.25
0.15
0.15
0.03
0.15
0.15
0.001
0.001
0.002
0.001
1.25
0.15
1.25
0.15
0.60
0.30
0.60
0.60
0.60
0.15
5.00
0.30
0.60
0.15
5.00
0.30
0.025
0.002
A. fumigatus
0.009
solvents were purified by distillation. The GC–MS analyses were
performed on a Hewlett-Packard 6890 N gas chromatograph
equipped with fused silica capillary column HP-5MS and coupled
with a 5975B mass selective detector from the same company as
detailed under Section 3.4. Preparative medium pressure liquid
chromatography (MPLC) separations were made on pre-packed
sufficient quantity of distilled water to make a paste (25 ml). No
separation of the roots was made due to the small size of the plants
(around 5 cm in height) and its minor contribution to the bulk of
the plant material. According to a modified procedure reported by
Vaughn and Berhow (2005), Et2O (10 ml) was added to each
sample, the all-glass vessels sealed, and the flasks placed in an
incubator shaker set at 25 8C and 200 rpm for 8 h. Following
hydrolysis, sodium chloride (6 g) was added and mixed thorough-
ly. The ether layer was then separated from the brine solution and
filtered through a Whatman No. 1 filter paper and the residual
plant mass was extracted an additional three times with excess
Et2O. The combined crude ether extracts were dried over dry
magnesium sulfate, evaporated to dryness on a rotary evaporator
silica gel (40–63
mm) polypropylene cartridges under gradient
conditions using hexane–ether as eluent at a flow rate of 2.5 ml/
min using a Bu¨chi system (C 610, Bu¨chi Labortechnik, Switzerland).
The IR measurements were carried out using a Thermo Nicolet
model 6700 FTIR instrument (Waltham, USA) – ATR: attenuated
total reflectance. The 1H and 13C NMR spectra have been recorded
on a GEMINI-200 spectrometer operating at 200 and 50.3 MHz,
respectively. All NMR spectra were measured at 25 8C in CDCl3
with tetramethylsilane (TMS) as the internal standard. Chemical
´
at 30 8C, and redissolved in Et2O (1 ml) (Radulovic et al., 2008).
shifts are reported in ppm (
d
) and referenced to TMS. The purity
3.4. Gas chromatography/mass spectrometry (GC–MS)
and identity of the synthesized compounds was checked by TLC,
GC–MS and spectroscopic techniques (MS, IR, 1H and 13C NMR) and
confirmed by comparison of their spectral data to those existent in
the literature (Asghari et al., 2010; Kiasat and Fallah-Mehrjardi,
2008; Kitamura et al., 1990; Stevens et al., 2009; Vaughn et al.,
1996).
The chemical composition of H. petraea autolysates were
analyzed by GC and GC–MS immediately upon completion of the
hydrolysis and separation procedure described above. The GC–MS
analyses (three repetitions) were performed on a Hewlett-Packard
6890 N gas chromatograph equipped with a fused silica capillary
column HP-5MS (5% phenylmethylsiloxane, 30 m ꢀ 0.25 mm, film
3.2. Plant material
thickness 0.25 mm, Agilent Technologies, USA) and coupled with a
5975B mass selective detector from the same company. The
injector and interface were operated at 250 8C and 320 8C,
respectively. Oven temperature was raised from 70 to 315 8C at
a heating rate of 5 8C minꢁ1 and then isothermally held for 10 min.
The plant samples (aerial and underground parts) of H. petraea
utilized in this work were collected from natural populations from
rocky and calcareous soils: (i) in Prizrenska Bistrica river gorge
(Prizren, Serbia) at the end of March 2007 (sample hp1), and (ii) on
Stara Planina Mountain (Southeast Serbia) in April 2007 (sample
hp2). Voucher specimens were deposited at the Herbarium
collection of the Faculty of Science and Mathematics, University
As a carrier gas helium at 1.0 ml minꢁ1 was used. The samples, 1
ml
of the solutions in Et2O (10 mg in 1 ml of Et2O), were injected in a
pulsed split mode (the flow was 1.5 ml/min for the first 0.5 min
and then set to 1.0 ml/min throughout the remainder of the
analysis; split ratio 40:1). The mass selective detector was
operated at the ionization energy of 70 eV, in the 35–650 amu
range and scanning speed of 0.32 s. GC (FID) analyses were carried
out under the same experimental conditions using the same
column and same gas chromatograph type as described for the GC–
MS. The percentage composition was computed from the total ion
chromatogram peak areas without the use of correction factors.
Qualitative analysis of the autolysate constituents was based on
the comparison of their linear retention indices relative to
retention times of C7–C32 n-alkanes on the HP-5MS column
(Van den Dool and Kratz, 1963) with those reported in the
ˇ
of Nis, under the accession numbers MD0207 (hp1) and MD0307
(hp2).
3.3. Isolation of hydrolysis products
Hydrolysis of GLSs was catalyzed by the endogenous myr-
osinase (thioglucoside glucohydrolase, EC 3.2.3.1) according to the
´
procedure of Radulovic et al. (2008).
Autolysis experiments (three repetitions for each sample) were
performed on homogenized fresh plant material (10 g of samples –
aerial and underground parts together), which were mixed with a

N.S. Radulovic´ et al. / Phytochemistry Letters 5 (2012) 351–357
355
literature (Adams, 2007), and by comparison of their mass spectra
with those of authentic standards, as well as those from Wiley 6,
NIST05, MassFinder 2.3, and a homemade MS library with the
spectra corresponding to pure substances, and wherever possible,
by GC co-injection with an authentic sample.
(ar. C–O–al. C), 1174, 1029 (ar. C–O–al. C), 815, 751, 668, 545; 1H
NMR spectral data (CDCl3): 3.79 (3H, s, CH3), 4.61 (2H, s, H-7),
6.87 (2H, m, H-3, H-5), 7.21 (2H, m, H-2, H-6); 13C NMR spectral
data (CDCl3): 48.1 (CH2), 55.2 (CH3O), 114.2 (C-3, C-5), 126.2
(C-1), 128.7 (C-2, C-6, NCS), 159.5 (C-4); EIMS, 70 eV, m/z (rel.
int.): 179 (9), 122 (8), 121 (100), 91 (6), 89 (3), 78 (11), 77 (11),
63 (3), 52 (3), 51 (5).
d
d
3.5. General procedure for the synthesis of benzylic isothiocyanates 1–
4 (Tsogoeva et al., 2005)
3.6. General procedure for the synthesis of benzylic thiocyanates 5–8
The benzyl isothiocyanates 1–4 were synthesized by the
reaction of the corresponding amine and carbon disulfide in the
presence of N,N0-dicyclohexylcarbodiimide (DCC) according to a
modified procedure reported by Tsogoeva et al. (2005).
The benzyl thiocyanates 5–8 were prepared by the reaction of
the corresponding aryl chlorides with potassium rhodanide in
dimethylformamide (DMF) at room temperature (Suzuki et al.,
1979).
A solution of the appropriate aryl chloride (15.8 mmol) and
potassium thiocyanate (31.5 g, 32 mmol) in DMF (150 ml) was
stirred at room temperature for 4 h, according to a modified
procedure reported by Suzuki et al. (1979). The reaction mixture
was then diluted with water (300 ml) and extracted with Et2O
(3ꢀ 50 ml). The combined ether extracts were washed with
water and dried over dry MgSO4. Evaporation of the solvent gave
the desired thiocyanates of sufficient purity for spectral analysis.
Carbon disulfide (12.5 ml) and DCC (6.3 g, 30.6 mmol) were
added to a solution of the appropriate amine (33 mmol) in dry
CH2Cl2 (40 ml) at ꢁ10 8C. With stirring, the reaction mixture was
allowed to warm slowly to room temperature over a period of 3 h
and then was stirred for a further 5 h at room temperature. The
reaction was monitored by TLC. After the separation of the
precipitated thiourea by filtration, the solvent was removed under
vacuum. The residue was taken up in ether and more of the
precipitated thiourea was removed by filtration. Evaporation of the
solvent gave a crude product which was further purified by MPLC
to afford the desired isothiocyanates of sufficient purity for
spectral analysis.
3.6.1. Benzyl thiocyanate (5)
White crystals; 2.04 g (89%), Rt (HP-5MS) 13.63 min, RI 1342;
FTIR (ATR) cmꢁ1: 3030 (ar. C–H), 2992, 2181 (SCBB N), 2050, 1598
(ar. C55C), 1492, 1454, 1426, 1243, 1203, 1159, 1074, 1027, 918,
804, 767–565 (ar. C–H and ar. C–C); 1H NMR spectral data (CDCl3):
3.5.1. Benzyl isothiocyanate (1)
Yellowish liquid; 3.73 g (75%), Rt (HP-5MS) 14.25 min, RI 1362;
FTIR (ATR) cmꢁ1: 3063 (ar. C–H), 3031 (ar. C–H), 2166 (vs, N55C55S),
2067 (N55C55S), 1495, 1454, 1346, 1301, 1200, 1072 (C–N), 813,
d
4.15 (2H, s, H-7), 7.25–7.44 (5H, m, Ar-H); 13C NMR spectral data
(CDCl3): 38.3 (CH2), 111.9 (SCN), 128.8 (C-4), 128.9 (two
d
696 (N55C55S), 573; 1H NMR spectral data (CDCl3):
7), 7.25–7.45 (5H, m, Ar-H); 13C NMR spectral data (CDCl3):
d
4.72 (2H, s, H-
48.6
overlapped signals, Ar-H), 129.1 (two overlapped signals, Ar-H),
134.3 (C-1); EIMS, 70 eV, m/z (rel. int.): 149 (3), 92 (8), 91 (100), 89
(4), 65 (15), 63 (6), 51 (4), 50 (2), 45 (2), 39 (7).
d
(CH2), 126.9 (C-2, C-6), 128.4 (C-4), 129.0 (C-3, C-5, NCS), 134.3 (C-
1); EIMS, 70 eV, m/z (rel. int.): 149 (23), 92 (7), 91 (100), 90 (2), 89
(6), 65 (15), 63 (6), 51 (5), 50 (3), 39 (5).
3.6.2. 2-Methoxybenzyl thiocyanate (6)
Yellow liquid; 0.24 g (84%), Rt (HP-5MS) 18.53 min, RI 1540;
FTIR (ATR) cmꢁ1: 2941, 2637, 2366, 2149 (SCBB N), 1600 (ar. C55C),
1492, 1462, 1438, 1291, 1247 (ar. C–O–al. C), 1180, 1107, 1025 (ar.
3.5.2. 2-Methoxybenzyl isothiocyanate (2)
Yellow liquid; 0.81 g (69%), Rt (HP-5MS) 19.22 min, RI 1569;
FTIR (ATR) cmꢁ1: 2927, 2853, 2170 (vs, N55C55S), 2071 (N55C55S),
1693, 1602 (ar. C55C), 1492, 1462, 1342, 1246 (ar. C–O–al. C), 1287,
1118, 1026 (ar. C–O–al. C), 750 (C–S), 671 (N55C55S); 1H NMR
C–O–al. C), 883, 853, 751, 641; 1H NMR spectral data (CDCl3):
d
3.87 (3H, s, CH3), 4.17 (2H, s, H-7), 6.89–6.99 (2H, m, H-3, H-5),
7.26–7.39 (2H, m, H-4, H-6); 13C NMR spectral data (CDCl3):
33.8
d
spectral data (CDCl3):
7.01 (2H, m, H-3, H-5), 7.24–7.37 (2H, m, H-4, H-6); 13C NMR
spectral data (CDCl3): 44.3 (CH2), 55.3 (CH3O), 110.4 (C-3), 120.6
(C-1), 122.4 (C-5), 127.9 (C-4),1 128.2 (C-6),1 129.7 (NCS), 156.5 (C-
2); EIMS, 70 eV, m/z (rel. int.): 179 (13), 122 (9), 121 (100), 93 (8),
92 (8), 91 (97), 78 (13), 77 (12), 65 (16), 51 (12).
d
3.85 (3H, s, CH3), 4.69 (2H, s, 7-H2), 6.80–
(CH2), 55.4 (CH3O), 110.7 (C-3), 112.8 (SCN), 120.7 (C-1), 122.9 (C-
5), 130.4 (C-4, C-6), 157.2 (C-2); EIMS, 70 eV, m/z (rel. int.): 179 (9),
122 (9), 121 (100), 93 (10), 92 (8), 91 (98), 78 (14), 77 (9), 65 (14),
51 (9).
d
3.6.3. 3-Methoxybenzyl thiocyanate (7)
Yellow liquid; 0.22 g (98%), Rt (HP-5MS) 19.31 min, RI 1572;
FTIR (ATR) cmꢁ1: 2936, 2836, 2356, 2153 (SCBB N), 1600 (ar. C55C),
1489, 1453, 1435, 1265 (ar. C–O–al. C), 1153, 1037 (ar. C–O–al. C),
3.5.3. 3-Methoxybenzyl isothiocyanate (3)
Yellow liquid; 0.76 g (67%), Rt (HP-5MS) 19.92 min, RI 1598;
FTIR (ATR) cmꢁ1: 2929, 2852, 2202 (vs, N55C55S), 2089 (N55C55S),
1692, 1601 (ar. C55C), 1490, 1452, 1336, 1261 (ar. C–O–al. C), 1150,
1039 (ar. C–O–al. C), 773 (C–S), 700 (N55C55S), 544; 1H NMR
875, 782, 735, 700, 595, 568; 1H NMR spectral data (CDCl3):
d
3.82
(3H, s, CH3), 4.13 (2H, s, H-7), 6.81–6.98 (3H, m, H-2, H-4, H-6),
7.23–7.34 (1H, m, H-5); 13C NMR spectral data (CDCl3):
38.3
d
spectral data (CDCl3):
6.98 (3H, m, H-2, H-4, H-6), 7.29 (1H, t, H-5); 13C NMR spectral data
(CDCl3): 48.5 (CH2), 55.2 (CH3O), 112.3 (C-4), 113.7 (C-2), 118.9
d
3.81 (3H, s, CH3), 4.68 (2H, s, H-7), 6.78–
(CH2), 55.2 (CH3O), 111.9 (SCN), 114.3 (C-4),1 114.4 (C-2),1 121.1
(C-6), 130.1 (C-5), 135.7 (C-1), 159.9 (C-3); EIMS, 70 eV, m/z (rel.
int.): 179 (33), 121 (100), 91 (30), 89 (6), 78 (16), 77 (13), 65 (7), 63
(7), 51 (8), 39 (6).
d
(C-6), 129.3 (C-5), 130.0 (NCS), 135.6 (C-1), 159.9 (C-3); EIMS,
70 eV, m/z (rel. int.): 179 (32), 122 (9), 121 (100), 91 (28), 78 (15),
77 (14), 65 (8), 63 (6), 51 (8), 39 (6).
3.6.4. 4-Methoxybenzyl thiocyanate (8)
Yellow liquid; 2.02 g (82%), Rt (HP-5MS) 20.04 min, RI 1603;
FTIR (ATR) cmꢁ1: 2936, 2836, 2356, 2153 (SCBB N), 1600 (ar. C55C),
1489, 1453, 1435, 1265 (ar. C–O–al. C), 1153, 1037 (ar. C–O–al. C),
3.5.4. 4-Methoxybenzyl isothiocyanate (4)
Yellowish liquid; 2.76 g (77%), Rt (HP-5MS) 20.53 min, RI
1623; FTIR (ATR) cmꢁ1: 3291, 2928, 2835, 2183 (vs, N55C55S),
2075 (N55C55S), 1685, 1610 (ar. C55C), 1510, 1438, 1341, 1246
875, 782, 735, 700, 595, 568; 1H NMR spectral data (CDCl3):
d
3.79
(3H, s, CH3), 4.12 (2H, s, H-7), 6.88 (2H, m, H-3, H-5), 7.27 (2H, m, H-
2, H-6); 13C NMR spectral data (CDCl3):
38.1 (CH2), 55.2 (CH3O),
112.1 (SCN), 114.3 (C-3, C-5), 126.1 (C-1), 130.2 (C-2, C-6), 159.8
d
1
Interchangeable.

356
N.S. Radulovic´ et al. / Phytochemistry Letters 5 (2012) 351–357
(C-4); EIMS, 70 eV, m/z (rel. int.): 135 (1), 121 (100), 107 (21), 94
(1), 79 (3), 77 (7), 69 (1), 64 (1), 51 (5), 41 (1).
(CN), 122.0 (C-1), 129.1 (C-2, C-6), 159.2 (C-4); EIMS, 70 eV, m/z
(rel. int.): 147 (100), 146 (44), 132 (36), 116 (21), 107 (21), 104 (16),
91 (11), 78 (10), 77 (38), 51 (12).
3.7. General procedure for the synthesis of phenylacetonitriles 9–12
(Adams and Thal, 1922)
3.8. In vitro antimicrobial activity
The phenylacetonitriles 9–12 were prepared from the corre-
sponding chlorides with potassium cyanide in refluxing ethanol
(Adams and Thal, 1922).
3.8.1. Test microorganisms
The synthesized compounds 1–5, 7–9 were tested against a
panel of microorganisms (isolates), including Gram-positive
Staphylococcus aureus (two strains isolated from human material
sample), Bacillus cereus (food isolate), S. lutea (food isolate), Gram-
negative Salmonella enteritidis (food isolate), Klebsiella pneumoniae
(human sample isolate), Escherichia coli (human sample), Shigella
sp. (human sample), yeast Candida albicans (human sample isolate)
and mould A. fumigatus (house dust isolate). Cultures of the
bacterial species were maintained on Nutrient agar while the
fungal cultures on Sabouraud dextrose agar at the appropriate
optimal temperature (37 and 30 8C, respectively).
A mixture of powdered sodium cyanide (2.6 g, 53 mmol) and
water (3 ml) was warmed on a water bath in order to dissolve most
of the cyanide. Then, under reflux, a solution of the appropriate aryl
chloride (34.8 mmol) in 95% ethanol (4 ml) was added dropwise
over a period of 30 min. After heating under reflux for a further
3.5 h (the reaction was monitored by TLC), the resulting mixture
was cooled to room temperature, filtered with suction to remove
most of the sodium chloride, and the precipitate was washed with
a small portion of ethanol. After evaporation of ethanol, the residue
was extracted with Et2O (3ꢀ 50 ml). The combined Et2O extracts
were dried over dry MgSO4 and concentrated at reduced pressure.
The resulting crude product was further purified by MPLC to afford
the desired nitriles of sufficient purity for spectral analysis.
3.8.2. Screening of antimicrobial activity
Antimicrobial activity was evaluated using a broth micro-
dilution method (NCCLS, 2003). The minimum inhibitory
concentrations (MIC) determination was performed by a serial
dilution method in 96-well microtiter plates. Microorganisms
were cultured at 37 8C in Mueller Hinton agar (MHA) for bacteria
and Sabouraud dextrose agar (SDA) for fungi (30 8C). After 18 h
of cultivation, bacterial suspensions were made in Mueller
Hinton broth and their turbidity was standardized to 0.5
McFarland. Absorbance of every suspension was adjusted to a
standard one using a McFarland Densitometer (DEN-1B, Biosan).
The final density of the bacterial and yeast’s inoculums was
5 ꢀ 105. Suspension of the mould was made in Sabouraud
dextrose broth and its turbidity was confirmed by viable
counting in Thoma chamber with 1 ꢀ 104 as the final size of
the inoculums. Stock solutions of the compounds were prepared
in 0.05% aq. Tween 80 (Hammer et al., 1999) in the concentra-
tion range from 0.0005 to 20.00 mg/ml. The highest concentra-
tion, 0.025% (v/v) in any well, of Tween 80 had no effect of the
growth of the microorganisms. After making the dilutions of the
test substances, the inoculum was added to all wells and the
plates were incubated at 37 8C during 24 h (bacteria) or at 30 8C
for 48 h (fungal strains). Tetracycline and nystatin served as
positive controls, while the solvent (aqueous 0.05% Tween 80, v/
v), showing no visible effect, was used as the negative one. One
non-inoculated well, free of the antimicrobial agents, was also
included to ensure medium sterility. The bacterial growth was
3.7.1. 2-Phenylacetonitrile (9)
Yellow liquid; 3.45 g (85%), Rt (HP-5MS) 8.44 min, RI 1136; FTIR
(ATR) cmꢁ1: 2940, 2837, 2354, 2250 (CBB N), 1600 (ar. C55C), 1493,
1463, 1416, 1292, 1249, 1180, 1108, 1026, 832, 752, 696, 642, 593;
1H NMR spectral data (CDCl3):
Ar-H); 13C NMR spectral data (CDCl3):
d
3.71 (2H, s, H-7), 7.24–7.42 (5H, m,
23.4 (CH2), 117.8 (CN),
d
127.8 (C-3, C-5), 127.9 (C-4), 129.0 (C-2, C-6), 129.8 (C-1); EIMS,
70 eV, m/z (rel. int.): 117 (100), 118 (9), 116 (42), 91 (9), 90 (47), 89
(29), 63 (12), 51 (13), 50 (9), 39 (9).
3.7.2. 2-(2-Methoxyphenyl)acetonitrile (10)
Yellow liquid; 310 mg (67%), Rt (HP-5MS) 13.82 min, RI 1350;
FTIR (ATR) cmꢁ1: 2939, 2837, 2249 (CBB N), 1600 (ar. C55C), 1591,
1495, 1463, 1412, 1291, 1247 (ar. C–O–al. C), 1163, 1108, 1026 (ar.
C–O–al. C), 751, 567; 1H NMR spectral data (CDCl3):
d
3.68 (2H, s, H-
7), 3.86 (3H, s, CH3), 6.79–7.00 (2H, m, H-3, H-5), 7.22–7.37 (2H, m,
H-4, H-6); 13C NMR spectral data (CDCl3):
18.6 (CH2), 55.4
d
(CH3O), 110.3 (C-3), 118.0 (CN), 118.5 (C-1), 120.7 (C-5), 129.1 (C-
4), 129.4 (C-6), 156.6 (C-2); EIMS, 70 eV, m/z (rel. int.): 147 (100),
132 (60), 116 (19), 107 (56), 104 (27), 91 (23), 89 (14), 78 (14), 77
(47), 51 (19).
3.7.3. 2-(3-Methoxyphenyl)acetonitrile (11)
Yellow liquid; 277.5 mg (69%), Rt (HP-5MS) 14.43 min, RI 1373;
FTIR (ATR) cmꢁ1: 2940, 2837, 2354, 2251 (CBB N), 1683, 1601 (ar.
C55C), 1489, 1455, 1414, 1284, 1259 (ar. C–O–al. C), 1148, 1108,
1039 (ar. C–O–al. C), 879, 778, 742, 690, 544; 1H NMR spectral data
determined by adding 20
m
l of 0.5% 2,3,5-triphenyltetrazolium
´
chloride (TTC) aq. solution (Radulovic et al., 2010). MIC was
defined as the lowest concentration of the sample that inhibited
visible growth (red colored pellet on the bottom of the wells
after the addition of TTC). To determine the minimal
bactericidal/fungicidal concentrations (MBC/MFC), broth was
taken from each well without visible growth and inoculated in
MHA for 24 h at 37 8C for bacteria or in SDA for 48 h at 28 8C
(mould) and 30 8C (yeast). The MBC/MFC was defined as the
lowest concentration of a tested sample at which the inoculated
microorganisms were 99.9% killed. All experiments were done in
duplicate and repeated three times.
(CDCl3):
overlapping signals, H-2, H-4, H-6), 7.28 (1H, dd, J4,5 = 8 Hz,
J5,6 = 7 Hz, H-5); 13C NMR spectral data (CDCl3):
23.5 (CH2),
d 3.71 (2H, s, H-7), 3.81 (3H, s, CH3), 6.73–6.91 (3H,
d
55.2 (CH3O), 113.4 (C-4),1 113.5 (C-2),1 117.7 (CN), 120.0 (C-6),
128.3 (C-5), 130.1 (C-1), 160.0 (C-3); EIMS, 70 eV, m/z (rel. int.):
147 (100), 146 (19), 132 (27), 117 (22), 116 (24), 104 (20), 90 (17),
77 (66), 63 (17), 51 (17).
3.7.4. 2-(4-Methoxyphenyl)acetonitrile (12)
Yellow liquid; 450 mg (58%), Rt (HP-5MS) 14.91 min, RI 1392;
FTIR (ATR) cmꢁ1: 3002, 2936, 2837, 2250 (CBB N), 1612 (ar. C55C),
1510, 1463, 1416, 1303, 1245 (ar. C–O–al. C), 1178, 1109, 1029 (ar.
3.9. Statistical analysis
To determine whether there is
a statistically significant
C–O–al. C), 906, 810, 755, 705, 580; 1H NMR spectral data (CDCl3):
d
difference among the obtained results for antifungal and
antibacterial activity assays, variance analyses were carried out
using the SPSS 10.0 software package. Values of p < 0.05 were
considered to be significantly different.
3.65 (2H, s, H-7), 3.80 (3H, s, CH3), 6.89 (2H, overlapping signals, H-
3, H-5), 7.20 (2H, overlapping signals, H-2, H-6); 13C NMR spectral
data (CDCl3):
d 22.6 (CH2), 55.2 (CH3O), 114.5 (C-3, C-5), 118.4

N.S. Radulovic´ et al. / Phytochemistry Letters 5 (2012) 351–357
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Acknowledgment
This work was supported by the Ministry of Education and
Science of Serbia [Project No. 172061].
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