Industrial Crops and Products 76 (2015) 416–421

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Industrial Crops and Products

journal homepage: www.elsevier.com/locate/indcrop

Chemical diversity and influence of plant age on the essential oil from
Lippia sidoides Cham. germplasm
Clesivan Pereira dos Santos a , Tereza Cristina de Oliveira b , Jéssika Andreza Oliveira Pinto a
, Saymo Santos Fontes a , Elizangela Mércia Oliveira Cruz a ,
Maria de Fátima Arrigoni-Blank a , Thiago Matos Andrade a , Iara Lisboa de Matos a ,
Péricles Barreto Alves a , Renato Innecco c , Arie Fitzgerald Blank a,∗
a
Universidade Federal de Sergipe, Cidade Universitária Professor José Aloísio de Campos, Avenida Marechal Rondon s/n, Rosa Elze, São Cristóvão, Sergipe
CEP 49100-000, Brazil
b
Embrapa Tabuleiros Costeiros, Av. Beira Mar, n◦ 3.250, Bairro Jardins, Aracaju, Sergipe CEP 49025-040, Brazil
c
Universidade Federal de Ceará, Campus do Pici, Avenida da Universidade 2853, Benfica, Fortaleza, Ceará CEP 60356-001, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Pepper-rosmarin (Lippia sidoides Cham.) is a medicinal and aromatic species that is native to northeastern
Received 9 March 2015 Brazil and has pronounced pharmacological, agronomical and economic importance. The objective of this
Received in revised form 13 July 2015 study was to evaluate the chemical diversity of L. sidoides genotypes from the Active Germplasm Bank of
Accepted 14 July 2015
the Federal University of Sergipe, and the influence of plant age on the content and chemical composition
Available online 26 July 2015
of its essential oil. Ten genotypes were analyzed in the years 2006 and 2012. The major compound of
the genotypes was thymol, except for genotype LSID104, which present carvacrol as major compound.
Keywords:
Age influenced the contents of essential oils in L. sidoides genotypes, and higher values were observed
Pepper-rosmarin
Genotype
in two-year-old plants. Variations were observed in the proportions of the chemical constituents of the
Chemical constituents essential oils of the genotypes because of the aging process. There was low genetic control for the essential
Thymol oil content; however, the chemical polymorphism in relation to the compounds thymol and carvacrol
Carvacrol was caused by genotypes. Two clusters were detected by multivariate analysis for both ages. Cluster I
was represented by the genotype LSID104, whereas cluster II was represented by genotypes LSID002,
LSID003, LSID004, LSID005, LSID006, LSID102, LSID103, LSID105 and LSID301.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction
The genus Lippia consists of approximately 200 species of
herbs, shrubs and small trees that are often aromatic (Terblanché
and Kornelius, 1996) and typically present chemical diversity in
the essential oils. The major identified compounds are thymol,
carvacrol, geranial, linalool, ␳-cymene, carvone, neral, limonene,
␤-caryophyllene, caryophyllene oxide, myrcene and ␥-terpinene,
which confer wide medicinal attributes to these species (Soares and
Tavares-Dias, 2013). Because of its biological activities, the essential
oil of Lippia sidoides is the only marketed oil of this genus.
Pepper-rosmarin (L. sidoides Cham. — Verbenaceae) is a shrubby
species that is native to semiarid northeastern Brazil and widely
used in traditional medicine. The plant occurs as a small tree or a
∗ Corresponding author. Fax: +55 79 21056494.
E-mail address: arie.blank@gmail.com (A.F. Blank).
deciduous, upright, densely branched and rather brittle shrub that
is 2–3 m tall. The leaves are 2–3 cm long, aromatic, spicy, simple and
petiolated. The yellowish-white flowers are small (1–2 mm) and
clustered in spikes with a short axis on the leaf axils. The fruits are
gathered in quadrangular sets around generally long axis (Lorenzi
and Matos, 2002).
The essential oil of L. sidoides has high pharmacological and
commercial value, and thymol or carvacrol monoterpenes are the
major constituents in the leaves (Almeida et al., 2010), which
present insecticidal, fungicidal, leishmanicidal, larvicidal, acaricidal
and anti-inflammatory activities (Carvalho et al., 2003; Cavalcanti
et al., 2010; Farias-Junior et al., 2012; Veras et al., 2012; Carvalho
et al., 2013; Lima et al., 2013). These compounds are responsible for
excellent antiseptic action against fungi and bacteria (Botelho et al.,
2007; Albuquerque et al., 2008); thus, L. sidoides leaves are widely
used in traditional medicine and phytotherapy programs in sev-
eral Brazilian regions because the plant provides powerful external
antiseptic properties (Matos and Oliveira, 1998).

http://dx.doi.org/10.1016/j.indcrop.2015.07.017
0926-6690/© 2015 Elsevier B.V. All rights reserved.
 C.P.d. Santos et al. / Industrial Crops and Products 76 (2015) 416–421
Because of its medicinal and economic importance, L. sidoides
provides an alternative source of income for local communities in
semiarid northeastern Brazil (Figueiredo et al., 2009). Because of
the trade of its essential oil, there has been increased interest in
the cultivation of this species. However, factors that influence the
content and yield of its essential oils and their chemical compo-
sition need to be study, such as climate and environment, harvest
season and time, post-harvest processing, developmental stage and
plant age.
The medicinal quality of the plant is primarily determined
by genetics. However, the plant does not behave the same way
throughout its life and undergoes changes that may cause vari-
ation in the concentration of its active components; changes in
the metabolite production and chemical composition are an effi-
cient response mechanism in L. sidoides to changing environments,
thus enabling their adaptation and survival (Chagas et al., 2011).
Therefore, when chemical variations are detected, it is important
to analyze and measure the amount of observed phenotypic vari-
ability is determined by genetic factors and edaphoclimatic factors,
which is indispensable information for the cultivation of and future
genetic improvements to L. sidoides.
Variations in plant age and nutritional availability are the most
significant factors influencing the chemical composition and con-
tent of essential oils (Martins et al., 2006). The plant’s age and
developmental stage may influence the total amount of secondary
metabolites as well as the relative proportion of these compounds
throughout the life of the plant. Younger plant tissue usually has
increased biosynthetic activity because of the increased production
of secondary metabolites, such as essential oils (Moraes, 2009).
Studies have shown that the aging process of plants may cause
a reduction in the content of essential oils and major compounds
(Matos and Innecco, 2002; Figueiredo et al., 2009; Oliveira et al.,
2012), whereas other studies have detected variations in the chem-
ical behavior of plants because of the age and development stage
and shown that age can promote increased levels of essential oils
and major constituents (Santos and Innecco, 2004). Therefore, such
studies are indispensable for determining the optimal conditions
for cultivating and harvesting aromatic species because such infor-
mation ensures that essential oils have the required quality and
safety because the market requires quality essential oils of a pre-
determined composition (Carvalho Filho et al., 2006).
The objective of this study was to evaluate the chemical diversity
of L. sidoides genotypes from the Active Germplasm Bank of the
Federal University of Sergipe, and the influence of plant age on the
content and chemical composition of its essential oil.
2. Materials and methods
2.1. Plant material and experimental design
The Active Germplasm Bank (AGB) of L. sidoides was established
at the Research Farm of the Federal University of Sergipe, called
“Campus Rural da UFS”, which is located in the municipality of São
Cristóvão, Sergipe State, Brazil, at latitude 11◦ 00 S and longitude
37◦ 12 W (Table 1).
The assay was conducted in a randomized block design, in a
split-plot scheme, with two replications. Each replication consists
of three plants with cultivation spacing of 1.0 × 1.0 m between
plants and blocks. In the plots 10 genotypes were tested (accessions
LSID002, LSID003, LSID004, LSID005, LSID006, LSID102, LSID103,
LSID104, LSID105, and LSID301 — see Table 1 for origin) and in the
split plots age of plants (2 and 8 years old plants, harvested in the
month June of the years 2006 and 2012) was tested. Defoliation
was performed manually between 2 pm and 4 pm, and leaves were
dried in a forced air oven at 40 ◦ C for five days (Ehlert et al., 2006).
417
2.2. Extraction and analysis of essential oils
The essential oils were extracted by hydrodistillation using a
modified Clevenger apparatus. Each sample consisted of 75 g of
dried leaves distilled for 140 min (Ehlert et al., 2006). The extracted
essential oil was properly assessed, collected and stored in amber
vials at −20 ◦ C until analysis.
The qualitative analysis of chemical composition was obtained
by gas-chromatography/mass-spectrometry (GC/MS; Shimadzu
QP5050A) with the following operating conditions: capillary
column DB-5 (5%-phenyl-95%-dimethylpolysiloxane) fused silica
capillary column (30 m × 0.25 mm i.d. × 0.25 ␮m film thickness),
connected to a detector operating at an electron impact of
70 eV; carrier helium gas at a flow rate of 1.2 mL/min; temper-
ature program of 50 ◦ C for 2 min, increase of 4 ◦ C/min–200 ◦ C,
15 ◦ C/min–300 ◦ C, and then 300 ◦ C held constant for 15 min; detec-
tor temperature (or interface) of 280 ◦ C; volume of 0.5 ␮L injected
into ethyl acetate (sample dilution: 10 mg/mL); and rate partition
of the injected volume of 1:100.
The quantitative analysis of the constituents was performed in a
Shimadzu GC-17A gas chromatograph equipped with a flame ion-
ization detector (FID) under the following operating conditions:
ZB-5 MS column (5%-phenyl-95%-dimethylpolysiloxane) fused sil-
ica capillary column (30 m × 0.25 mm i.d. × 0.25 ␮m film thickness),
using the same conditions as the GC–MS. The quantification of each
constituent was performed by normalization of the area (%), and
the compound concentrations were calculated by area and placed
in order of elution by the GC.
Identification of individual components of the essential oil was
performed by computerized matching of the acquired mass spectra
with those stored in NIST21, NIST107 and WILEY8 mass spec-
tral library of the GC–MS data system. A mixture of hydrocarbons
(C9 H20 C19 H40 ) was injected under these same conditions and
identification of constituents was then performed by comparing
the spectra obtained with those of the equipment data bank and
by the retention index, calculated for each constituent as previ-
ously described (Adams, 2007). Retention indices were obtained
with equation proposed by Van Den Dool and Kratz (1963).
2.3. Statistical analysis
The content and chemical composition data of the essential oils
from the L. sidoides genotypes were subjected to an analysis of vari-
ance (ANOVA), and the means were compared by the Scott-Knott
test at 5% probability, using the software Sisvar® .
The following genetic and phenotypic parameters were
estimated: genetic variance (Sg2 ), environmental variance (Se2 ),
genotype × environment variance (Sge 2 ), broad-sense heritability
2
(h ), coefficient of genetic variation (CVg ) and coefficient of
environmental variation (CVe ) for the content and chemical con-
stituents of L. sidoides essential oil were calculated with the
software Genes® . The following mathematical model was used:
Yij = m + Gi + Aj + GAij + ij , where-in Yij : mean of phenotypic value
of variable Y measured in the genetic material i, in the year j; m:
overall parametric mean of the studied data; Gi : effect of the ith
accession; Aj : effect of the jth year; GAij : effect of the interaction
between the ith accession and the jth year; ij : standard error of the
mean associated with the observation Yij .
The formulas of the estimated parameters are:
MSG − MSR
Sg2 =
ar
Se2 = MSR
418 C.P.d. Santos et al. / Industrial Crops and Products 76 (2015) 416–421

Table 1
Identification and geographical origin of Lippia sidoides genotypes maintained in the Active Germplasm Bank of the Federal University of Sergipe.

Code Scientific name Origin Geographical data Voucher N.

LSID002 L. sidoides Mossoró, Rio Grande do Norte State, Brazil 5◦ 07 26.7 S; 37◦ 24 14.6 W 8219
LSID003 L. sidoides Mossoró, Rio Grande do Norte State, Brazil 5◦ 08 28.3 S; 37◦ 23 58.0 W 8220
LSID004 L. sidoides Quixeré, Ceará State, Brazil 5◦ 05 03.5 S; 37◦ 58 43.9 W 8221
LSID005 L. sidoides Limoeiro do Norte, Ceará State, Brazil 5◦ 09 47.8 S; 38◦ 06 31.0 W 8222
LSID006 L. sidoides Tabuleiro do Norte, Ceará State, Brazil 5◦ 14 05.4 S; 38◦ 11 35.0 W 8223
LSID102 L. sidoides Poço Redondo, Sergipe State, Brazil 9◦ 58 07.6 S; 37◦ 51 49.2 W 8224
LSID103 L. sidoides Poço Redondo, Sergipe State, Brazil 9◦ 58 08.6 S; 37◦ 51 50.3 W 8225
LSID104 L. sidoides Poço Redondo, Sergipe State, Brazil 9◦ 58 09.2 S; 37◦ 51 50.3 W 8226
LSID105 L. sidoides Poço Redondo, Sergipe State, Brazil 9◦ 58 12.9 S; 37◦ 51 49.2 W 8227
LSID301 L. sidoides Recife (UFRPE), Pernambuco State, Brazil 8◦ 01 05.0 S; 34◦ 56 48.0 W 8228

  Pandeló et al. (2012) studied the biosynthesis of L. alba essential

2
Sg2
h = × 100
MSG/ar
  
100 Sg2
CVg =
m
  
100 Se2
CVe =
m
where in: MSG = mean square of genotype; MSR = mean square of
the residuals; ar = number of replicates; m = overall experimental
mean.
Two multivariate analyses were performed, clustering and prin-
cipal component analysis (PCA), using the software Statistica.
Subsequently, a dissimilarity matrix was constructed based on
the chemical constitution of the essential oils from each genotype
based on their Euclidean distances. The dissimilarity matrix was
simplified with dendrograms using Ward’s clustering method.
3. Results and discussion
The results showed that the content of L. sidoides essential
oils varied significantly among genotypes within each age group
(Table 2). In two-year-old plants, the highest essential oil content
was observed in the genotype LSID105 (7.40%), whereas the lowest
value was observed in LSID006 (4.90%) (Table 2). In the eight-
year-old plants, the highest essential oil content was observed
for genotype LSID102 (6.52%), whereas the lowest content was
observed in LSID002 (3.58%) (Table 2).
When the age of the genotypes (two and eight years) was com-
pared for essential content, a significant variation was observed
between the two ages for most of the genotypes, with higher con-
tent obtained in the two-year-old plants (Table 2). The greatest
variations were detected for the genotypes LSID004 (6.40–4.17%),
LSID105 (7.40–5.18%), and LSID002 (5.60–3.58%). The genotypes
LSID102, LSID103 and LSID104 did not present significant varia-
tion in the content of essential oils based on the age of the plant
(Table 2).
Figueiredo et al. (2009) found higher levels of L. sidoides essential
oils in the first harvest, at 180 days, in an experiment testing differ-
ent periods of harvest. In this study, the authors observed that the
concentration of essential oils decreased linearly with an increase
in the number of days after the first harvest, indicating that young
plants have a higher oil content but lower plant mass production.
Additionally, Oliveira et al. (2012) found a marked reduction in
the content of essential oils for the species Mentha piperita var. cit-
rata as the plants aged. According to Matos and Innecco (2002),
a proportional reduction occurs in the activity of metabolic path-
ways as the plant ages that reduces the synthesis of secondary
metabolites.
oils based on the expression of three candidate genes (LATPS12,
LATPS23 and LATPS25) that encode terpene synthases, and they
evaluated different times of leaf development and observed that
the maximum yield of essential oils in young leaves occurred at the
fourth node compared with leaves at the second and eighth node.
The aging of plant tissues is followed by a loss of vigor in physio-
logical processes; therefore, an intense reduction can occur in the
production of essential oils. Furthermore, biosynthetic processes
are more intense in young tissues, which was expected and also
observed in this study.
The distinct presence of two chemotypes was observed among
genotypes within each studied age: thymol chemotype and car-
vacrol chemotype. The LSID104 genotype presented a high content
of carvacrol in the two age groups (55.39 and 72.39% for 2 and
8 year-old plants, respectively) with a low content of thymol
(Table 2). The carvacrol content of this genotype increased from
55.39% to 72.39% in the eight-year old plants, and increased car-
vacrol was also found for the other genotypes in the same time
period (Table 2).
For the other genotypes, thymol was the major compound.
Among the two-year-old plants, genotype LSID103 had the high-
est content of thymol (83.20%), and genotype LSID104 had the
lowest content (8.41%) (Table 2). Among the eight-year-old plants,
genotype LSID301 had the highest content of thymol (75.79%), and
genotype LSID104 had the lowest content (8.88%) (Table 2). Age sig-
nificantly influenced the thymol content of most genotypes, with
a varied chemical behavior resulting from the aging process of the
plants. The genotype LSID105 had the greatest reduction in thymol
content, from 77.39% in the two-year-old plants to 62.60% in the
eight-year-old plants. However, the genotype LSID102 presented
a significant increase in thymol content with age, from 56.65% to
68.80%, for 2 and 8 year-old plants, respectively (Table 2). Statis-
tically significant variations in thymol content were not observed
with age in the genotypes LSID104 and LSID301. Thus, the Active
Germplasm Bank in this study has genotypes with major com-
pounds thymol and carvacrol.
For the secondary compounds myrcene, ␣-terpinene, ␳-cymene,
terpinen-4-ol, ␥-terpinene, thymol methyl ether, caryophyllene
oxide and (E)-caryophyllene, there was a significant variation
within and between ages (Table 2). The highest secondary com-
pound content recorded among genotypes between the 2 and 8
year-old plants, respectively, was as follows: myrcene in LSID102
(2.59%) and LSID105 (2.65%); ␣-terpinene in LSID104 (1.46%) and
LSID301 (0.87%); ␳-cymene in LSID102 (23.26%) and LSID102
(8.82%), terpinen-4-ol in LSID005 (0.87%) and LSID105 (1.86%); ␥-
terpinene in LSID104 (9.49%) and LSID104 (3.60%); thymol methyl
ether in LSID105 (8.13% and 15.0%); (E)-caryophyllene in LSID301
(6.84%) and LSID002 (7.92%); caryophyllene oxide in LSID301
(1.54%) and LSID003 (2.21%) (Table 2).
Between 2 and 8 year-old plants, the genotype LSID301 pre-
sented an increase of myrcene content from 0.87% to 1.28% and
 C.P.d. Santos et al. / Industrial Crops and Products 76 (2015) 416–421 419

Table 2
Means of the essential oil content and main chemical compounds of the essential oils from 10 Lippia sidoides genotypes according to ages of the plants (2 and 8 years-old).

Genotypes Compounds

Content (%) Myrcene ␣-Terpinene ␳-Cymene ␥-Terpinene Terpinen-4-ol Thymol methyl Thymol Carvacrol (E)-Caryophyllene Caryophyllene
ether oxide

2-year-old plants
LSID002 5.60 cA 0.85 bA 0.51 cA 7.24 cA 0.69 dB 0.74 aB 0.65 cB 77.95 bA 0.30 bB 4.74 bB 1.19 aA
LSID003 5.70 cA 0.83 bA 0.39 cA 8.44 cA 0.63 dB 0.67 aB 0.36 cB 79.30 aA 0.42 bB 5.23 bB 1.13 aB
LSID004 6.40 bA 0.91 bA 0.48 cA 8.13 cA 0.71 dA 0.69 aA 0.57 cB 79.60 aA 0.32 bB 4.86 bB 0.98 aB
LSID005 5.75 cA 1.03 bA 0.71 cA 7.70 cA 1.74 cA 0.87 aB 0.66 cB 76.22 bA 0.42 bB 5.21 bA 1.18 aB
LSID006 4.90 cA 0.85 bA 0.40 cA 8.00 cA 0.57 dB 0.67 aA 0.51 cB 80.91 aA 0.16 bB 4.81 bA 0.97 aB
LSID102 6.15 cA 2.59 aA 0.91 bA 23.26 aA 3.57 bA 0.69 aA 0.50 cB 56.65 cB 0.19 bB 0.42 cA 0.08 bA
LSID103 6.70 bA 2.57 aA 0.38 cA 8.18 cA 1.45 cA 0.59 aB 0.74 cB 83.20 aA 0.20 bB 0.32 cA 0.00 bA
LSID104 5.50 cA 2.44 aA 1.46 aA 13.81 bA 9.49 aA 0.81 aA 3.96 bA 8.41 dA 55.39 aB 0.08 cA 0.00 bA
LSID105 7.40 aA 2.29 aA 0.23 cB 7.68 cA 0.41 dB 0.72 aB 8.13 aB 77.39 bA 0.34 bB 0.27 cA 0.35 bA
LSID301 7.35 aA 0.87 bB 0.40 cB 6.67 cA 0.59 dB 0.78 aA 0.58 cA 76.70 bA 0.21 bA 6.84 aA 1.54 aA

8-year-old plants
LSID002 3.58 cB 0.57 cA 0.49 aA 5.07 cB 1.58 cA 1.76 aA 1.39 eA 69.33 bB 2.04 bA 7.92 aA 1.93 aA
LSID003 4.31 cB 0.78 cA 0.60 aA 5.54 cB 2.07 bA 1.77 aA 1.23 eA 72.29 aB 2.42 bA 7.55 aA 2.21 aA
LSID004 4.17 cB 0.45 cB 0.38 aA 4.13 cB 1.25 cA 0.94 cA 1.07 eA 73.56 aB 2.57 bA 6.12 bA 1.80 aA
LSID005 4.31 cB 0.57 cB 0.43 aB 4.69 cB 1.68 cA 1.36 bA 1.07 eA 69.74 bB 2.56 bA 6.32 bA 2.04 aA
LSID006 3.92 cB 0.56 cA 0.37 aA 3.93 cB 1.43 cA 1.12 cA 2.41 dA 71.56 a B 2.16 bA 5.74 bA 1.96 aA
LSID102 6.52 aA 0.58 cB 0.32 aB 8.82 aB 2.34 bB 1.05 cA 9.82 bA 68.80 bA 2.39 bA 0.79 cA 0.22 bA
LSID103 6.23 aA 2.42 aA 0.57 aA 6.62 bA 2.06 bA 1.24 cA 1.62 eA 73.28 aB 1.92 bA 0.93 cA 0.51 bA
LSID104 6.12 aA 0.61 cB 0.41 aB 4.42 cB 3.60 aB 0.91 cA 3.70 cA 8.88 dA 72.39 aA 0.16 cA 0.20 bA
LSID105 5.18 bB 2.65 aA 0.51 aA 7.00 bA 1.91 bA 1.86 aA 15.0 aA 62.60 cB 1.96 bA 0.89 cA 0.37 bA
LSID301 5.58 bB 1.28 bA 0.87 aA 6.45 bA 2.25 bA 0.18 dB 0.79 eA 75.79 aA 0.00 bA 5.09 bB 1.03 bA
CV-a (%) 5.72 18.51 26.54 11.28 16.11 16.09 14.42 2.64 3.39 10.54 28.53
CV-b (%) 6.14 12.59 21.13 11.04 17.80 21.69 5.41 2.06 7.84 14.87 34.73

Means followed by the same small letters in the columns and capital letters between ages of genotypes do not differ by the Scott-Knott test at 5% probability. CV-a — coefficient
of variation in the plots. CV-b — coefficient of variation in the split-plots.

␣-terpinene content from 0.40% to 0.87%; genotype LSID102 pre-
sented a decrease of ␳-cymene content from 23.26% to 8.82%;
genotype LSID104 presented a decrease of ␥-terpinene content
from 9.49% to 3.60%; genotype LSID105 presented an increase of
terpinen-4-ol content from 0.72% to 1.86% and thymol methyl ether
from 8.13% to 15.00%; genotype LSID002 presented an increase
of (E)-caryophyllene content from 4.74% to 7.92%; and genotype
LSID003 presented an increase of caryophyllene oxide content from
1.13% to 2.21% (Table 2).
For species Hyptis suaveolens (Martins et al., 2006) and Alpinia
zerumbet (Murakami et al., 2009), age also influenced the propor-
tion of volatile constituents in essential oils. According to Moraes
(2009), the age and developmental stage of the plant can redirect
the metabolic pathway and lead to the biosynthesis of different
compounds that might cause an increase of certain compounds.
In the ANOVA, significant differences were observed in the
source of genotype variation for all of the assessed compounds.
However, for the variation source ‘year’ and genotype × year inter-
action, a significant variation was found for all compounds except
for ␥-terpinene and caryophyllene oxide (Table 3).
The genetic variance of essential oil content (0.37) was less than
the effect of the genotype × environment interaction (GE) (0.47),
demonstrating that most of the variation in content is caused by the
environment. Heritability was moderate (39.37%), which indicated
that most of the phenotypic variation was caused by environmen-
tal variation (Table 3). However, high heritability (98.76%) for the
essential oil content was reported for Ocimum basilicum L., which
indicated strong genetic control and low environmental influence
(Blank et al., 2010).
The production and variability of essential oil content is primar-
ily determined by the genetic constitution of each genotype in each
region based on the absence of genetic exchange between these
areas (Martins et al., 2006). However, the production of essential
oils is a response mechanism and interaction of plants to a series
of external factors that influence their adaptation and survival;
therefore, production is predominantly determined by changes in
the environment and there is limited genetic control, which was
observed for L. sidoides.
The genetic variance was high and above the variance of interac-
tion (GE) for the major compounds thymol (428.19) and carvacrol
(387.91) and secondary compounds, such as thymol methyl ether,
(E)-caryophyllene and caryophyllene oxide. The coefficients of
genetic variation (CVg ) were also high, indicating that the genetic
variability in the content of these compounds is determined by
genetic factors with limited influenced by the environment (years)
(Table 3). However, the contents of myrcene, ␣-terpinene, ␳-
cymene, ␥-terpinene, terpinen-4-ol presented genetic variance
that was lower than 4.44 and below the variances of GE interac-
tions, indicating that these traits are strongly influenced by the
environment because of the plant age (Table 3).
High heritability was observed for thymol, thymol methyl ether,
(E)-caryophyllene and caryophyllene oxide and carvacrol (96.99%),
which indicated that the phenotypic variability of these compounds
is a result of genotypic variations that are most likely caused by
distinct alleles; therefore, these traits can be transmitted to future
generations (Table 3). However, moderate to low heritability (lower
than 48.77%) was observed for myrcene, ␣-terpinene, ␳-cymene,
␥-terpinene, and terpinen-4-ol (2.45%), which indicated that the
phenotypic variability of these compounds is primarily a result of
environmental factors (years); therefore, a limited amount of vari-
ability will be transmitted (Table 3). Blank et al. (2010) observed
a high heritability (71.45%) of the primary compound linalool in
O. basilicum L. populations. The chemical polymorphism among
genotypes for the major constituents thymol and carvacrol was
most likely an expression of genotypes with different alleles that
occurred during the biosynthesis of thymol and carvacrol.
According to the multivariate analysis performed for the chemi-
cal constituents of the essential oils of the genotypes, a dendrogram
was constructed that represented each age. At both assessed ages,
the formation of two distinct groups with dissimilarity (≥90%) was
detected and primarily represented by the compounds thymol and
carvacrol (Fig. 1).
420 C.P.d. Santos et al. / Industrial Crops and Products 76 (2015) 416–421

Table 3
Analysis of variance of both ages and estimation of genetic parameters, such as genetic variance (Sg2 ), environmental variance (Se2 ), variance of genotype/environment inter-
2
action (Sge ), broad-sense heritability (h2 ), coefficient of genetic variation (CVg ) and coefficient of environmental variation (CVe ) for essential oil content and the concentration
of chemical constituents of Lippia sidoides essential oils.

VS DF EOC MR TE CM TP TO TE TM CA CR CO

Genotype (G) 9 2.56** 2.02** 0.10* 37.15** 11.53** 0.24** 47.72** 1768.36** 1575.50** 31.48** 1.81**
Years (A) 1 13.24** 2.28** 0.08* 180.11** 0.01ns 2.46** 45.96** 255.17** 105.36** 7.66** 2.33**
G×E 9 1.05** 0.67** 0.20** 19.36** 5.05** 0.27** 10.50** 55.57** 23.84** 1.62** 0.25ns
Residue 20 0.43 0.22 0.04 7.42 0.26 0.06 0.45 10.61 0.65 0.98 0.18
Sg2 – 0.37 0.33 0.02 4.44 1.62 0.00 9.30 428.19 387.91 7.43 0.38
Se2 – 0.10 0.04 0.01 0.76 0.09 0.02 0.08 1.99 0.20 0.24 0.08
Sge
2
– 0.47 0.31 0.09 9.29 2.48 0.12 5.20 26.79 11.82 0.74 0.08
h2 (%) – 39.37 48.77 18.04 30.63 38.63 2.45 63.72 93.70 96.99 88.30 69.34
CVg (%) – 11.01 45.13 28.53 27.06 63.51 8.59 111.28 30.83 265.43 73.37 63.19
CVe (%) – 5.70 15.62 23.25 11.26 15.24 16.28 10.81 2.10 6.11 49.84 29.88

Variation source (VS), degree of freedom (DF), essential oil content (EOC), myrcene (MR), ␣-terpinene (TE), ␳-cymene (CM), ␥-terpinene (TP), terpine-4-ol (TO), thymol methyl
ether (TE), thymol (TM), carvacrol (CA), E-caryophyllene (CR), caryophyllene oxide (CO). * 5% Significance level by F-test; ** 1% significance level by F-test. ns: Not significant
by F-test.

Fig. 1. Clustering of Lippia sidoides accessions at two (A) and eight years (B) old
plants based on Ward’s clustering method. Fig. 2. Distribution of the chemical constituents of the essential oil of Lippia sidoides
in relation to according to a principal component analysis at two (A) and eight years
(B) old plants.

Cluster I of L. sidoides was represented by the genotype LSID104,

which was grouped alone in both ages (Fig. 1), and cluster II was
represented by genotypes LSID002, LSID003, LSID004, LSID005,
LSID006, LSID102, LSID104, LSID105 and LSID301, showing consid-
erable chemical similarity (Fig. 1). In the two-year-old plants, the
most divergent genotypes were LSID104 and LSID103, with 93.53%
of dissimilarity, whereas the most similar genotypes were LSID003
and LSID004, with 99.36% of similarity. In the eight-year-old plants,
the most divergent genotypes were LSID104 and LSID301, with
98.78% of dissimilarity, whereas the most similar genotypes were
LSID002 and LSID005, with 98.15% of similarity (Fig. 1).
The principal component analysis identified the compounds
that provided the greatest contribution to variation among the
genotypes at both assessed ages (Fig. 2). The first principal com-
ponent at two-years-old plants explained 57.00% of the total
variation, and was positively correlated with ␥-terpinene (r = 0.90),
␣-terpinene (r = 0.93), and carvacrol (r = 0.88) and negatively corre-
 C.P.d. Santos et al. / Industrial Crops and Products 76 (2015) 416–421
lated with thymol (r = −0.91) (Fig. 2A). In the eight-year-old plants,
the first principal component explained 41.36% of the variation,
and was positively correlated with myrcene (r = 0.76), p-cymene
(r = 0.83) and thymol methyl ether (r = 0.80) and negatively corre-
lated with caryophyllene oxide (r = −0.87), and (E)-caryophyllene
(r = −0.84) (Fig. 2B).
The second principal component explained 23.13% and 26.94%
of the total variance of harvests performed at two and eight-
years-old plants, respectively (Fig. 2). In the two-years-old
plants, the second principal component was positively corre-
lated with myrcene (r = 0.95), and negatively correlated with
(E)-caryophyllene (r = −0.97) and caryophyllene oxide (r = −0.97)
(Fig. 2A). In the eight-years-old plants, the second principal
component was positively correlated with thymol (r = 0.96), and
negatively correlated with carvacrol (r = −0.99) and ␥-terpinene
(r = −0.84) (Fig. 2).
Considering the similarities of the chemical constituents of the
essential oils of these 10 genotypes, the clusters were classified
as follows: cluster 1 included LSID002, LSID003, LSID004, LSID005,
LSID006, LSID102, LSID103, LSID105 and LSID301, which contained
the thymol as major compound (Table 2). Cluster 2 was formed
by genotype LSID104 and comprised carvacrol as major compound
(Table 2).
The correlation between thymol and carvacrol was negative and
very strong (−0.95 and −0.99 in two and eight-years-old plants),
suggesting that these constituents that are present in the essen-
tial oil of L. sidoides are closely correlated, indicating that, possibly,
when selecting a genotype with high thymol content the carvacrol
content will be low.
The major compound of the L. sidoides genotypes was thy-
mol, except for genotype LSID104, which present carvacrol as
major compound. Age influenced the contents of essential oils in
L. sidoides genotypes, and higher values were observed in two-
year-old plants. Variations were observed in the proportions of the
chemical constituents of the essential oils of the genotypes because
of the aging process. There was low genetic control for the essen-
tial oil content; however, the chemical polymorphism in relation
to the compounds thymol and carvacrol was caused by genotypes.
Two clusters were detected by multivariate analysis for both ages.
Cluster I was represented by the genotype LSID104, whereas clus-
ter II was represented by genotypes LSID002, LSID003, LSID004,
LSID005, LSID006, LSID102, LSID103, LSID105 and LSID301.
Acknowledgements
The authors thank CNPq, FAPITEC/SE, CAPES and FINEP for their
financial support of this work.
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