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Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Anti-Pseudomonas aeruginosa activity of hemlock
(Conium maculatum, Apiaceae) essential oil
Michela Di Napoli, Mario Varcamonti, Adriana Basile, Maurizio Bruno,
Filippo Maggi & Anna Zanfardino
To cite this article: Michela Di Napoli, Mario Varcamonti, Adriana Basile, Maurizio
Bruno, Filippo Maggi & Anna Zanfardino (2018): Anti-Pseudomonas�aeruginosa activity
of hemlock (Conium�maculatum,�Apiaceae) essential oil, Natural Product Research, DOI:
To link to this article: https://doi.org/10.1080/14786419.2018.1477151
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Published online: 21 May 2018.
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NATURAL PRODUCT RESEARCH, 2018
Anti-Pseudomonas aeruginosa activity of hemlock (Conium
maculatum, Apiaceae) essential oil
MichelaDi Napolia, MarioVarcamontia, AdrianaBasilea, MaurizioBrunob,
FilippoMaggic and AnnaZanfardinoa
aDepartment of Biology, University of Naples “Federico II”, Naples, Italy; bDepartment of Biological, Chemical
and Pharmaceutical Science and Technology (STEBICEF), University of Palermo, Palermo, Italy; cSchool of
Pharmacy, University of Camerino, Camerino, Italy
Conium maculatum is a nitrophilous weed belonging to the Apiaceae
family and occurring in hedgerows, pastures, waste ground, along rivers
and roadsides. Little is known on the chemistry and bioactivity of other
secondary metabolites occurring in the plant. In the present work, we
have analysed the chemical composition and antimicrobial activity
of the essential oils hydrodistilled from leaves and inorescenes of C.
maculatum growing in Sicily, Italy. The composition of essential oils was
achieved by gas chromatography-mass spectrometry (GC-MS) analysis,
whereas the inhibitory eects on the growth of two Gram negative
strains, namely Escherichia coli and Pseudomonas aeruginosa were
assessed by two dierent analysis. The essential oils exhibited dierent
chemical proles (1-butylpiperidine and myrcene in the inorescenes),
(mostly (E)-caryophyllene in the leaves). The latter oil was particularly
active in inhibiting the growth of P. aeruginosa. These results shed light on
the possible application of hemlock essential oils as antimicrobial agents.
© 2018 Informa UK Limited, trading as Taylor & Francis Group
Conium maculatum; essential
oil; chemical composition;
Received 12 March 2018
Accepted13 May 2018
CONTACT Filippo Maggi ﬁlippo.firstname.lastname@example.org; Anna Zanfardino email@example.com
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2018.1477151.
2 M. DI NAPOLI ET AL.
Conium maculatum L., also known as ‘cicuta’, is a nitrophilous weed belonging to the Apiaceae
(Umbelliferae) family and occurring in hedgerows, pastures, waste ground, along rivers and
roadsides (Vetter 2004). The generic name comes from the Greek ‘Konas’, meaning ‘to whirl’
and alluding to the convulsions produced by its consumption, whereas the specic name
derives from the Latin ‘maculatum’ alluding to the purple spots occurring on the stems (Vetter
2004). Hemlock is a biennial or, in favorable conditions, perennial plant with an erect stem,
120–180 cm tall, endowed with irregular purple spots, and alternate, tripinnate leaves.
Flowers are small and white, grouped in umbels with 12–16 rays which appear from June to
September. The fruit is an anise-like schizocarp, formed by two greyish-brown achenes bear-
ing ve ridges (Vetter 2004). The plant emits a mousy odour and has a bitter taste. The
notoriety of hemlock is linked to its historical use as poisoning, having been the cause of
the death of the famous Greek philosopher Socrates in 399 BC (De Boer 1950). Frequent
poisonings are reported in livestock that can be killed by respiratory paralysis (Panter et al.
1985). The hemlock toxicity is given by the presence of piperidine alkaloids (Vetter 2004).
These secondary metabolites, derived through the acetate pathway, act as chemical defense
against herbivore attacks (Panter et al. 1988a). Notably, eight main piperidine alkaloids are
produced by hemlock (Panter et al. 1985), with γ-conicein and coniine as the most abundant
and toxic compounds (Panter et al. 1988a, 1988b, 1988c). The alkaloid content in hemlock
is quite variable, depending on the plant organ, development stage and environmental and
climatic conditions, with the fruits usually being the richest part (López et al. 1999). Piperidine
alkaloids are generally accumulated in the secretory ducts occurring in the various organs
of hemlock along with essential oils (Corsi and Biasci 1998). Whereas many reports have
investigated the biosynthesis and structures of the former, only a few reports referred to the
volatile components of hemlock (Masoudi et al. 2006; Radulovic et al. 2008). Therefore, as a
continuation of our studies on the chemistry and bioactivity of medicinal and aromatic
plants occurring in Sicily (Casiglia et al. 2017; Pavela et al. 2017), here we report the antimi-
crobial properties of the essential oil obtained from inorescences and leaves of C. macula-
tum growing in Sicily against the two Gram negative strains.
2. Results and discussion
2.1. Essential oil analysis
A total of sixty volatile components were identied in the essential oils from inorescences
and leaves of C. maculatum, accounting for 80.7 and 82.2% of the total composition, respec
tively (Table S1). The two oils showed signicant dierences in the chemical prole as
detected by GC-MS (Figure S1). The essential oil from inorescences was characterized by
four chemical classes, namely monoterpene hydrocarbons (32.7%), volatile alkaloids (26.4%),
oxygenated sesquiterpenes (15.9%) and sesquiterpene hydrocarbons (15.2%). The major
components were 1-butylpiperidine (26.4%), myrcene (24.0%), (E)-caryophyllene (8.8%), (Z)-
β-ocimene (7.4%) and germacrene D (5.3%). It is worth to note the presence of some
unknown oxygen-containing sesquiterpenes having pseudo-molecular ion peak at m/z 214
[M + H]+ whose mass fragmentation is depicted in Figure S2. Also, the presence of com-
pounds involved in the attraction of pollinators (Pecetti and Tava 2000), such as the esters
octyl hexanoate (1.2%) and benzyl hexanoate (0.7%) was found in this essential oil and
NATURAL PRODUCT RESEARCH 3
conrmed what reported in a previous report (Radulovic et al. 2008). Finally, several alkanes
deriving from the hydrolysis of waxy coating of owers (Afshar et al. 2015) were found, with
n-tricosane (0.9%) and n-pentacosane (0.5%) as the most abundant ones. Interestingly, we
detected for the rst time the massive presence of a volatile alkaloid, i.e. 1-butylpiperidine
(Figure S3), a saturated piperidine alkaloid (Vetter 2004). On the other hand, the poisonous
coniine was not found, probably because it was linked to cations inside the vacuole of plant
cells, hence non-volatile under distillation. We assume that this volatile alkaloid may allow
plant to defend itself from herbivore feeding (Birkett et al. 2004). The essential oil from
hemlock leaves was dominated by sesquiterpenes hydrocarbons (64.2%) with oxygenated
sesquiterpenes (8.9%), monoterpene hydrocarbons (6.5%) and esters (4.7%), By far, the major
compound was (E)-caryophyllene (54.8%) (Figure S3) with minor contributions of germac-
rene D (4.2%), α-humulene (3.6%), myrcene (3.1%) and (Z)-β-ocimene (2.2%). Surprisingly,
here the alkaloid 1-butylpiperidine was detected in scant levels (0.1%) supporting its involve-
ment in the ower’s function. Also in this case we detected appreciable amounts (15.3%) of
two unknown compounds having pseudo-molecular ion peak at m/z 214 [M + H]+ (Figure
S2). Finally, we also found detectable amounts of esters, such as octyl hexanoate (1.5%) and
phenyl ethyl octanoate (1.2%), as well as alkanes like n-nonacosane (0.8%), n-pentacosane
(0.8%) and n-heptacosane (0.6%). Two reports described the chemical composition of hem-
lock essential oils from spontaneous plants growing in Serbia and Iran (Masoudi et al. 2006;
Radulovic et al. 2008). In both cases, the chemical proles reported were quite dierent from
those found in Sicilian hemlock. In the essential oil obtained from the Serbian population
the major volatile compounds were germacrene D (27.2%), (Z)-β-ocimene (14.3%), myrcene
(9.3%), (E)-β-ocimene (7.7%) and (E)-nerolidol (7.1%) in inorescences, and germacrene D
(41.0%), (E)-β-ocimene (22.3%) and (Z)-β-ocimene (7.1%) in leaves (Radulovic et al. 2008).
On the other hand, the essential oil obtained from the aerial parts of Iranian hemlock showed
germacrene D (46.1%), (E)-caryophyllene (15.3%) and (E,E)-α-farnesene (10.1%) as the major
compounds (Masoudi et al. 2006). Therefore, the present work highlighted the presence of
new essential oil chemotypes for hemlock, characterized by 1-butylpiperidine in inores-
cences and (E)-caryophyllene in leaves. Notably, to the best of our knowledge, the alkaloid
1-butylpiperidine has never reported in C. maculatum so far. We assume that the geograph-
ical isolation of the Sicilian population with respect to the continental ones gave rise to its
2.2. Eect of C. maculatum essential oils on bacterial survival
The essential oils extracted from C. maculatum leaves and inorescences were tested against
two Gram negative strains: Escherichia coli DH5α and Pseudomonas aeruginosa PAOI which
represent a model strain and an opportunistic pathogen for plants, respectively. As shown
in Figure S4 A, the oil extracted from leaves was able to inhibit the P. aeruginosa growth,
forming an inhibition halo (about 250 AU/mL) almost comparable to the antibiotic colistin,
a polymyxin that acts on the bacterial membrane of Gram negative microorganisms used
as positive control in the experiment. Instead the other Gram negative indicator strain used
in this study, E. coli DH5α, was resistant to the treatment. The essential oil extracted from the
hemlock inorescences does not seem to have an equivalent antimicrobial activity. In Figure
S4 B is reported a quantitative analysis of inhibition halos. A more sensitive method to cal-
culate the eciency of antimicrobial activity is shown in Figure S5, where we demonstrate
4 M. DI NAPOLI ET AL.
that the activity of C. maculatum leaves extract is able to kill about seventy percent of P.
aeruginosa PAOI treated cells. In previous studies the antimicrobial activity of the major
volatile compound present in hemlock leaves, the (E)-caryophyllene, was demonstrated to
be directed against phytopathogens strains such as Pseudomonas syringae (Huang et al.
2012). In addition, caryophyllene rich essential oils are able to block or reduce the deterio-
ration of certain foodstus contaminated by Pseudomonas uorescens (Myszka et al. 2017).
Caryophyllene is able to enter inside the lipid bilayer of bacterial membrane causing loss of
its uidity and permeability, inhibition of respiration and alteration of transport processes
(Sikkema et al. 1994). Antimicrobial activities belonging to inorescences of hemlock were
not previously observed. Taking together results from this manuscript and from literature,
we can speculate that the antimicrobial molecules are produced as defense strategy in leaves
but not in the inorescences where attractor compounds for pollinators are instead
In this paper we demonstrate that C. maculatum essential oil is eective in preventing or
killing P. aeruginosa cells. Important future goals include the identication of antimicrobial
molecular mechanisms by which this oil counteracts bacterial growth. Thus, results of this
work give new insights into the potential application of hemlock essential oils as antimicro-
bial agent to combat pathogens dicult to eradicate and for the discovery of alternatives
All authors planned and carried out the experimental work, discussed the results and com-
mented on the manuscript.
No potential conict of interest was reported by the authors.
This work was supported by grant from MIUR-ITALY PRIN2015 ‘Top-down and Bottom-up approach in
the development of new bioactive chemical entities inspired on natural products scaolds’ (Project
Filippo Maggi http://orcid.org/0000-0003-1375-4744
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