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Phytotoxicity and cytotoxicity of essential oil from leaves of Plectranthus amboinicus, carvacrol and thymol in plant bioassays Patrícia Fontes Pinheiro, Adilson Vidal Costa, Thammyres de Assis Alves, Iasmini Nicoli Galter, Carlos Alexandre Pinheiro, Alexandre Fontes Pereira, Carlos Magno Ramos Oliveira, and Milene Miranda Praça Fontes J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b03049 • Publication Date (Web): 29 Sep 2015 Downloaded from http://pubs.acs.org on October 5, 2015

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Journal of Agricultural and Food Chemistry

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Phytotoxicity and cytotoxicity of essential oil from leaves of Plectranthus

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amboinicus, carvacrol and thymol in plant bioassays

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Patrícia Fontes Pinheiro,1* Adilson Vidal Costa,1 Thammyres de Assis Alves,2 Iasmini

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Nicoli Galter,2 Carlos Alexandre Pinheiro,1 Alexandre Fontes Pereira,3 Carlos Magno

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Ramos Oliveira,4 Milene Miranda Praça Fontes2

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1

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Universitário, s/n, 29500-000, Alegre, ES, Brazil

Department of Chemistry and Physics, Federal University of the Espírito Santo, Alto

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2

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s/n, 29500-000, Alegre, ES, Brazil

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3

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Cruzeiro, s/n, Bauxite District, 35400-000, Ouro Preto, Minas Gerais, Brazil

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4

15

Universitário, s/n, 29500-000, Alegre, ES, Brazil

Department of Biology, Federal University of the Espírito Santo, Alto Universitário,

Department of Food, Nutrition School, Federal University of Ouro Preto, Morro do

Department of Plant Production, Federal University of the Espírito Santo, Alto

16 17

*Author to whom correspondence should be addressed. Telephone: (55) 028 35528661.

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Fax: (55) 028 35528655. E-mail: [email protected]

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1

ACS Paragon Plus Environment


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ABSTRACT: The essential oil of Plectranthus amboinicus and its chemotypes,

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carvacrol and thymol, were evaluated on the germination, root and aerial growth of

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Lactuca sativa and Sorghum bicolor, and in acting on cell cycle of meristematic root

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cells of L. sativa. The main component found in the oil by analysis in

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gaschromatography-mass spectrometry (GC-MS) and gas chromatography flame

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ionization detector (GC-FID) was carvacrol (88.61% in area). At a concentration of

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0.120% (w v-1), the oil and its chemotypes retarded or inhibited the germination and

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decreased root and aerial growth in monocot and dicot species used in the bioassays. In

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addition, all substances caused changes in the cell cycle of the meristematic cells of L.

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sativa, with chromosomal alterations occurring from the 0.015% (w v-1) concentration.

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The essential oil of P. amboinicus, carvacrol and thymol have potential for use as

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bioherbicides.

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KEYWORDS: allelopathy, mutagenicity, volatile constituents of P. amboinicus

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INTRODUCTION

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The growing demand for food as a consequence of increased populations purred

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the modernization of agriculture in the 20th century. The use of mechanized agricultural,

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fertilizers and pesticides have been instrumental in increasing productivity and success

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in this field.1,2

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Agrochemicals, or pesticides, have become part of pre and post-harvest handling

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in most agricultural sectors. Herbicides in particular are used to control weeds and

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represent about 50% of all pesticides used in Brazil.3,4

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Despite the economic benefits of herbicide use, toxicological studies have

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demonstrated adverse effects on humans, causing DNA alterations, cancer and

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teratogenic action because of their possible role in causing or promoting tumors.5

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In March 2015, the International Agency for Research on Cancer (IARC)6

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reported that the herbicide glyphosate is a probable carcinogenic agent. Of concern is

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the fact that since 2009, Brazil has been the leading consumer of agrochemicals,

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including glyphosate7. Thus, it has become necessary to find alternative methods for

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reducing its use.

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In addition to modifying genetic material, herbicides have become less efficient,

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since over time their use selects for resistant varieties, reducing effectiveness8. In order

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to minimize these problems, other weed control methods should be studied and

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improved.9

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There are several natural compounds with potential for use in weed control.

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Volatile constituents of certain plants, known as essential oils, affect the growth of other

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species by the allelopathic effect10,11.

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Among the plant families producing essential oils are the Lamiaceae, which

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includes the species Plectranthus amboinicus, commonly known as Mexican mint. The 3



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compounds carvacrol and thymol are chemotypes found in the essential oil of this

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species.12

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Kordali et al. found a potent phytotoxic effect of the essential oil of Origanum

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acutidens and of the phenols carvacrol and thymol on seed germination and plantlet

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growth of the species Amaranthus retroflexus, Chenopodium album and Rumex

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crispus.13 The essential oil of Lippia sidoides, containing thymol as its main component

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(84.90%), presented negative allelopathic effects on the culture of Lactuca sativa.14

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Considering the allelopathic potential of essential oils and their isolated

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components, this work aimed to investigate the effects of P. amboinicus essential oil,

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carvacrol and thymol on the germination and root growth of L. sativa and S. bicolor, as

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well as to evaluate their action on the cell cycle of meristematic root cells of L. sativa.

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MATERIALS AND METHODS

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General Experimental Procedures. The solvents used in the experiments

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(pentane and dichloromethane) and 2% acetic orcein were obtained from Vetec (Rio de

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Janeiro, RJ, Brazil) and thymol, carvacrol and a mixture of linear alkanes (C9 and C26)

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were obtained from Sigma Aldrich. The chromatographs used in essential oil analysis

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were gas chromatography coupled to mass spectrometer (GC-MS) model QP2010 Plus

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(Shimadzu - Tokyo, Japan) and gas chromatography equipped with a flame ionization

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detector (GC-FID) model GC-2010 (Shimadzu - Tokyo, Japan). Seeds of S. bicolor

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were obtained from LG Sementes (Goianésia - GO, Brazil) and seeds of L. sativa from

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Feltrin Sementes (Farroupilhas - RS, Brazil).

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Plant Material Samples. Leaves of P. amboinicus were collected during the

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morning in Alegre (ES, Brazil). The exsiccation (n. 21590) was deposited in the

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herbarium (VIES) at the Federal University of Espírito Santo (UFES). Plants were

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classified by the botanist A.C. Tuler. 4


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Essential Oil. Essential oil was extracted using 500 g of fresh leaves from P.

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amboinicus, cut into small pieces and placed in a round-bottom 5-liter flask coupled to a

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Clevenger (modified). The flask was half filled with distilled water and hydrodistillation

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was performed for3 h after the water was boiled. During this time, the hydrolate (150

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mL) was collected and subsequently subjected to liquid-liquid extraction using pentane

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(3 x 40 mL). The organic phase was dried with anhydrous sodium sulfate and filtered.

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The filtrate was concentrated under reduced pressure in a rotatory evaporator to obtain

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the essential oil.

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To determine the composition of the P. amboinicus essential oil, gas

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chromatography coupled to mass spectrometry (GC-MS) was used. A capillary column

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of fused silica Rtx-5MS (30 m long, 0.25 mm internal diameter) was used with helium

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as the carrier gas. The temperatures were 220°C for the injector and 300°C for the

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detector. The initial column temperature was 60°C, programmed for an increase of 3°C

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per minute until reaching the maximum temperature of 240°C. To determine the

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chemical constituents of the P. amboinicus essential oil, the obtained mass spectra was

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compared with reference data from the equipment database, using data from other

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sources and the Kovats index (KI).15 To determine the KI, a mixture of linear alkanes

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(C9 and C26) was injected into the chromatograph, under the same conditions used in the

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referenced essential oil analysis. Retention indices (KI) were calculated using equation

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1.

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KI = 100Z + 100[(logt’RX) - (logt’RZ)] (Equation 1)

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(logt’RZ +1) - (logt’RZ)

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where:

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X is the analyzed compound; 5



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Z is the number of carbon atoms of the hydrocarbon with retention time immediately

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preceding the retention time of X;

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t’RX is the adjusted retention time of X;

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t’RZ is the adjusted retention time of Z;

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t’RZ +1 is the adjusted retention time of the hydrocarbon with retention time

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immediately preceding the retention time of X.

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For component quantification, P. amboinicus essential oil was analyzed on a gas

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chromatograph equipped with a flame ionization detector (GC-FID). The stationary

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phase was the capillary column Rtx-5MS (30 m long and 0.25 mm internal diameter).

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Nitrogen was used as carrier gas. Temperature programming was the same as previously

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reported for GC-MS analysis. The temperatures of the injector and the detector were

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240°C and 250°C. A 10 mg sample of essential oil was diluted in 1 mL

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dichloromethane and 1 µL of the mixture was injected.16

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Biological Assay. To perform the biological tests, solutions of essential oil from

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P. amboinicus leaves were prepared in dichloromethane at the concentrations of 0%

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(dichloromethane), 0.015%, 0.030%, 0.060% and 0.120% (w v-1). The compounds

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carvacrol and thymol were applied at the same concentrations as the essential oil. The

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herbicides glyphosate and boral were used as positive controls. Seeds of Sorghum

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bicolor (monocot) and of L. sativa (dicot) were used as plant models. For the

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germination tests, 2 mL of each solution were added to Petri dishes with 9 cm diameter

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containing filter paper. Twenty-five seeds of S. bicolor and of L. sativa were used for

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each treatment, with five repetitions. The dishes were sealed with plastic foil and placed

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in the germination chamber (BOD) at 24±2°C, where they were kept for the duration of

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the experiment.

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Phytotoxicity. The number of germinated seeds was evaluated from 8 to 48 h, at

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8 h intervals. The germination speed index (GSI) was obtained according to the

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formula:

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(N1*1) + (N2-N1)*1/2 + (N3-N2)*1/3 + ... (Ny-(Ny-1))*1/y,

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where Ny represents the number of seeds germinated in a given period and Y represents

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the total number of time intervals. The percentage of germinated seeds (GR) and the

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root length (RL) were obtained after 48h. The aerial parts of the plantlets were

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measured after 120 h to determine the aerial growth (AG). All measurements were taken

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with a digital caliper.

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Cytotoxicity. After 48 h of exposure, ten roots from each dish were collected

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and fixed in an ethanol: acetic acid solution (3:1/ v v-1), and stored at -4ºC for at least 24

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h. For cytogenetic analysis, the slides were prepared using the squash technique and

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stained with 2% acetic orcein.3 Approximately 5,000 meristematic cells were evaluated

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per treatment, observing and quantifying the different stages of mitotic division. The

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mitotic index (MI) was obtained by dividing the number of cells in the process of

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division (prophase, metaphase, anaphase and telophase) by the total number of cells

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evaluated in each treatment. The frequencies of chromosome and nuclear alterations

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were calculated by dividing the number of alterations (chromosomal or nuclear,

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respectively) by the total number of cells analyzed.17

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Statistical Analysis. The experiment was done in a Completely Randomized

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Design (CRD) using a 3x5+2 factorial scheme with 3 treatments (carvacrol, thymol and

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essential oil), 5 concentrations (0.120%, 0.060%, 0.030%, 0.015% w v-1 and 0%) and 2 7



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additional treatments (boral and glyphosate). When significant, the obtained

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cytotoxicity data were subjected to analysis of variance and the means were compared

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by the Dunnett’s test at 5% significance. The phytotoxicity data when significant were

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the quantitative factors (concentrations of the solutions used) applied to analysis of

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regression, with the models to be chosen based on the significance of the regression

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coefficients, using the Student’s t test at 5% probability and the coefficient of

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determination (R²). For the significant qualitative factors related to the 'solutions'

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variables, the Dunnett’s test was used at 5% significance to compare the treatments. For

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statistical analysis of qualitative factors (Dunnett, 5% significance) was used the Genes

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program18 and quantitative factors (regression) was used Sisvar program19.

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RESULTS AND DISCUSSION

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Essential Oil. The yield of essential oil from the leaves of P. amboinicus was

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0.12% (ww-1) compared to plant dry mass. Bandeira et al. found a 0.43% essential oil

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content in the leaves of this species.20 Four compounds were identified in the essential

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oil (Table 1). Two chemotypes have been reported for this species, one rich in thymol

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and the other in carvacrol. Here, carvacrol was found to be the main constituent

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(88.61%). These compounds are isomers and belong to the class of aromatic

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monoterpenes.12

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Carvacrol was also identified as the main constituent (77.16%) in the essential

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oil of P. amboinicus leaves by Joshi et al, while Oliveira et al. found thymol to be the

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main component of this oil (75.54%).21, 22

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Genetic factors are responsible for determining the chemical composition of

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essential oils. Other factors however may also generate changes in secondary metabolite

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production.23 Biotic and abiotic factors may interfere with the quality and quantity of the 8


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secondary products resulting from the plant metabolism at a given time. The main biotic

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factors are plant-microorganism, plant-insect and plant-plant interactions, as well as age

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and development stage. Abiotic factors include luminosity, temperature, rainfall,

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nutrition, season and time of collection, and harvest and post-harvest techniques. These

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factors may present correlations among themselves and do not act in isolation. In a

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study with Acorus calamus L., Kumari et al. observed that the addition of UV-B

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complementary indices caused a significant carvacrol increase in the plant’s essential oil

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composition.24

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Biological Assay. Plectranthus amboinicus essential oil and its chemotypes in

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pure form, thymol and carvacrol, in dichloromethane solution at concentrations of

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0.120%, 0.060%, 0.030% and 0.015% (w v-1) and the negative control (0.000%) were

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tested with respect to germination and growth of L. sativa root (dicot) and S. bicolor

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(monocots). In all treatments, the solutions with higher concentrations had the most

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significant effects in the referenced tests.

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The data regarding GR (percentage of germination) and GSI (germination speed

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index) for L. sativa and S. bicolor are described in Table 2. For all the different

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solutions tested in L. sativa at the concentration of 0.120%, the values of GR presented

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a significant difference compared to the positive controls (Figure 1a). The solution of

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carvacrol at 0.060% presented a significant difference compared to the positive controls.

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For GSI, only the thymol and carvacrol solutions at 0.015% did not differ from the

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negative control treatment (0.000%), while the essential oil presented differences at all

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tested concentrations. This way, germination was retarded but did not cease (Figure 2a).

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The GR values for S. bicolor in all treatments at the concentrations of 0.120%

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and 0.060% were lower than the negative control, although germination occurred in all

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tests (Figure 1b). The GSI data for all treatments at all concentrations were different 9



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from the positive and negative controls, with only the P. amboinicus essential oil at

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0.015% not differing from the boral herbicide (Figure 2b).

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The allelopathic effects may be less intense on the final germination percentage

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and more intense on the germination speed, as shown by Oliveira et al. when

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considering the allelopathic potential of aqueous extracts from mulungu bark (Erythrina

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velutina Willd. – Leguminosae) on L. sativa.25

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The results obtained referring to germination and GSI can be explained by the

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fact that the allelopathic effect in plants is most often expressed not by the amount of

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germinated seeds, but rather by the retardation of their germination.26 Monoterpenoids

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may have allelopathic activity, which interferes with germination and plant

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development. Moreover, these substances are able to affect physiological processes,

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such as photosynthesis, chlorophyll synthesis, lipid accumulation in the cytoplasm and

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reduction of organelles because of ruptured membranes.27

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In a study by Romero et al. on the effect of natural monoterpenes on mycelium

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growth and conidium germination in Corynespora cassiicola, the authors found that

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thymol and carvacrol as those that present the highest effects on the reduction of

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germination.28

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According to Yamagushi et al., eucalyptus and guacatonga (Casearia sylvestris),

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which contain monoterpenes in their composition, may be considered potentially

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allelopathic by reducing and inhibiting germination and GSI in various vegetables

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(mustard, cabbage, broccoli, kale, turnip, rocket, lettuce, tomato and radish).29

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The values for root length (RL) and aerial length (AL) after germination of

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L. sativa and S. bicolor treated with different concentrations of P. amboinicus essential

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oil, thymol and carvacrol, are presented in Table 3. The RL of L. sativa decreased

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progressively with increased concentrations of all tested solutions (Figure 3a). RL was

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not reduced when using P. amboinicus essential oil or carvacrol at 0.015%.

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All solutions tested at 0.120% were more effective at reducing root grow thin L.

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sativa than the herbicides glyphosate and boral. Moreover, a decrease in aerial growth

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was observed in all treatments as concentrations increased (Figure 4a).

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For S. bicolor, all treatments at all tested concentrations showed significant

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reductions in growth compared to the negative control (Table 3). Figures 3b and 4b

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represent root and aerial growth. These aspects were not significant for any essential oil

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concentration or for thymol at 0.030%.

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Alves et al. tested Lippia sidoides essential oil on L. sativa seeds and noted there

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was a phytotoxic effect, with thymol being the main component present in this oil.30

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Monoterpenes may show allelopathic effects; the vapors of these substances can cause

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anatomical and physiological alterations in plantlets.31 According to Formagio et al.,

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inhibiting root growth is an important ecological aspect, as it reduces the competitive

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pressure of the plant, enabling the neighboring species to establish dominance

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features.32 The inhibitory effect of germination, of the root and aerial growth

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characteristic of the allelopathic agents, may be related to their interference with cell

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division, membrane permeability and enzyme activation.33,34

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The cytogenetic study was used to analyze the alterations caused by P.

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amboinicus essential oil, thymol and carvacrol on the L. sativa cell cycle. The same

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concentrations were used as in the phytotoxic test, except for 0.120% because it totally

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inhibited seed germination in all tested solutions. The results of the cell divisions are

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shown in Tables 4 and 5.

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The percentage of interphases was identical to the negative controlin the

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treatments with carvacrol at all tested concentrations. On the contrary, a decrease 11



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occurred using the thymol and P. amboinicus essential oil solutions (Table 4). This

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result is directly related to the MI, where all treatments differed from the negative

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control. The carvacrol solutions caused MI to decrease, however, while the other

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treatments (thymol and essential oil) caused this index to increase.

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In the treatment of L. sativa with the herbicide boral, a very high index of

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condensed nuclei and a very low MI were observed (Table 5). The MIis used to evaluate

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the cytotoxicity of allelopathic agents. MI values significantly lower than the control

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indicate that the alterations are caused by the action of chemical components on the

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growth and development of the exposed organisms. On the other hand, if the MI

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presents values higher than the control because of increased cell division, this may be

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harmful to the organism as it may lead to disordered cell proliferation and eventually

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chromosome abnormalities.35

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The presence of chromosome abnormalities was greater in all treatments

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compared to the controls, whereas nuclear alterations only differed from the controls in

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the treatments with P. amboinicus essential oil and thymol (Table 5). The detected

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alterations were lost chromosome, sticky chromosome, C-metaphase and chromosome

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polyploidization (Figure 5).

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Compared to the chromosomal alterations found, what most differentiated the

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controls was the percentage of sticky chromosomes (Table 5) that found significant

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differences between the controls and all treatments except the carvacrol.

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The presence of sticky chromosomes is generally attributed to the toxic effects

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of chemical agents on the organization of chromatin. Since chromatin organization is

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affected, chromosome bridges may arise, as observed in this work (Table 5).

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In view of these results, it was suggested that the essential oil of P. amboinicus,

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carvacrol and thymol have potential for use as bioherbicides and may help to minimize

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the damage caused by intensive herbicide use on biodiversity and human health.

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ACKNOWLEDGMENTS

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We thank the National Council for Scientific and Technological Development (CNPq)

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for financial support (484183/2013-3).

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(19) Ferreira, D.F. SisVar®: Sistema de análise de variância para dados balanceados,

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versão 4.0. Lavras: DEX/UFLA, 2000. (Software estatístico).

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(20) Bandeira, J.M.; Barbosa, F.F.; Barbosa, L.M.P.; Rodrigues, I.C.S.; Bacarin, M.A.;

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Peters, J.A.; Braga, E.J.B. Composição do óleo essencial de quatro espécies do gênero

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Plectranthus. Rev. Bras. Plantas Med. 2011, 13, 2, 157-164.

Adams,

R.

P.

Identification

of

essential

oil

components

by

gas

15



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(21) Joshi, R. K; Badakar, V.; Kholkute, S.D. Carvacrol rich essential oils of Coleus

340

aromaticus (Benth.) from Western Ghats Region of North West Karnataka, India. Ad.

341

Envi. Biol. 2011, 5, 6, 1307-1310.

342

(22) Oliveira, R. A.; Sá, I. C.; Duarte, L. P.; Oliveira, F. F. Constituintes voláteis de

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Mentha pulegium L. e Plectranthus amboinicus (Lour.) Spreng; [Volatile constituents

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of Mentha pulegium L. and Plectranthus amboinicus (Lour.) Spreng]. Rev. Bras.

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Plantas Med. 2011, v.13, n.2, pp. 165-169.

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(23) Morais, L. A. S. Influência dos fatores abióticos na composição química dos óleos

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essenciais. Hort. Bras. 2009, 27, 2, S4050-S4063 (Suplemento –CD ROM).

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(24) Kumari, R.; Agrawal, S. B.; Singh, S.; Dubey, N. K. Supplemental ultraviolet-B

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induced changes in essential oil composition and total phenolics of Acorus calamus L.

350

(sweet flag). Ecotox. Environ. Safe. 2009, 72, 2013-2019.

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(25) Oliveira, A. K.; Coelho, M. F. B.; Maia, S. S. S.; Diógenes, F. E. P.; Medeiros-

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Filho, F. M. Alelopatia de extratos de diferentes órgãos de mulungu na germinação de

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alface. Hort. Bras. 2012, 30, 480-483.

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(26) Iganci, J.R.V.; Bobrowski,V.L.; Heiden, G.; Stein, V.C.; Rocha, B.H.G. Efeito do

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Extrato Aquoso de Diferentes Espécies de Boldo sobre a Germinação e Indice Mitótico

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de Allium cepa L. Arq. Inst. Biol., São Paulo, 2006, v.73, n.1, pp.79-82.

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(http://200.144.6.109/docs/arq/V73_1/iganci.PDF)

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(27) Grosso, C.; Coelho, J. A.; Urieta, J. S.; Palavra, A. M. F.; Barroso, J. G. Herbicidal

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activity of volatiles from coriander, winter savory, cotton lavender, and thyme isolated

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by hydrodistillation and supercritical fluid extraction. J. Agri. Food Chem. 2010, 58, 20,

361

11007-11013. 16


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(28) Romero, A. L.; Oliveira, R. R.; Romero, R. B. Efeito de monoterpenos naturais no

363

crescimento micelial e germinação de conídios de Corynespora cassiicola. Pesq.

364

Agropec. 2013, 18, 1, 3-7.

365

(29) Yamagushi, M. Q.; Gusman, G. S.; Vestena, S. Efeito alelopático de extratos

366

aquosos de Eucalyptus globulus Labill. e de Casearia sylvestris Sw. sobre espécies

367

cultivadas. Sem.: Ciênc.Agr. 2011, 32, 4, 1361-1374.

368

(30) Alves, M. C. S.; Medeiros-Filho, S.; Innecco, R.; Torres, S. B. Alelopatia de

369

extratos voláteis na germinação de sementes e no comprimento da raiz de alface. Pesq.

370

Agropec. Bras. 2004, 39, 11, 1083-1086.

371

(31) Poser, G.L.; Menut, C.; Toffoli, M.E.; Sobral, M.; Bessiere, J.M.; Lamaty, G.;

372

Henriques, A.T. Aromatic plants from Brazil: 4. Essential oil composition and

373

allelopathic effect of the Brazilian Lamiaceae Hesperozygis ringens (Benth.) Epling and

374

Hesperozygis rhododon Epling. J. Agri. Food Chem. 1996, 44, 1829-1832.

375

(32) Formagio, A. S. N.; Masetto, T. E.; Vieira, M. C.; Zárate, N. A. H; Matos, A. I. N.

376

Potencial alelopático e antioxidante de extratos vegetais. Biosci. J. 2014, 30,

377

supplement 2, 629-638.

378

(33) Rodrigues, L.R.A.; Rodrigues, T.J.D.; Reis, R.A. Alelopatia em plantas forrageiras.

379

Guaíba: FUNEP/Jaboticabal, 1999, 18p.

380

(34) Rosado, L.D.S.; Rodrigues, H.C.A.; Pinto, J.E.B. P.; Custódio, T.N.; Pinto, L.B.B.;

381

Bertolucci, S.K.V. Alelopatia do extrato aquoso e do óleo essencial de folhas do

382

manjericão “Maria Bonita” na germinação de alface, tomate e melissa. Rev. Bras. Pl.

383

Med. 2009, 11,.4, 422-428.

17



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384

(35) Leme, D.M.; Marin-Morales, M. A.Allium cepa test in environmental monitoring:

385

A review on its application. Mutation Res. 2009, 682, 1, 71-81.

386

387

388

389

390

391

392

393

394

395

396

397

398

399

400

401

402 18


Page 19 of 32


403

Figure captions

404

Figure 1. Graphs of GR (percentage of germination) for Lactuca sativa (a) and

405

Sorghum bicolor (b) treated with solutions of Plectranthus amboinicus essential oil,

406

thymol and carvacrol.

407

Figure 2. Graphs of GSI (germination speed index) for Lactuca sativa (a) and Sorghum

408

bicolor (b) treated with solutions of Plectranthus amboinicus essential oil, thymol and

409

carvacrol.

410

Figure 3. Graphs of RG (root growth) for Lactuca sativa (a) and Sorghum bicolor (b)

411

treated with solutions of Plectranthus amboinicus essential oil, thymol and carvacrol.

412

Figure 4. Graphs of AG (aerial growth) for Lactuca sativa (a) and Sorghum bicolor (b)

413


414

Figure 5. Abnormally dividing cells of L. sativa after exposure to carvacrol solution

415

(0.060%, w v-1). (A) stickiness metaphase; (B) anaphase bridge; (C) condensed nucleus;

416

(D and E) laggard chromosomes; (F) micro-c-metaphase. Bar = 5µm.

417

418

419

420

421

422

423

424 19



Page 20 of 32

425

Tables

426

Table 1. Chemical constituents of essential oil from leaves of Plectranthus amboinicus No.

Compound

IR

GC peak area (%)

1

NI

__

2.74

2

NI

__

1.62

3

Oct-1-en-3-ol

909

1.79

4

Carvacrol

1306

88.61

5

Eugenol

1360

1.59

6

Z-Caryophyllene

1399

2.39

7

NI

1496

1.26

427

The compounds were listed in order of elution in the Rtx-5MScolumn. RI: retention

428

index compared to linear alkanes (C9 and C26). Peak area percentages are calculated in

429

GC (gas chromatography) Rtx-5MScolumn. NI=not identified.

430

431

432

433

434

435

436

437

20


Page 21 of 32


438

Table 2. Germination parameters for Lactuca sativa and Sorghum bicolor treated with

439

different concentrations of Plectranthus amboinicus essential (EO), thymol and

440

carvacrol L. sativa Solutions

Concentrations (% m v-1)

GR

S. bicolor GSI

0,120 0,060 0,030 0,015 0,120 0,060 0,030 0,015 0,120 0,060 0,030 0,015

GR

GSI

441

0 0 23.2 85.6abcd 6.63c 80bcd EO 88abd 6.88c 83.2abcd 88.8abd 8.37 93.6abcd 0 0 54.4 70.4bcd 4.97c 86.4abcd Thymol 82.4abcd 6.48c 90.4abcd 89.6abcd 9.53abd 89.6abcd 0.8 0.4 21.6 10.4 0.8 68.8d Carvacrol 81.6abcd 6.75c 84.8abcd 89.6abd 9.53abd 89.6abcd Water 91.2 a 10.48a 96.8a Glyphosate 88.8 b 10.4b 94.4b Boral 72.0 c 6.48c 93.6c Dichloromethane 89.6d 10.41d 84.0d GR=percentage of germination; GSI=germination speed index.

1 4.22 5.28d 5.7cd 2.98 5.25d 5.25d 5.44d 1.19 4.76 5.69abcd 6.3abcd 6.81a 6.84b 6.57c 6.14d

442

* Means followed by the same letter in the column do not differ by Dunnett’s test (P >

443

0.05).

444 445 446 447 448

21



Page 22 of 32

449

Table 3. Parameters for root and aerial growth of Lactuca sativa and Sorghum bicolor

450

treated with different concentrations of Plectranthus amboinicus essential oil (EO),

451

thymol and carvacrol L. sativa Solutions

Concentrations (% m v-1) 0,120 0,060 0,030 0,015 0,120 0,060 0,030 0,015 0,120 0,060 0,030 0,015

RG (cm)

S. bicolor

AG (cm)

RG (cm)

0 bc 0 bc 1.63bc 2.31bcd 2.69bd 0 bc 0.18bc 0.34bc 2.36bcd 0 bc 0 bc 2.13bcd 7.87ad 8.71a 3.28b 2.15c

1.3bc 2.41b 2.68b 3.08 1.49bc 2.05bc 2.57b 3.01 0.7bc 3.33 5.05 5.22 7.38a 1.78b 1.26c 7.26d

AG (cm)

452

0 0 EO 4.81 5.71 7.7ad 0 Thymol 2.68bc 3.78b 7.04d 0.02 Carvacrol 0.42 4.44 b Water 91.2 a Glyphosate 88.8 b Boral 72.0 c Dichloromethane 89.6d RG=root growth; AG=aerial growth.

2.78ad 2.59ad 3.09ad 4.26ad 1.06bc 2.87a 2.94ad 2.64 0.52bc 4.07ad 5.67ad 5.63ad 4.91a 0b 0c 4.97d

453

* Means followed by the same letter in the column do not differ by Dunnett’s test (P >

454

0.05).

22


Page 23 of 32


455

Table 4. Normal cell division parameters from cytogenetics of Lactuca sativa treated with different concentrations of Plectranthus amboinicus

456

essential oil (EO) and thymol and carvacrol

Solutions

Concentrations

I%

P%

M%

A%

T%

MI%

0,060%

85.52

49.08abcd

22.89ad

11.42

18.36ad

8.08ad

0,030%

83.1

45.13abcd

24.5ad

13.2

19.73ad

8.54d

0,015%

82.72

45.51abcd

23.79ad

11.81

23.83d

9.22

0,060%

85.24

51.37abcd

23.03ad

8.88

19.73ad

8.7

0,030%

86.16

50.28abcd

23.19ad

10.6

19.45ad

8.86d

0,015%

86.38

45.36abcd

23.17ad

12.62ad

24.11ad

9.38d

0,060%

93.96ab

53.29ad

21.51ad

10.99

8.49b

4.36

0,030%

93.24a

45.72ad

22.84ad

14.04ad

14.52ad

5.82

0,015%

92.5ad

41.8ad

26.05ad

18.66ad

11.51ad

6.12

Water

92.6a

41.44a

23.71a

17.39a

17.81a

7.36a

Glyphosato

95.06b

80.8b

8.98b

3.43b

2.46b

2.64b

Boral

69.62c

40c

0c

0c

0c

0.04c

Dichloromethane

91,76d

44.45d

21.98d

16.78d

18.63d

8.1d

EO

Thymol

Carvacrol

(% w v-1)

23



Page 24 of 32

457

I%=percentage of interphases; P%=percentage of prophases; M%=percentage of metaphases; A%=percentage of anaphases; T%=percentage of

458

telophases; MI%=percentage values of MI.

459

*Means followed by different letters differed significantly according to Dunnett’s test (P > 0.05); means followed by the same letterwere similar

460

according to the same test.

461

24


Page 25 of 32


462

Table 5. Chromosome and nuclear alterations observed in root meristems of L. sativa treated with different concentrations of Plectranthus

463

amboinicus essential oil (EO), thymol and carvacrol

Solutions

Concentrations

C-Poly%

0.48

0.08abcd

0.2

0.46

0.26abd

0.24d

0.3

0.26b

0.62

0.22abcd

0.16ad

0.22

6.06

0.18abc

0.7

0.36bd

0.14ad

0.14

1.44

4.98

0.16abcd

0.76

0.28abd

0.14ad

0.08abcd

0,015%

1.14

4.24

0.1abcd

0.82

0.08abcd

0.12abcd

0.02abcd

0,060%

0.56abd

1.68b

0.02abcd

0.12abcd

0.4bd

0.02abcd

0abcd

0,030%

0.7d

0.94abd

0.02abcd

0.18abd

0.34abd

0.1abcd

0.06abcd

0,015%

0.84d

1.38abd

0.1abcd

0.2acd

0.34abd

0.12abcd

0.08abcd

Water

0.22a

0.04a

0.02a

0.08ª

0.1a

0.02a

0a

Glyphosato

0.22b

2.3b

0.04b

0.02b

0.16b

0b

0b

0c

30.34c

0c

0c

0c

0c

0c

0.48d

0.14d

0d

0.22d

0.16d

0.1d

0d

Thymol

Carvacrol

Boral Dichloromethane

NA%

Lost%

Sticky%

0,060%

1.28

6.4

0.2abcd

0.32d

0,030%

1.54

8.36

0.28

0,015%

1.48

8.06

0,060%

1.52

0,030%

C-

Bridge%

EO

CA%

(% w v-1)

metaphase%

464

25



Page 26 of 32

465

CA%=percentage of chromosome alterations; NA%=percentage of nuclear alterations; Lost%=percentage of lost chromosomes;

466

Sticky%=percentage of sticky chromosomes; C-metaphase%=percentage of c-metaphases; Bridge%=percentage of bridges; C-Poly%

467

=percentage of chromosome polyploidization.*Means followed by different letters present significant difference according to Dunnett’stest (P >

468

0.05); means followed by the same letter present similarity according to the same test.

26


Page 27 of 32


469

Figure graphics

470

120,00

471

100,00

472

80,00

Carvacrol(CAR) = 0,000057x 2 - 0,1562x + 103 R² = 0,8706 Thymol(THYM) = -0,0767x + 100,9 R² = 0,91

Essential oil (EO) = -0,0001x 2 + 0,0538x + 85,945 R² = 0,9891 Glyphosate (GLY) = 88,80 Boral = 72,00

473 474

60,00

40,00

475 20,00

(a)

476 0,00 0

477

200

400

600

800

1000

CAR

THYM

EO

GLY

Polinômio (CAR) Poly. (CAR)

Linear Linear(THYM) (THYM)

Polinômio Poly. (EO)(EO)

Linear (GLY)

1200

1400

Boral

-20,00

478

Linear (Boral) Linear (Boral)

479 100,00

480 481

Glyphosate (GLY) = 94,40 Bora l = 93,70

90,00 80,00 70,00

482

Carvacrol (CAR) = 86,769 +0,003x - 0,00004x² R² = 0,99

60,00 50,00

483

Thymol (THYM) = 84,738 + 0,032x - 0,00004x² R² = 0,99

40,00

Essential oil (EO) = 85,587 + 0,028x - 0,00006x² R² = 0,98

30,00

484

20,00 10,00

(b)

485 0,00 0

486 487

0,02

0,04

0,06

0,08

0,1

0,12

0,14

CAR

THY THYM

EO

GLY

Boral


Polinômio (THY) Poly. (THYM)

Polinômio (EO) Poly. (EO)

Linear Linea(GLY) r (GLY)

Linea r (Bora Linear (Boral)l)

488

Figure 1. Graphs of GR (percentage of germination) for Lactuca sativa (a) and

489

Sorghum bicolor (b) treated with solutions of Plectranthus amboinicus essential oil,

490

thymol and carvacrol.

491 492

27



Page 28 of 32

493 Carvacrol (CAR) = 11,490-0,021x+0,00001x² R = 0,94 Thymol (THYM) = 10,165 - 0,008x R² = 0,97

14,00

494 495 496

12,00

Essential oil (EO) = 10,076-0,008x R² = 0,94

10,00

Glyphosate (GLY) = 10,40

8,00

Boral = 6,48 6,00

497 4,00

498 499

2,00

(a) 0,00 0

500

0,02

0,04

0,06

0,08

0,1

0,12

0,14

CAR

THYM

EO

GLY

Boral


Linear Linear(THYM) (THYM)

Linear Linear(EO) (EO)

Linear Linear(GLY) (GLY)

Linear Linear(Boral) (Boral)

-2,00

501 502 503

8,00

Carvacrol(CAR) = 6,249 - 0,0006x - 0,000003x² Thymol(THYM) = 5,860 - 0,0007x - 0,000001x² R² = 0,99 R² = 0,94

Essential oil (EO) = 6,411 - 0,004x R² = 0,98

7,00

Glyphosate (GLY) = 6,84 Boral = 6,56

6,00

504 5,00

505 4,00

506

3,00

507

2,00

508

1,00

(b) 0,00

509 510 511

0

0,02

0,04

0,06

0,08

0,1

0,12

0,14

CAR

THYM

OIL

GLY

BORAL

Poly. (CAR) Polinômio (CAR)

Linear(THYM) (THYM) Linear

Linear(OIL) (EO) Linear

Linear(GLY) (GLY) Linear

Linear(BORAL) (Boral) Linear

512

Figure 2. Graphs of GSI (germination speed index) for Lactuca sativa (a) and Sorghum

513

bicolor (b) treated with solutions of Plectranthus amboinicus essential oil and thymol

514

and carvacrol.

515

516 28


Page 29 of 32


517

10,00

Carvacrol(CAR) = 9,311 - 0,0196x + 0,00001x² R² = 0,95 8,00

Thymol (THYM) = 8,42-0,013x+0,000006x² R² = 0,96

518

Essential oil (EO) = 8,44 - 0,00069x R² = 0,98

6,00

519 4,00

Glyphosa te (GLY) = 3,28

520

Boral = 2,15

2,00

(a) 0,00

521

0

0,02

0,04

0,06

0,08

0,1

0,12

0,14

-2,00

522

CAR

THYM

OIL

GLY

BORAL

Poly. (CAR) Polinômio (CAR)

Poly. (THYM) Polinômio (THYM)

Poly. (EO)(OIL) Polinômio

Linear (GLY) Linear

Linear (Bora l) Linear (BORAL)

523

524

8,00

Ca rvacrol(CAR) = 6,594 - 0,005x R² = 0,96

7,00

Thymol(THYM) = 6,192 - 0,013x + 0,000008x² R² = 0,80 6,00

525

Essential oil (EO)= 6,136 - 0,0118x + 0,000007x² R² = 0,78

5,00

4,00

526 3,00

2,00


527

Boral = 1,26 1,00

(b) 0,00

528

529

0

0,02

0,04

0,06

0,08

0,1

0,12

0,14

CAR

THYM

EO

GLY

Bora l

Linea r(CAR) (CAR) Linear

Linear (THYM) Poly. (THYM)

Polinômio Poly. (EO)(EO)

Linear Linear(GLY) (GLY)

Linear l) Linear(Bora (Boral)

530

Figure 3. Graphs of RG (root growth) for Lactuca sativa (a) and Sorghum bicolor (b)

531


532

533

534

535

29



Page 30 of 32

536 6,00

537

Carvacrol (CAR) = 5,582- 0,012x + 0,000006x2 R² = 0,86 Thymol (THYM) = 4,237-0,012x+0,000008x² R² = 0,91

5,00

Essential oil (EO)=4,204-0,006x - 0,0000003x 2 R² = 0,93

4,00

538 3,00

539

2,00

1,00


540

Boral = 0,00

0,00 0

541

-1,00

542

-2,00

0,02

0,04

0,06

0,08

0,1

0,12

0,14

(a) CAR

THYM

OIL

GLY

BORAL


Polinômio (THYM) Poly. (THYM)

Polinômio Poly. (EO)(OIL)


Linear Linear(BORAL) (Boral)

543 544 545

Carvacrol (CAR) = 5,273 + 0,001X - 0,000004x² R² = 0,97

6,00

Thymol(THYM) = 4,017 - 0,0024x R² = 0,72

546 5,00

Essentia l oil (EO) = 4,981 - 0,006x + 0,00004x² R² = 0,97

547 4,00

548 3,00

549 2,00

550 1,00

551

(b)

Glyphosate (GLY) = 0,00 Bora l = 0,00

0,00

552 553

0

0,02

0,04

0,06

0,08

0,1

0,12

0,14

CAR

THYM

OIL

GLY

BORAL


Linear (THYM) Linear (THYM)

Poly. (EO) Polinômio (OIL)


Linear(BORAL) (Bora l) Linear

554 555

Figure 4. Graphs of AG (aerial growth) for Lactuca sativa (a) and Sorghum bicolor (b)

556


30


Page 31 of 32


557 558

Figure 5. Abnormally dividing cells of L. sativa after exposure to carvacrol solution

559

(0.060%, w v-1). (A) stickiness metaphase; (B) anaphase bridge; (C) condensed nucleus;

560

(D

and

E)

laggard

chromosomes;

(F)

micro-c-metaphase.

Bar

=

5µm.

31



561

Page 32 of 32

TOC Graphic

562

563

564

565

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Plectranthus amboinicus, Carvacrol - American Chemical

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