Biocidal Compounds from Mentha sp. Essential Oils and Their Structure–Activity Relationships

Article abstract of DOI:10.1002/cbdv.201600270

Essential oils from Greek Mentha species showed different chemical compositions for two populations of M.?pulegium, characterized by piperitone and pulegone. Mentha spicata essential oil was characterized by endocyclic piperitenone epoxide, piperitone epo

Full text of DOI:10.1002/cbdv.201600270

Received Date: 28-Jul-2016
Revised Date: 18-Oct-2016
Accepted Date: 19-Oct-2016
Article Type: Full Paper
Biocidal compounds from Mentha sp essential oils and their structure-activity
relationships
Omar Santana-Méridas a), Azucena González-Coloma *b), Maria Fe Andrés b), Veroniki P.
Vidali c), Moschos G. Polissiou d), and Athanasios C. Kimbaris e)
a) Parque Científico y Tecnológico de Castilla-La Mancha, Paseo de la Innovación 1, 02006,
Albacete, Spain
b) Instituto de Ciencias Agrarias, CSIC, Serrano 115, 28006 Madrid, Spain (phone: +34 91
745 25 00; e-mail: azu@ica.csic.es)
c) Laboratory of Synthesis of Natural Products and Bioorganic Chemistry, Institute of
Nanoscience and Nanotechnology, NCSR “Demokritos”, P.O. Box 60228, GR-153 10 Ag.
Paraskevi, Athens, Greece
d) Laboratory of Chemistry, Department of Food Science & Human Nutrition, Agricultural
University of Athens, Iera Odos 75, 11855, Athens, Greece
e) Laboratory of Chemistry & Biochemistry Department of Agricultural Development
Democritus University of Thrace 193, Pantazidou str 68200, N. Orestiada, Greece
Essential oils from Greek Mentha species showed different chemical compositions for two
populations of M. pulegium, characterized by piperitone and pulegone. M. spicata essential
oil was characterized by endocyclic piperitenone epoxide, piperitone epoxide, and carvone.
This article has been accepted for publication and undergone full peer review but has
not been through the copyediting, typesetting, pagination and proofreading process,
which may lead to differences between this version and the Version of Record. Please
cite this article as doi: 10.1002/cbdv.201600270
This article is protected by copyright. All rights reserved.

The bioactivities of these essential oils and their components have been tested against insect
pests (Leptinotarsa decemlineata, Spodoptera littoralis and Myzus persicae), root-knot
nematodes (Meloydogine javanica) and plants (Lactuca sativa, Lolium perenne, Solanum
lycopersicum). The structure-activity relationships of these compounds has been studied
including semi-synthetic endocyclic trans-carvone epoxide, exocyclic carvone epoxide, a new
exocyclic piperitenone epoxide and trans pulegone epoxide. L. decemlineata feeding was
affected by piperitenone and piperitone epoxide. S. littoralis was affected by piperitone
epoxide and pulegone. The strongest nematicidal agent was piperitenone epoxide, followed
by piperitone epoxide, piperitenone and carvone. Germination of S. lycopersicum and L.
perenne was significantly affected by piperitenone epoxide. This compound and carvone
epoxide inhibited L. perenne root and leaf growth. Piperitenone epoxide also inhibited the
root growth of S. lycopersicum. The presence of a C-1 epoxide resulted in strong antifeedant,
nematicidal and and phytotoxic compounds regardless of the C-4 substituent. New natural
crop protectants could be developed through appropriate structural modifications in the p-
menthane skeleton.
Keywords. - p-menthane monoterpene, epoxide, antifeedant, nematicidal, SAR.
Introduction
Plant essential oils (EOs) are complex mixtures of monoterpenes, sesquiterpenes and aromatic
compounds. EOs have antimicrobial, nematicidal, anti-parasite, insecticidal, medicinal and
cosmetic applications [1]. EOs can play an important role in crop protection and have been
proposed as an environmentally friendly alternative to synthetic pesticides [2-4].
The genus Mentha (mint) has been known since ancient times for its medicinal and
aroma-therapeutic properties. It is one of the important members of the Lamiaceae family
represented by about 19 species and 13 natural hybrids [5]. Monoterpenes of the p-menthane
group are the main constituents of Mentha spp. essential oils and have attracted interest for
This article is protected by copyright. All rights reserved.

their chemistry and biological properties [6]. In this connection, the current methods for the
commercial production of specific-high-value p-menthane monoterpenes are chemical
synthesis or isolation from natural sources [7].
The essential oil of M. pulegium (pennyroyal oil) contains between 25 and 90 %
pulegone [8-9]. The main component of M. spicata (spearmint) essential oil, widely used as
spice, is (R)-carvone and can account up to 65 % of its essential oil composition [10].
Furthermore, EOs from M. pulegium rich in pulegone and M. spicata rich in carvone showed
significant insect antifeedant (M. pulegium) and nematicidal (M. spicata) effects [11].
Chemical variations have been reported for Greek populations of M. pulegium containing
high percentages of piperitone and piperitenone [12]. Additionally, four chemotypes have
been described for Greek populations of M. spicata, being the piperitone epoxide/piperitenone
epoxide type the most common one [13].
Here we report on the chemical composition of EOs from Greek M. pulegium (two
chemotypes) and M. spicata. Additionally, we tested their insect (Leptinotarsa decemlineata
Say, Spodoptera littoralis Boisd. and Myzus persicae Sulzer) antifeedant effects, their
nematicidal activity against root-knot nematode (Meloydogine javanica (Treub) Chitwood)
and their phytotoxic activity (Lactuca sativa L., Lolium perenne L. and Solanum
lycopersicum Mill). The bioactivities of the major components of the essential oils have also
been tested and their structure-activity relationships studied by comparison with semi-
synthetic epoxy-derivatives including endocyclictrans-carvone epoxide (2), exocyclic
carvone epoxide (3), the new exocyclic piperitenone epoxide (6) and trans pulegone epoxide
(8).
Results and Discussion
Yields and chemical composition of the oils are shown in Table 1. The two different
populations of M. pulegium showed different chemical profiles for their EOs. EOPul1 was
characterized by piperitone (96.5 %) and the minor presence of limonene (0.9 %), 3-octanol
This article is protected by copyright. All rights reserved.

(0.2 %) and menthone (0.1 %). In EOPul2 pulegone was the most abundant component (74.4
%), followed by isomenthone (22.2 %), piperitenone (1.2 %), menthone (0.6 %), piperitone
(0.5 %) and 3-octanol (0.3 %). The major components of wild M. spicata EO (EOSP) were
piperitenone epoxide (52.5 %) and piperitone epoxide (24.7 %) followed by carvone (14.0%),
limonene (3.9 %) menthone (1.5%) and linalool (1.2 %) among other minor compounds.
M. pulegium has been reported as being a chemical polymorph species, both in
qualitative and/or quantitative composition. A correlation has been established between oil
composition and origin for 38 Greek populations of M. pulegium [8]. M. spicata plants with
essential oils rich in piperitenone and piperitone epoxides are the most common chemotype
found in Greek wild populations, as shown here.
Table 2 shows the insect antifeedant effects of the essential oils, their major
components and derivatives. All the essential oils tested were effective antifeedants to L.
decemlineata and M. persicae followed by S. littoralis. Similar antifeedant effects have been
reported for M. pulegium (pulegone chemotype) and M. spicata (carvone chemotype) EOs
from Morocco against S. littoralis and M. persicae [11].
Compounds 1-10 exhibited species-related antifeedant effects (Table 2). Overall, L.
decemlineata was the most sensitive to all these compounds, followed by S. littoralis and M.
persicae (Table 2). L. decemlineata feeding was strongly affected by piperitenone (4) and
piperitone epoxide (10), followed by compounds 3, 5, 9, 2, 1, 7 and 8. S. littoralis was
affected by piperitone epoxide (10) and R-pulegone (7), followed by 2, 5 and 8. The lowest
antifeedant activity was observed against M. persicae, being 3 and 5 the most active
compounds (Table 2). The insecticidal properties of pulegone 7 have been studied, including
antifeedant activity against S. littoralis, M. persicae [11][14] among other insect species [15],
toxicity [6][16-17], insect growth inhibition and related effects [18]. Pulegone epoxide,
This article is protected by copyright. All rights reserved.

carvone, carvone epoxide, piperitenone oxide and piperitone have been reported as a strong
antifeedants, toxic, and repellent against chewing and sucking insects [19-22].
The three Mentha spp. oils induced high mortality of infective juveniles (J2) of M.
javanica (Table 3). Compounds 1-10 exhibited structure-dependent nematicidal effects. The
most active compound was piperitenone epoxide (5) with similar LC50 and LC90 values (0.04
and 0.05 mg/ml respectively), followed by piperitone epoxide (10), piperitenone (4) and R-(-
)-carvone (1). M. pulegium EO has been reported as very active against M. incognita [23-24].
On the contrary, an M. pulegium EO from Morocco, rich in pulegone (similar to EOpul2 but
without iso-menthone), resulted inactive against M. javanica [11]. None of the major
components of the M. pulegium EOs (piperitone and pulegone) tested here showed
nematicidal effects against M. javanica, indicating that the presence of minor components can
modulate the activity of the EO as suggested [2]. On the other hand, high in vitro activity has
been reported for M. spicata oil, rich in carvone, against M. javanica [2][11][25] and M.
incognita [9]. Similarly, carvone has been reported as having in vitro nematicidal effect
against M. javanica [25].
The phytotoxic effects of essential oils and pure compounds were tested on
monocotyledonous and dicotyledoneous plant species (Fig. 2). At the end of the experiment
(168 h), germination of S. lycopersicum was significantly affected by M. pulegium EO and
compound 5 (38.5% and 53.8% respect to the control respectively), while L. perenne
germination was also affected by 5 (37.5% respect to the control). M. spicata EO, its main
component 5 and compound 3 showed the strongest inhibition of L. perenne root and leaf
growth. Compound 5 also inhibited the root growth of S. lycopersicum and stimulated L.
sativa root growth.
Essential oils from Mentha spp. have been previously described for their ability to
affect the plant germination and/or the development of roots and hypocotyls [11] [26-27]. A
wide range of volatile p-menthane monoterpenes released by these species are able to inhibit
This article is protected by copyright. All rights reserved.

both germination and plant growth of many species and could be considered for its potential
use as commercial natural herbicides [6] [28-30]. Overall, phytotoxic activity is often
attributed to synergistic effects of all components of the essential oil through different
processes including enzyme inhibition, chlorophyll degradation, electrolyte leakage and
membrane damage [26].
The structure-activity relationships (SARs) of compounds 1-10 are summarized in
Table 4. Among the compounds with a C-6 ketone (1-3), the C-1 / C-9 substituents
determined their effects. A C-9 epoxide resulted in a strong L. decemlineata antifeedant effect
in the presence of 1 (3), followed by 9 / C-1 epoxide (2) or 1 (1), which also showed
nematicidal effect. Among the compounds with a C-3 ketone (4-10), the C-1 / C-9
substituents determined their effects. The presence of a 4 (4) resulted in the strongest L.
decemlineata antifeedant with nematicidal effect. The presence of a C-1 epoxide resulted in
strong antifeedant, nematicidal (5 and 10) and phytotoxic (5) compounds regardless of the C-
4 substituent. The loss of the 4 unsaturation (9), the presence of a C-4 epoxide (6) or the loss
of the 1 unsaturation (7 and 8) decreased these activities.
The α, β - unsaturated ketones (carvone, pulegone, piperitone, piperitenone) are
generally more active than other ketones, suggesting that this chemical functionality can play
an important role in their biocidal properties [31]. Enantioselectivity, occurrence of
stereoisomers, the degree of saturation in the functional group of the side chain, variations in
the position of the conjugated C-C double bond and/or it replacement by an epoxide group
alters the biological activity of these compounds and play an important role for its insecticidal
and phytotoxic properties [6] [19] [21] [32] as we have shown here. In this connection, new
natural crop protectants could be developed through appropriate structural modifications in
the p-menthane skeleton. However, risk assessment must be taken into account since
This article is protected by copyright. All rights reserved.

pulegone can be oxidized by cytochrome P450 to reactive and toxic metabolites such as
menthofuran [33].
Acknowledgements
This work has been partially supported by grant CTQ2012-38219-C03-01 and CTQ2015-
64049-C3-1-R, (MINECO, Spain). O. Santana gratefully acknowledges the European Social
Fund and Fundación Parque Científico y Tecnológico de Albacete (PCYTA, Spain).
Supporting Information
Additional Supporting Information may be found in the online version of this article: Specific
spectroscopic data for synthesized and isolated compounds are available as Supporting
Information.
Experimental Part
Instrumentation
NMR spectra were recorded on a Brucker AMX−500 instrument, at 25 °C. The optical
rotation was measured on a Perkin-Elmer polarimeter. Melting point was measured on a
GALLENKAMP melting point apparatus. FT-IR spectra was recorded on a Nicolet 6700 FT-
IR spectrometer operating in the region 4000 – 400 cm-1 equipped with a deuterated triglycine
sulphate detector, a Nichrome source and KBr beam splitter, with a total of 100 scans with 4
cm−1 resolution.
Plant Material
Two different species of the genus Mentha were used: M. pulegium (pennyroyal) and M.
spicata (spearmint). Flowering plant material was collected from various natural habitats in
Greece between July and August 2013. Two populations of M. pulegium were collected from
Orestiada (North-East of Greece; Latitude: 41°24'05.5"N, Longitude: 26°33'13.7"E and
named as Pul1) and Samothraki Island (North-East of Greece, Latitude: 40°29'47.7"N,
This article is protected by copyright. All rights reserved.

Longitude: 25°36'30.3"E and named Pul2). M. spicata (SP) was collected from Sparti (South
of Greece, Peloponisos, Latitude: 37°01'38.7"N; Longitude: 22°25'59.3"E). All plant
materials were air dried and stored for further use. Voucher specimens were deposited in the
Herbarium of the Agricultural University of Athens with numbers 1436 (M. spicata-SP), 1437
(M. pulegium-Pul2) and 1438 (M. pulegium-Pul1).
Chemicals
All reagents used were of analytical grade. R-(+)-pulegone, 3-octanol, R-(-)-carvone,
terpinen-4-ol, mesityl oxide, methyl vinyl ketone, (-)-menthone, hydrogen peroxide solution
(30 %) and m-chloroperbenzoic acid were purchased from Aldrich (Steinheim, Germany). S-
(-)-limonene, thymol and β-caryophyllene were purchased from Sigma (St. Louis, USA).
Piperitone and iso-menthone were purchased from Extra Synthese (Genay, France). Diethyl
ether (BHT free) was purchased from SDS (Cedex, France). Pentane, methanol, chloroform
and ethyl acetate was bought from Lab-Scan (Dublin, Ireland). Potassium and sodium
hydroxide, tetrahydrofuran (THF), silica gel 60A (35-70 mesh) and TLC plates (silica gel 60,
F254) were purchased from Merck (Darmstadt, Germany).
Extraction of the essential oils
Aerial parts (leaves and flowers) from Pul1, Pul2 and SP were powdered in an electrical
blender and 200 g of each plant sample placed into a 2 L round bottomed flask with 1 L of
deionized distilled water and submitted to hydrodistillation for 3 h using a Clevenger
apparatus. The obtained essential oils, named as EOpul1, EOpul2 and EOsp respectively,
were dried over anhydrous magnesium sulfate, filtrated and stored in labeled sterile screw
capped bottles at -22 oC until use. The yield of each essential oil was determined on average
of three replicates and expressed as ml of essential oil/100 g of dry material (Table 1).
This article is protected by copyright. All rights reserved.

Essential oils and pure compounds analysis by gas chromatography
Essential oils and pure compounds were analyzed using a Hewlett Packard II 5890 gas
chromatography (GC) system, equipped with a FID detector and HP-5ms capillary column
(30 m x 0.25 mm, film thickness 0.25 μm). Injector and detector temperatures were set at 220
oC and 290 oC, respectively. GC oven temperature was programmed from 60 oC to 240 oC at a
rate of 3 oC /min and held isothermally for 10 min. Helium was the carrier gas at a flow rate
of 1 ml/min. Diluted samples (1/100 in diethyl ether, mgl-1) of 1.0 μl were injected manually
and in the splitless mode. Quantitative data were obtained electronically from FID area
percent data without the use of correction factors. Qualitative analysis of the essential oils was
performed using the same conditions with GC, in a Hewlett Packard II 5890 gas
chromatograph equipped with Hewlett Packard II 5972 mass selective detector in the electron
impact mode (70 eV). The retention indices (RI) of compounds were determined relative to
the retention times of n-alkanes (C8-C20) with linear interpolation, using the Van Den Dool
and Kratz equation [34]. Identification of the major essential oil components was based on the
comparison of their retention indices with those of authentic compounds by coelution and MS
analysis. For the other components, tentative identification involved matching retention
indices and recorded mass spectra with those obtained from NIST 98 and Wiley 275 libraries
as well as from Adams CD computer library [35].
Isolation of piperitenone and piperitone epoxides
Four (4) g of M. spicata EO (EOSP) were fractionated by column chromatography on silica
gel and eluted with a pentane/diethyl ether gradient (100-80 % pentane) of increasing
polarity. Column fractions were monitored by thin layer chromatography (TLC) with
pentane/ethyl acetate (90/10). Fractions with similar Rf values on the TLC plates were pooled.
Spots were detected under UV lamp and afterwards by spraying with a mixture of 1% vanillin
and 5% sulfuric acid solution (in ethanol) and heating at 120 oC on a hot plate. The chosen
fractions merged thus affording two sub-fractions while elution solvents removed in vacuo by
a rotary flash evaporator. The distillation was stopped when the volume of solvents was
This article is protected by copyright. All rights reserved.

reduced to ~10 ml and completed by flushing through nitrogen thus resulting in two yellowish
oils identified as endocyclic (+)-piperitenone epoxide (5) (1.7 g, mixture of cis and trans
diastereoisomers) and piperitone epoxide (10) (0.5 g). Both compounds were found pure (>
98.5 %) according to GC-MS analysis. Structural determination was carried out by GC-MS,
1H and 13C NMR analysis and the results (included as supporting information) were in
agreement with previous reports [36-37].
Synthetic procedures
Synthesis of piperitenone.
Piperitenone (4) was prepared as previously reported [38]. Purity of the isolated product
(97.3%) was estimated according to GC-MS analysis. Its structural characterization (included
as supplementary information) was carried out by mass spectral analysis and NMR
experiments and the results were in agreement with previous reports [35] [39].
Endocyclic alkaline epoxidation
The synthesis was based on the method described by Nilsson et al. [40]. A solution of
hydroxide in methanol (2.0 M, 8.0 mL) was added slowly to a stirred solution of R-(-)-
carvone (2.3 g, 15 mmol) and hydrogen peroxide (35 %, 2.0 mL). The mixture was stirred at
room temperature under N2 for 1.5 h and then it was extracted with diethyl ether (3 x 50 mL).
The combined organic phases were washed with water until neutral, dried with anhydrous
MgSO4 and concentrated under vacuum. The crude product subjected to flash
chromatography on a silica gel 60A (35-70 mesh) column with a pentane/diethyl ether
gradient (100-80 % pentane). Fractions with similar Rf values on the TLC plates were pooled.
Spots detected under UV lamp and afterwards by spraying with a mixture of 1% vanillin and
5% sulfuric acid solution (in ethanol) and heating at 120 oC on a hot plate. The chosen
fractions were merged in one and elution solvents were removed in vacuo in a rotary flash
evaporator. The condensation was stopped when the volume of solvents was reduced to ~10
This article is protected by copyright. All rights reserved.

ml and completed by flushing through nitrogen resulting in pure endocyclic trans-carvone
epoxide (2) (1.8 g, 71%) as a colorless liquid.
Exocyclic epoxidations
The synthesis was based on the method described by von Poser et al. [41]. A starting material
[R-(-)-carvone or piperitenone or R-(+)-pulegone] (19.7 mmol) was added to a chloroform
solution of m-chloroperoxybenzoic acid (12 g in 150 mL) and the reaction mixture was left
for 24 h at room temperature with occasional stirring. This reaction mixture washed with 10
% Na2CO3 aqueous solution followed by water until neutral, dried over anhydrous MgSO4
and finally the solvent was removed under reduced pressure. The crude products, resulting
from the three different starting materials, were subjected to flash chromatography on a silica
gel 60A (35-70 mesh) column with a pentane/ethyl acetate gradient (100-85 % pentane).
Fractions with similar Rf values on the TLC plates were pooled. Spots were detected under
UV lamp and afterwards by spraying with a mixture of 1% vanillin and 5% sulfuric acid
solution (in ethanol) and heating at 120 oC on a hot plate. The chosen fractions merged to one
and a rotary flash evaporator used to remove elution solvents. The condensation was stopped
when the volume of solvents was reduced to ~10 ml and completed by flushing through
nitrogen resulting in pure exocyclic carvone epoxide (3) (2.2 g, 67%) as a yellowish liquid,
2,2,6-trimethyl-1-oxaspiro[2.5]oct-5-en-4-one (6) (1.7 g, 52%) as a yellowish solid and trans-
pulegone epoxide (8) (1.1 g, 33%) as a white solid, respectively. In the case of R-(+)-
pulegone the epoxidation procedure resulted in a mixture of the two diastereoisomeric
pulegone oxides (cis and trans), as expected according to literature data [23]. Nevertheless,
our effort to isolate pure both of isomers accomplished only in the case of the trans
diastereoisomer (8). Products purity (> 97.5 %) was confirmed by GC-MS analysis.
Isolated and synthesized compounds were analyzed by GC-MS, 1H and 13C NMR and
their spectral data were validated by previously reported data 2 [35][40], 3 [42], 5 [35-36], 8
[43] and 10 [10][37], with the exception of compound 6 for which no available data were
This article is protected by copyright. All rights reserved.

found in the literature. Data for compound 6 are provided here while for known compounds 2,
3, 5, 8 and 10 are given in the supplementary information of this paper. To the best of our
knowledge, this is the first report on the synthesis of the 2,2,6-trimethyl-1-oxaspiro[2.5]oct-5-
en-4-one (exocyclic- piperitenone epoxide).
2,2,6-Trimethyl-1-oxaspiro[2.5]oct-5-en-4-one (6): M.p.: 46 oC. FT-IR (KBr disc)
cm-1 2964 (asymetric strech C-H), 2883 (symetric strech C-H), 1672 (C=O strech), 1627
(C=C strech), 1467 (asymmetric bending –CH3), 1426 (=CH bending in plane), 1377
(symmetric bending –CH3), 1288 (epoxide C-O strech), 1206 (skeletal of C(CH3)2), 1185
(skeletal of C(CH3)2), 1146 (-CH3 deformation), 1113 (C-O stretching), 1079 (C-O-C
stretching), 1046 (C-C olefinic of –OC(CH3)2), 991 (=CH out of plane), 945 (asymmetric
stretching of ring), 891 (epoxide ring), 875 (CH2 out-of-plane deformation), 827 (C(CH3)2
rocking), 733 (-CH2-deformation). 1H NMR (500 MHz, CDCl3):  = 1.21 (brs, 3H; CH3-9),
1.43 (brs, 3H; CH3-8), 1.92 (m, 1H; H-5b), 1.95 (brs, 3H; CH3-10), 2.38 (m, 2H; H-6b, H-5a),
2.53 (m, 1H; H-6a), 5.9 (brs, 1H; H-2) ppm.; 13C NMR (125 MHz):  = 19.1, 20.3, 24.0, 27.7,
31.0, 63.6, 67.1, 127.6, 161.9, 194.8 ppm; EI-MS (70 eV), m/z (%): 45 (1); 51 (39), 53 (75);
65 (33), 67 (70); 77 (47), 79 (100); 95 (52), 97 (65); 109 (31), 111 (43); 123 (44), 126 (39);
133 (1), 135 (1); 151 (37), 152 (3); 166 (70), 167 (9).
Insect antifeedant assays
Spodoptera littoralis, Leptinotarsa decemlineata and Myzus persicae insect colonies were
reared on artificial diet and their respective host plants (Solanum tuberosum and Capsicum
annum) and maintained at 22 ± 1◦C, >70% relative humidity, with a photoperiod of 16:8 h
(L:D) in a growth chamber. The bioassays were conducted with newly emerged S. littoralis
sixth-instar larvae, L. decemlineata adults, and M. persicae apterous adults. Feeding or
settling inhibition (%FI or %SI) were calculated as FI= [1-(T/C)] x 100, where T and C are
the consumption of treated and control leaf disks, respectively, or as %SI= [1-(%T/%C)] x
This article is protected by copyright. All rights reserved.

100 where %C and %T are percent aphids settled on control and treated leaf disks,
respectively, as previously described [44]. The antifeedant effects (FI/SI) were analyzed for
significance by the non-parametric Wilcoxon signed-rank test. EC50 values (effective dose to
obtain 50% feeding inhibition) were determined for pure compounds with FI/SI values > 75%
from linear regression analysis (%FI/SI on log dose).
Nematidal activity
Meloydogine javanica population maintained on Solanum lycopersicum plants (var.
Marmande) in pot cultures at 25±1°C and at >70% relative humidity was used. Second-stage
juveniles (J2) hatched within a 24 h period from egg masses handpicked from infected tomato
roots were used. The experiments were carried out in 96-well microplates (Becton,
Dickinson) as described [2]. The organic extracts and pure compounds were tested at initial
concentrations of 1.0 and 0.5 mg/mL (final concentration in the well) and diluted serially if
needed. The number of dead juveniles was recorded after 72 h. All treatments were replicated
four times. The data were determined as percent mortality corrected according to Scheider-
Orelli’s formula. Effective lethal doses (LC50 and LC90) were calculated for the active pure
compounds by Probit analysis (five serial dilutions, 0.5-0.01 mg/mL).
Phytotoxic activity
The experiments were conducted with Lactuca sativa, Lolium perenne and Solanum
lycopersicum seeds (100 seeds / test) in 12-well microplates as described previously [45]. The
essential oils and pure compounds were tested at initial concentrations of 0.4 and 0.2 mg/mL
(final concentration in the well) and diluted serially if needed. Germination was monitored for
6 (L. sativa, S. lycopersicum) or 7 days (L. perenne) and the root length measured at the end
of the experiment (25 plants were selected randomly for each experiment, digitalized and
measured with the application Image J, http//rsb.info.nih.gov./ij/). A no parametric analysis of
variance (ANOVA) was performed on radical length data. Juglone (JU) (5 µg/µL) was
included as a positive control.
This article is protected by copyright. All rights reserved.

References
[1] A. González-Coloma, M. Reina, C. E. Diaz, B. M. Fraga, O. Santana-Meridas, `Natural
product-based biopesticides for insect control´. Elsevier Inc, 2013, M. A. Walthman, in:
`Reference Module in Chemistry, Molecular Sciences and Chemical Engineering´, Ed. J.
Reedijk, J., Elsevier, 2013, doi:0.1016/B978-0-12-409547-2.02770-0.
[2] M. F. Andrés, A. González-Coloma, J. Sanz, J. Burillo, P. Sainz, Phytochem. Rev. 2012,
11, 371.
[3] M. Goindarajan, R. Sivakumar, M. Rajeswary, K. Yogalakshmi, Exp. Parasitol. 2013,
134, 7.
[4] M. B. Isman, S. Miresmailli, C. Machial, Phytochem. Rev. 2011, 10, 197.
[5] C. Brickell, J. D. Zuk, The American Horticultural Society: A-Z Encyclopedia of Garden
Plants. DK Publishing, Inc., New York, NY, USA, 1997, p. 668.
[6] N. Sekine, S. Shibutani, J. Wood Sci. 2013, 59, 229.
[7] B. M. Lange, `Biosynthesis and Biotechnology of high-value p-menthane monoterpenes,
including menthol, carvone, and limonene´, Springer Berlin Heidelberg, 2015, in `Advances
in Biochemical Engineering/Biotechnology´. Springer Berlin Heidelberg. 2015, DOI:
10.1007/10_2014_289. 2015.
[8] S. Kokkini, E. Hanlidou, R. Karousou, T. Lanaras, J. Essent. Oil Res. 2004, 16, 588.
[9] P. Caboni, M. Saba, G. Tocco, L. Casu, A. Murgia, A. Maxia, U. Menkissoglu-Spiroudi,
J. Agric. Food Chem. 2014, 61, 9784.
[10] S. Kokkini, R. Karousou, T. Lanaras, T, Biochem. Syst. Ecol. 1995, 23, 425.
[11] O. Santana, M. F. Andrés, J. Sanz, J. N. Ehrramani, L. Abdeslam, A. González-Coloma,
Nat. Prod. Commun. 2014, 9, 1109.
[12] A. Michaelakis, D. Papachristos, A. Kimbaris, M. Polissiou, Hellenic Plant Prot. 2011,
J. 4, 35.
[13] S. Kokkini, D. Vokou, Econ. Bot. 1989, 43, 192.
[14] K. Dancewicz, B. Gabryz, I. Dams, J. Chem. Ecol. 2008, 34, 530.
[15] M. Szczepanik, I. Dams, C. Wawrzenczyk, Entomol. Exp. Appl. 2008, 128, 337.
This article is protected by copyright. All rights reserved.

[16] G. Franzios, M. Mirotsou, E. Hatziapostolou, J. Kral, Z. G. Scouras, P. Mavragani-
Tsipidou, J. Agric. Food Chem. 1997, 45, 2690.
[17] N. Zekri, S. Amalich, A. Boughdad, M. A. Al Belghiti, T. Zair, Mediterr. J. Chem. 2013,
2, 607.
[18] S. J. Yu, Zool. Stud. 2000, 39, 243.
[19] M. Szczepanik, A. Grudniewska, B. Zawitowska, C. Wawrzeńczyk, Pest Manag. Sci.
2014, 70, 953.
[20] A. K. Tripathi, V. Prajapati, A. Ahmad, K. K. Aggarwal, S. P. S. Khanuja, J. Med.
Entomol. 2004, 41, 691.
[21] T. Cardoso-Lima, T. Kayne-Mariano da Silva, F. Lima-Silva, J. M. Barbosa-Filho, M. O.
Mayo-Marques, R. La Corte Santos, S. C. de Holanda Cavalcanti, D. Pergentino de Sousa,
Chemosphere 2014, 104, 37.
[22] R. Pavela, K. Kaffková, K. Kumšta, Plant Protect. Sci. 2014, 50, 36.
[23] R. Pandey, A. Kalra, S. Tandon, N. Mehrotra, H. N. Singh, S. Kumar, J. Phytopathol.
2000, 148, 501.
[24] N. G. Ntalli, S. Vargiu, U. Menkissoglu-Spiroudi, P. Caboni, J. Agric. Food Chem. 2010,
58, 11390.
[25] Y. Oka, S. Nacar, E. Putievsky, U. Ravid, Z. Yaniv, Y. Spiegel, Phytopathology 2000,
90, 710.
[26] E. Rolli, M. Marieschi, S. Maietti, G. Sacchetti, R. Bruni, Ind. Crops Prod. 2014, 60,
280.
[27] M. A. A. Souza, M. J. Lemos, D. M. C. Brito, M. S. Fernandes, R. N. Castro, S. R.
Souza, AJPS 2014, 5, 3311.
[28] L. De Martino, E. Mancini, L. F. R. de Almeida, V. De Feo, Molecules 2010, 15, 6630.
[29] F. Aranitia, A. Lupinia, A. Sorgona`a, G. A. Stattib, M. R. Abenavolia, Nat. Prod. Res.
2013, 27, 1651.
[30] Y. Sunohara, Y. Baba, S. Matsuyama, K. Fujimura, H. Matsumoto, Protoplasma 2015.
doi:10.1007/s00709-014-0739-4.
This article is protected by copyright. All rights reserved.

[31] N. A. Gañán, J. S. Dambolena, R. E. Martini, S. B. Bottini, J. Supercrit. Fluids 2015, 98,
1-11.
[32] S. R. L. Santos, M. A. Melo, A. V. Cardoso, R. L. C. Santos, D. P. de Sousa, S. C. H.
Cavalcanti, Chemosphere 2011, 84, 150.
[33] J. M. Verdes, A. Moraña, V. Dehl, A. Ruiz-Díaz, E. Dellacasa, F. Dutra, in `Poisoning
by plants, mycotoxins, and related toxins´, Ed. F. Riet-Correa, J. Pfister, A. L. Schild, T.
Wierenga, CAB International, 2011, p. 535. doi: 10.1079/9781845938338.0535.
[34] H. van den Dool, P. A. Dec. Kratz, J. Chromatogr. A. 1963, 11, 463.
[35] R. P. Adams, `Identification of Essential Oil components by Gas Chromatography / Mass
Spectroscopy´, 4th edn. Allured Publishing Corporation, Carol Stream, 2007.
[36] S. Ghoulami, A. Il Idrissi, S. Fkih-Tetouani, Tetr. Let. 2001, 42, 2107.
[37] J. A. Elings, H. E. B. Lempers, R. A. Sheldon, Eur. J. Org. Chem. 2000, 1905.
[38] J. J. Beereboom, J. Am. Chem. Soc. 1996, 31, 2026.
[39] N. J. Manenzhe, N. Potgieter, T. van Ree, T, Phytochemistry 2004, 65, 2333.
[40] A. M. Nilsson, M. A. Bergstrom, K. Luthman, J. L. G. Nilsson, A. T. A. Karlberg, Chem.
Res Toxicol. 2005, 18, 308-316.
[41] G. L. von Poser, C. Menut, M. E. Toffoli, P. Verin, M. Sobral, J. M. Bessiere, G.
Lamaty, A. T. Henriques, J. Agric. Food Chem. 1996, 44, 1829.
[42] B. Kang, M. Kim, J. Lee, Y. Do, S. Chang, J. Org. Chem. 2006, 71, 6721.
[43] T. M. Feeley, M. K. Hargreaves, J. Chem. Soc. (C). 1970, 1745.
[44] E. Burgueño-Tapia, L. Castillo, A. González-Coloma, P. Joseph-Nathan, J. Chem. Ecol.
2008, 34, 766.
[45] C. Moiteiro, M. Joao, O. M. Curto, N. Mohamed, M. Bailen, R. Martínez-Díaz, A.
González-Coloma, J. Agric. Food Chem. 2006, 54, 3566.
This article is protected by copyright. All rights reserved.

Tables
Table 1. Chemical composition (abundance: % area) of essential oils isolated from two Mentha
pulegium populations (EOPul1 and EOPul2) and M. spicata (EOSP).
Area [%]
Tentative identification
RI
Pub. RI
EOPul1
EOPul2
EOSP
3-Octanola)
991
988
0.2 ± 0.1
0.9 ± 0.2
0.3 ± 0.1
-
Limonenea)
1029
1102
1151
1167
1185
1195
1241
1244
1254
1256
1296
1344
1375
1426
1435
1024
1095
1148
1158
1174
1191
1233
1239
1249
1250
1289
1340
1366
1417
1430
-
-
3.9 ± 0.3
1.2 ± 0.2
1.5 ± 0.3
-
0.2 ± 0.1
0.4 ± 0.1
-
Linalool a)
Menthone a)
-
0.1 ± 0.0
0.6 ± 0.2
22.2 ± 2.3
iso-Menthone a)
Terpinen-4-ol a)
cis-Dihydro carvone b)
Pulegone a)
-
-
-
-
-
-
74.4 ± 1.5
Carvone a)
-
-
14.0 ± 1.3
-
Piperitone a)
0.5 ± 0.1
96.5 ± 1.2
Piperitone epoxide a)
Thymol a)
-
-
-
-
-
-
-
-
24.7 ± 1.1
0.5 ± 0.1
0.1 ± 0.0
52.5 ± 1.7
0.1 ± 0.0.
0.1 ± 0.0
99.2
Piperitenone a)
Piperitenone oxide a)
β-Caryophyllene a)
β-Copaene b)
1.2 ± 0.3
-
-
-
Total
97.7
2.1 ± 0.1
99.2
2.2 ± 0.2
Οil yield (ml/100 g dry wt) c)
1.9 ± 0.1
a) Comparison with pure standards; b) Tentative identification based on data obtained from NIST 98 and
Wiley 275 library of the GC-MS system and from Adams CD computer library [35]; c) Mean ± SD (n =
3); Components are listed in order of elution from an HP-5 MS column. Retention indices (RI), relative to
n-alkanes, are listed alongside published values (Pub. RI) [34]; Bold values have been used to indicate
major compounds.
This article is protected by copyright. All rights reserved.

Table 2. Insect antifeedant effects of essential oils (expressed as % FI / SI a) ± SE)
from Mentha spp. (M. pulegium, EOPul1, EOPul2; M. spicata, EOSP), isolated compounds and
derivatives (expressed as EC50b)).
L. decemlineata
S. littoralis
M. persicae
Essential oil /
Compound
EOPul1
EOPul2
EOSP
75.3 ± 9.7 c)
84.6 ± 9.4 c)
80.8 ± 4.6 c)
75.1 ± 2.7 c)
51.2 ± 8.8
~ 50
1.15 (0.2, 7.4)
~ 50
1.45 (0.2, 9.9)
5.0 (1.8, 13.5)
> 50
0.25 (0.1, 0.4)
3.35 (2.3, 14.3)
~ 50
89.9 ± 3.3 c)
87.6 ± 5.1 c)
74.0 ± 7.1 c)
13.9 (6.15, 27.1)
> 100
8.8 (3.0, 18.2)
~ 50
8.6 (3.0, 24.5)
~ 50
~ 50
> 50
24.5 (14.5, 40.4)
24.0 (16.4, 33.1)
83.0 ± 4.5 c)
0.39 (0.04, 8.2)
0.25 (0.1, 1.6)
0.004 (0.0, 0.6)
0.00001 (0.0, 0.1)
0.007 (0.0, 0.2)
0.13 (0.01, 1.4)
1.01 (0.1, 7.4)
3.98 (1.4, 11.0)
0.09 (0.02, 0.5)
0.0002 (0.0, 0.1)
1
2
3
4
5
6
7
8
9
0.18 (0.01, 3.0)
10
a)Percent feeding (FI) / setting (SI) inhibition (100 μg/cm2); b) EC50 and 95% confidence limits
(lower, upper), concentration needed to produce 50% feeding / setting inhibition in μg/cm2; c)
p<0.05, Wilcoxon paired rank test; n.t. Not tested.
Table 3. Effects of essential oils (1 µg/µl) from Mentha spp. (M. pulegium, EOPul1, EOPul2; M.
spicata, EOSP), isolated compounds and derivatives (0.5 µg/µl) on mortality of second stage
juveniles (J2) of Meloidogyne javanica
Essential oil
/Compound
EOPul1
LC50 [mg/ml]b)
(95% CLc)
LC90 [mg/ml]b)
(95% CLc)
J2 mortality [%]a)
87.13±2.96
99.60±0.40
0.68 (0.65-0.72)
1,19(1.12-1.27)
EOPul2
0.59 (0.56-0.62)
1.01 (0.95-1.08)
EOSP
99.66±0.34
0.51 (0.49-0.54)
0.23 (0.22-0.24)
0.95 (0.90-1.02)
0.35 (0.33-0.37)
100.00±0.00
1
3.32±1.21
32.40±5.79
100.00±0.00
100.00±0.00
5.82±1.23
n.c.
n.c.
n.c.
n.c.
2
3
4
5
6
0.15 (0.14-0.15)
0.04 (0.04-0.42)
n.c.
0.24 (0.23-0.25)
0.05 (0.05-0.06)
n.c.
This article is protected by copyright. All rights reserved.

2.30±2.00
4.60±0.84
n.c.
n.c.
n.c.
n.c.
7
8
13.70±2.25
100.00±0.00
n.c.
n.c.
9
0.12 (0.12-0.13)
0.21 (0.19-0.22)
10
a) Values are means of four replicates, Corrected according to Scheider-Orelli’s formula; b)
Mortality was observed 72 h after treatment, Five concentrations were used to obtain LC50 and
LC90, and four replicates were used for each treatment; c) CL denotes confidence limit, Values are
means of four replicates; n.c. Not calculated.
Table 4. Structure-activity relationships of compounds 1-10 based on EC50 / LC50 values for insect
antifeedant and nematicidal effects (Ld, Leptinotarsa decemlineata; Sl, Spodoptera littoralis; Mp,
Myzus. persicae; Mj, Meloidogyne javanica). The phytotoxic effects are
expressed qualitatively (effect on one target plant species, +).
Compound
C1
1
C3
H
C4
H
C6
C9
Ld
Sl
Mp
Mj Phyt
C=O epox
0.04
0.25
0.40
>50
8.8
>1
>1
+
-
-
-
-
-
-
3
2
9
epox
1
H
H
C=O
1.15 >100
1
9
H
H
4
C=O
>50 13.9 0.23
1
C=O
H2
H2
H2
H2
H2
H2
H2
H3
H3
H3
H3
H3
H3
H3
0.1x10-4 1.45 >50 0.15
4
1
C=O epox
0.13
>50
>50
>1
>1
6
1
C=O
H
H
0.9x10-1 >50 24.5
9
epox C=O
0.2x10-3 0.18 24.0 0.12
10
5
epox C=O
0.7x10-2 5.0
8.6
0.04 +++
>1
4
7
4
H
H
C=O
1.01
3.98
0.25 >50
3.35 >50
C=O epox
>1
-
8
This article is protected by copyright. All rights reserved.

Legends
Figure 1. Chemical structures of compounds 1-10.
Figure 2. Phytotoxic effects of EOs, isolated compounds and derivatives on Lolium perenne
(A), Latuca sativa, (B), and Solanum lycopersicum (C). Data represented as % of
control±SE; *p<0.05, ANOVA-LSD test).
Fig. 1.
This article is protected by copyright. All rights reserved.

A
*
*
*
*
*
*
*
pul1 pul2 M. s.
1
2
3
4
5
6
7
8
9
10
B
*
pul1 pul2 M. s.
1
2
3
4
5
6
7
8
9
10
C
*
*
*
*
pul1 pul2 M. s.
1
2
3
4
5
6
7
8
9
10
Fig. 2.
This article is protected by copyright. All rights reserved.