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Individual and joint activity ofterpenoids, isolated from Calaminthanepeta extract, on Arabidopsis thalianaFabrizio Araniti a , Elisa Graña b , Manuel J. Reigosa b , Adela M.Sánchez-Moreiras b & Maria Rosa Abenavoli aa Dipartimento di Agraria , Università Mediterranea di ReggioCalabria – Salita Melissari , I-89124 , Reggio Calabria , RC , Italyb Department of Plant Biology and Soil Science , University ofVigo , Campus Lagoas-Marcosende s/n, E-36310 , Vigo , SpainPublished online: 25 Aug 2013.

To cite this article: Natural Product Research (2013): Individual and joint activity of terpenoids,isolated from Calamintha nepeta extract, on Arabidopsis thaliana , Natural Product Research:Formerly Natural Product Letters, DOI: 10.1080/14786419.2013.827193

To link to this article: http://dx.doi.org/10.1080/14786419.2013.827193

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Individual and joint activity of terpenoids, isolated from Calamintha nepetaextract, on Arabidopsis thaliana

Fabrizio Aranitia, Elisa Granab, Manuel J. Reigosab, Adela M. Sanchez-Moreirasb and

Maria Rosa Abenavolia*

aDipartimento di Agraria, Universita Mediterranea di Reggio Calabria – Salita Melissari, I-89124 ReggioCalabria, RC, Italy; bDepartment of Plant Biology and Soil Science, University of Vigo, CampusLagoas-Marcosende s/n, E-36310 Vigo, Spain

(Received 11 April 2013; final version received 12 June 2013)

Four terpenoids, camphor, pulegone, trans-caryophyllene and farnesene, previouslyfound in Calamintha nepeta (L.) Savi methanolic extract and essential oils wereassayed on germination and root growth of Arabidopsis thaliana (L.) Heynh. None ofthe terpenes, singularly or in combination, was able to inhibit the germination process.Farnesene and trans-caryophyllene caused a strong inhibitory effect on root growth,and pulegone, at the highest concentrations, reduced lateral root formation. Althoughthe mixture of camphor– trans-caryophyllene with or without farnesene did not causeany effect on root growth, the addition of pulegone induced a marked synergisticactivity. Moreover, the addition, at low concentration, of farnesene to pulegone–camphor– trans-caryophyllene mixture further increased the inhibitory effect on rootelongation. These results suggested that the inhibitory effects caused by C. nepetamethanolic extract may depend on the combined action of different molecules.

Keywords: Arabidopsis thaliana; Calamintha nepeta; germination; phytotoxicity; rootgrowth; synergism

1. Introduction

Calamintha nepeta (L.) Savi is a natural perennial grass belonging to the Labiateae family,

growing spontaneously on the Mediterranean coast. In the last years, C. nepeta essential oils

have been largely studied because of their wide variability in chemical composition,

antimicrobial and fungicidal activities (Marongiu et al. 2010). Essential oils were characterised

by high contents of terpenes, one of the largest groups of secondary metabolites, such as

pulegone, b-caryophyllene, menthone and menthol, their major constituents (Marongiu et al.

2010; Araniti et al. 2012a). Terpenes play many ecological roles such as the attraction of

pollinating insects, the defense against herbivores and pathogens and are also involved in plant–

plant communication (Cheng et al. 2007). Further, the phytotoxic activity of pure monoterpenes,

main constituents of essential oils in plants, was widely demonstrated. For example, foliar

volatiles of Eucalyptus globulus and Eucalyptus citriodora inhibited seed germination of

Parthenium hysterophorus L. and caused in adult plants of same species a strong reduction in

chlorophyll content and cellular respiration accompanied by an increased water loss and plant

wilting (Kohli et al. 1998). The monoterpenes citronellol, citronellal, cineole and linalool

reduced seed germination, seedling length and biomass of Cassia occidentalis L. (Singh et al.

2002). However, it is argued that, in nature, a combination of these molecules is responsible for

q 2013 Taylor & Francis

*Corresponding author. Email: mrabenavoli@unirc.it

Natural Product Research, 2013

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the phytotoxic activity on many physiological processes. Despite the abundance of studies on

additive and/or synergic effects of terpenoids in pharmacological fields (Mulyaningsih et al.

2010), few data in plant–plant interactions have been reported. For example, it has been

demonstrated that camphor, pulegone and borneol exhibited a marked synergistic action when

used in combination (Asplund 1969). The mixture of parthenin and coronopilin, two

sesquiterpenes isolated from P. hysterophorus, has autotoxic effects on both seedlings and adult

plants (Picman & Picman 1984). Lydon et al. (1997) observed that the pure sesquiterpene

artemisin from Artemisia annua was less phytotoxic on Amaranthus retroflexus than total leaf

extract. Finally, Vokou et al. (2003), assaying 11 pairs of terpenoids mixtures, observed that the

level of inhibition of the mixtures was not comparable to that from a single active compound.

Recently, Araniti et al. (2012b) have shown that the aqueous extract and plant mulches of

C. nepeta Savi L. exhibited a strong phytotoxic potential on lettuce and Chenopodium album,

Sinapis alba and Echinochloa crus-galli, three of the most common weeds. Furthermore, they

indentified and quantified in both n-hexane fraction obtained from methanolic extract and in

C. nepeta essential oils (Araniti et al. 2013), four terpenes, camphor, trans-caryophyllene,

farnesene and pulegone, probably responsible for the inhibitory activity on germination and root

growth (Araniti et al. 2012a, 2013). In this study, the individual and/or combined effects of these

pure terpenoids were evaluated in in vitro bioassays, at different combinations and

concentrations, on seed germination and seedling growth of Arabidopsis thaliana to assess

their potential phytotoxicity and their joint activity.

2. Results and discussion

2.1. Bioassays of individual terpenoids

All the terpenoids did not influence, either individually or in combination, seed germination of

A. thaliana. The total germination index, GT, was not significantly modified by all compounds

(data not shown). These results appeared to be in disagreement with those observed with

methanolic and n-hexane extracts of C. nepeta (Araniti et al. 2012a) probably because

(i) different compounds present in the same fraction, but not tested here, could be responsible for

the inhibition; (ii) different compounds present in other fractions utilised in the bio-guided

fractionation method could be responsible for the inhibition and (iii) a strong species-specific

activity of compounds (Arabidopsis seeds could be less sensitive than lettuce). The species-

specificity of allelochemicals on seed germination was reported by several authors on many

species (Asplund 1969; Nishida et al. 2005). The effects of the terpenoids on the root elongation

were compound- and concentration-dependent. Although pulegone did not inhibit root growth of

seedlings (Figure 1A), at the highest concentrations (800 and 1200mM), it caused a marked

inhibition of lateral roots formation. Furthermore, camphor caused, only at the highest

concentrations, a weak inhibitory effect on root growth showing a reduction by 26% compared

to control (Figure 1B). However, both pulegone and camphor, at lowest concentrations

(0–200mM), stimulated root elongation of A. thaliana (Figure 1A and B). This stimulatory

phenomenon was known as ‘hormesis’. Kim (2008) demonstrated that essential oils extracted

from Agastache rugosa increased the root elongation of Platycarpum taraxacum. Coumarin

stimulated root growth of A. thaliana (L.) Heynh. (Abenavoli et al. 2008). In an ecosystem

context, ‘hormesis’ could make a plant more competitive for nutrient and water resources than

neighbour plants. Differently, trans-caryophyllene, at the highest concentrations (800 and

1200mM), reduced root growth by 45% compared to control (Figure 1C). Although the

mechanism of action of this allelochemical is not known yet, previous study pointed out the

disruption of cells’ mitotic activity (Sanchez-Moreiras et al. 2008). Moreover, at 800 and

1200mM, trans-caryophyllene caused chlorosis in leaves (Figure S2), and a strong reduction,

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higher than 50% when compared to control, in shoot fresh and dry weight (Figures S1 and S2).

Among all terpenoids, farnesene was the only compound that strongly inhibited root growth of

A. thaliana seedlings. Already at 200mM concentration, it reduced root elongation by 40%

compared to the control, reaching 88% of inhibition at 1200mM (Figure 1D). Moreover,

seedlings treated with this compound, at the same concentration, showed a loss in gravitropism,

a total absence of root hairs as well as a significant deformation of the root (corkscrew-shaped)

(Figure S3), a phenomenon well known in nature as ‘handedness’ (Whippo & Hangarter 2009).

In order to compare the effects of terpenoids, root growth data were fitted by non-linear

regression models selected on the basis of coefficient R 2 determination. The ED50 and the ED80,

parameters which define the effective dose causing 50% and 80% of total response, respectively,

were calculated. The obtained curves were characterised by a high statistical significance

(P # 0.001) and a correlation coefficient R 2 $ 0.90. Among all the compounds, only farnesene

was able to induce a root inhibition higher than 50%, showing ED50 and ED80 values by 323 and

780mM, respectively, whereas trans-caryophyllene inhibited shoot dry and fresh weight with

ED50 values of 954 and 760mM, respectively.

2.2. Synergic effect of terpenoids mixtures

After 15 days of treatment, the C–TC combination (Mixture A) (Table 1) caused a marked

reduction of root growth by 15% and 30% at 300/600 and 600/1200mM, respectively

(Figure 2A). Farnesene addition (Mixture B) slightly increased this inhibitory activity, at the two

highest concentrations, by 20% and 40%, respectively (Figure 2B). However, by comparison of

Figure 1. Dose–response curves of TRL of A. thaliana seedlings treated with different concentrations(0–1200mM) of (A) pulegone, (B) camphor, (C) trans-caryophyllene and (D) farnesene; N ¼ 5. Differentletters indicate significant differences at P , 0.05 (Tukey’s test).

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two mixtures (A vs B, or C–TC vs C–TC–F), it was evident that the farnesene addition did not

cause any significant effect (Figure 2C). At the lowest concentration, the P–C–TC combination

(Mixture C) determined a stimulation of root growth by 18% (hormetic effect), whereas, at the

highest one, it caused root inhibition equal to 23% when compared to control (Figure 2D).

Interestingly, although farnesene, when assayed alone, was unable to inhibit root growth at

concentrations lower than 100mM (Figure 1D), the addition of a very small amount of this

compound (2.4mM) tomixtureCcauseda significant root growth reductionby54%whencompared

to control (Figure 2E).Bycomparisonof the twocurves (Figure 2F), a significant differencebetween

the treatments (P–C–TC vs P–C–TC–F) was evident. In conclusion, the results indicated that the

addition of farnesene toMixture A did not determine either an additive or a synergistic but rather an

independent effect because the reduction in root growth was comparable to the inhibition observed

with individual compounds (Figure 2 vs Figure 1). On the contrary, the addition of pulegone and/or

farnesene toMixtureAcauseda synergistic action inboth treatments. Indeed, themixtureP–C–TC,

at the highest concentration, evidenced an inhibition of root growth by 23% (Figure 2D), which was

not evident in the experimentswith the individual compounds at the same concentrations (Figure 1).

Moreover, the addition to this mixture of very low concentration of farnesene (2.4mM) statistically

increased the inhibitory effect of the mixture from 23% to 53% supporting the hypothesis of a

synergistic action (Figure 2E and F). These results were in agreement with Fujita and Kubo (2003)

which reported that the inhibitory activity of trans-cinnamic acid on lettuce root growth was

enhanced 17-fold when it was applied in combination with the sesquiterpene polygodial.

Interestingly, as observed in Mixture C, polygodial alone, at all concentrations did not cause any

inhibition (Fujita & Kubo 2003). Some evidences of synergism were also reported with terpenoid

compounds by Asplund (1969) who demonstrated that the combination of camphor and pulegone

caused a reduction ofwheat germination at concentrations 100 times lower than those caused by the

individual compounds.

Table 1. Concentrations of terpenoid mixtures assayed in the in vitro experiments.

Concentrations (mM)

Treatments Pulegone Camphor trans-Caryophyllene Farnesene

Mixture A1 – – – –2 – 100 200 –3 – 300 600 –4 – 600 1200 –

Mixture B1 – – – –2 – 100 200 203 – 300 600 604 – 600 1200 120

Mixture C1 – – – –2 140 2 4 –3 560 8 16 –4 1120 16 32 –

Mixture D1 – – – –2 140 2 4 0.403 560 8 16 1.204 1120 16 32 2.40

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Figure 2. Effect of terpenoids mixtures on root growth of A. thaliana seedlings. Graph (A) Mixture A (Table1); (B) Mixture B (Table 1); (C) comparison of the effects between the two treatments: C–TC (Mixture A)versus C–TC–F (Mixture B); (D) Mixture C (Table 1); (E) Mixture D (Table 1); (F) comparison of theeffects between the two treatments: P–C–TC (Mixture C) versus P–C–TC–F (Mixture D). The mixturesconcentrations reported in Table 1 are expressed on graphs with values 1–2–3–4. Different letters indicatesignificant differences at P , 0.05 (Tukey’s test).

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3. Experimental

3.1. Bioassays of individual terpenoids

3.1.1. Germination bioassays

The chemicals used in the experiments, pulegone, camphor, trans-caryophyllene and farnesene

(SigmaAldrich,Madrid, Spain), were dissolved in EtOH to reach the following final concentrations:

0, 50, 100, 200, 400, 800 and 1200mM. A. thaliana L. (Heyn.) seeds, ecotype Columbia (Col-0),

were surface sterilisedbydipping inEtOH:TritonX-100 (50:0.01) and successively inNaClO:Triton

X-100 (0.5:0.01) solutions for 3min, rinsed in sterilised distilled water and then vernalised in 0.1%

agar solution at 48C for 48h.After this period, 24 seedswere placed into Petri dishes (100 £ 15mm)

containing 30ml agarised (0.8 %) Murashige–Skoog medium (Sigma Aldrich) and sucrose (1%),

pH 6.0. Thirtymicrolitres of each compound for each concentrationwere included into themedium.

EtOH(30ml)wasused as control.ThePetri disheswere thenverticallyplaced ina growth chamber at

22^ 28C with 55% relative humidity and 8/16h light (60mmolm22 s21)/dark photoperiod, for

15 days. Then, the germinated seeds were counted using any extrusion of the radicle as a criterion,

and the total germination index (GT %) as described by Chiapusio et al. (1997) was calculated.

3.1.2. Root elongation bioassays

After the radicle protrusion, 24 seedlings were placed into Petri dishes containing 30ml of

agarised Murashige–Skoog medium added with 30ml of each terpenoid at above all

concentrations. Growth conditions were identical to those applied for germination process. After

15 days, an image of roots for each treatment was captured by scanner (Epson Expression 800;

Regent Instruments, Quebec, Canada) and the total root length (TRL, cm) was measured using

the WinRhizo Pro System version 2002a software (Instruments Regent, Inc., Quebec, Canada).

3.2. Bioassays of terpenoid mixtures

3.2.1. Germination and root elongation bioassays

The joint activity of pulegone (P), camphor (C), trans-caryophyllene (TC) and farnesene (F) was

in vitro evaluated on germination and root growth. The choice of mixture concentrations was

carried out maintaining the same concentration ratio among compounds observed during the

characterisation and quantification of methanolic extract of C. nepeta (P:C:TC:F) (70:1:2:0.2).

Moreover, although at different concentrations, camphor and trans-caryophyllene were

constantly maintained in all mixtures, whereas pulegone and farnesene were added individually

and/or in combination (Mixture A: C–CT; Mixture B: C–TC–F; Mixture C: P–C–TC; Mixture

D: P–C–TC–F; Table 1).

3.3. Statistical analyses

A completely random design with five replications was adopted. GT and TRL data

were evaluated by ANOVA. Differences among group means were estimated by least significant

difference tests in the case of homoscedastic data, and by Tamhane’s T2 test in the case of

heteroscedastic data (P # 0.05). Tukey test with P # 0.05 was performed in order to evaluate

the synergistic effects. All dose–response curves were modelled by regression analysis with

SPSSw models selected on the basis of the determination coefficient R 2.

4. Conclusion

These results confirmed that allelopathic effects are generally due to the interaction of a wide

variety of molecules released by plants into the environment and that the effects observed in

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nature cannot be always related to a single compound. In this specific case, it is probable that the

effect found in C. nepeta aqueous and methanolic extracts in previous work (Araniti et al. 2013)

was derived from a combination of terpenoids and not due to the individual compounds.

Furthermore, the results suggested that the cooperative action among the compounds, in a

natural community or in field, could reduce the threshold concentration needed to cause the

phytotoxic effect, as observed with farnesene addition. Finally, considering that these

compounds were derived from the methanolic extract of C. nepeta, the results suggested that this

Mediterranean plant could be a very interesting source of natural molecules to use as bio-

herbicides in a sustainable agriculture.

Supplementary material

Supplementary material related to this article is available online, alongside Figures S1–S3.

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