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

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

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

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

(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,

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 LC₅₀and LC₉₀values (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

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 ⁴unsaturation (9), the presence of a C-4 epoxide (6) or the loss

of the ¹unsaturation (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

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^-1equipped with a deuterated triglycine

sulphate detector, a Nichrome source and KBr beam splitter, with a total of 100 scans with 4

cm⁻¹resolution.

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,

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).

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 R_fvalues 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

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 N₂for 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

MgSO₄and 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 R_fvalues 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

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

% Na₂CO₃aqueous 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 R_fvalues 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

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^-12964 (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 (-CH₃deformation), 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 (CH₂out-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

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. EC₅₀values (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 (LC₅₀and 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.

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.

[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.

[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´, 4^thedn. 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.

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-Octanol^a)

991

988

0.2 ± 0.1

0.9 ± 0.2

0.3 ± 0.1

-

Limonene^a)

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.

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 EC₅₀^b)).

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

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) EC₅₀and 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% CL^c)

LC₉₀[mg/ml]^b)

(95% CL^c)

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

5.82±1.23

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.

2.30±2.00

4.60±0.84

n.c.

7

8

13.70±2.25

100.00±0.00

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 LC₅₀and

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/ LC₅₀values 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

+

-

3

2

⁹

epox

¹

H

C=O

1.15 >100

1

⁹

H

⁴

C=O

>50 13.9 0.23

¹

C=O

H₂

H₃

0.1x10^-41.45 >50 0.15

4

¹

C=O epox

0.13

>50

>1

6

¹

C=O

H

0.9x10^-1>50 24.5

9

epox C=O

0.2x10^-30.18 24.0 0.12

10

5

epox C=O

0.7x10^-25.0

8.6

0.04 +++

>1

⁴

7

⁴

H

C=O

1.01

3.98

0.25 >50

3.35 >50

C=O epox

>1

-

8

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.

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.

Article Doi

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

DOI: 10.1002/cbdv.201600270

Source and publish data:

Authors:

Article abstract of DOI:10.1002/cbdv.201600270

Full text of DOI:10.1002/cbdv.201600270

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