Industrial Crops & Products 125 (2018) 236–240
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Industrial Crops & Products
journal homepage: www.elsevier.com/locate/indcrop
Chemical composition and antimicrobial activity of the essential oil of Lippia
lasiocalycina Cham. (Verbenaceae)
T
Wanessa Sales de Almeidaa, , Sidney Gonçalo de Limad, Humberto Medeiros Barretob,
Leila Maria de Sousa Andradeb, Lorenna Fonsecad, Candido Athayde Sobrinhoc,
Ananda Rosa Beserra Santosc, Maria Christina Sanches Muratoria
⁎
a
Food and Nutrition Postgraduate Program, Federal University of Piauí, CampusMinistro Petrônio Portela, 64049-550, Teresina, PI, Brazil
Postgraduate Program in Pharmaceutical Sciences, Federal University of Piauí, CampusMinistro Petrônio Portela, 64049-550 Teresina, PI, Brazil
c
Brazilian Agricultural Research Corporation, Av. Duque de Caxias, no. 5650 CEP: 64008-780, Teresina, PI, Brazil
d
Laboratory of Organic Geochemistry, Postgraduate Program in Chemistry, Federal University of Piauí, Campus Ministro Petrônio Portela, 64049-550 Teresina, PI, Brazil
b
A R T I C LE I N FO
A B S T R A C T
Keywords:
Piperitenone oxide
Candida albicans
Limonene
Lippia
The use of plant species of the genus Lippia in the treatment of diseases is an old practice, however, some species
still needs studies. Thus, the present work aimed to characterize chemically and to evaluate the antimicrobial
activity of the essential oil of Lippia lasiocalycina. The oil was extracted by hydrodistillation and the analysis of
the chemical constituents done using gas chromatography coupled to the mass spectrometer. Minimum inhibitory concentrations were determined by microdilution method against Staphylococcus aureus, Escherichia coli
and Candida albicans strains. The constituents present were identified (90.09%) and piperitenone oxide was
defined as the major compound (57.55%) followed by limonene (20.69%). The essential oil of Lippia lasiocalycina
presented activity against C. albicans strain, signaling for a potential application in the treatment of infections
caused by this yeast.
1. Introduction
The use of medicinal plants has been constant since the origin of
mankind. (Boukhatem et al., 2014). Among the substances of natural
origin, essential oils are very important both economically and scientifically, being versatile products with applicability in the most varied
sectors (Bernardos et al., 2015).
Essential oils are rich in bioactive substances (Medeiros et al.,
2011). Its chemical composition is complex and presents a wide variety
of constituents like monoterpenes, sesquiterpenes, and their derivatives
such as aldehydes and phenols. This composition varies between plant
species and seasons of the year. (Hajlaoui et al., 2010). In plants, the
essential oils are directly related to the processes of pollination, dissemination of seeds, and in defense against attacks of herbivores as well
as fungi and bacteria (Costa et al., 2015; Li et al., 2013).
The Verbenaceae family has approximately 36 genera of plants and
1000 plant species distributed in pantotropical regions. Brazil is the
country with the greatest diversity of taxon with 16 genera and about
290 species. The plants of this family usually present in the form of
herbs, shrubs, sub-shrubs and lianas (Costa et al., 2017). Among the
⁎
genus belonging to this family, we can highlight Lippia, constituted by
200 species that exhibits a striking appearance and pleasant odor
(Oliveira et al., 2006). The genus Lippia is widely used in folk medicine
in gastrointestinal disorders and respiratory diseases. The infusion and
the essential oil of various parts of plants is used as antifungal, antimicrobial, larvicide, and anesthetic agents (Linde et al., 2010).
In recent years, we have seen the emergence of a problem that
permeates the treatment of various diseases, the phenomenon of microbial resistance. This resistance has rapidly proliferated by involving
Gram-positive and Gram-negative bacteria, such as Staphylococcus and
Escherichia coli (Silveira et al., 2006) and opportunistic fungal species
like Candida albicans (Casto and Lima, 2011).
Although the advances in research on the chemical and pharmacological properties of the Lippia genus, there are still species that needs
clarifying studies such as Lippia lasiocalycina. To the best of our
knowledge, there have been just one literature sources reporting the
preliminary study of the chemical constituents of L. lasiocalycina alcohol extract (Funari et al., 2012). No information on the biological
activities or chemical composition of the essential oil is available.
Therefore, the chemical characterization and determination of the
Corresponding author.
E-mail address: wanessa.salmeida@yahoo.com.br (W.S. de Almeida).
https://doi.org/10.1016/j.indcrop.2018.09.007
Received 2 May 2018; Received in revised form 2 September 2018; Accepted 4 September 2018
0926-6690/ © 2018 Elsevier B.V. All rights reserved.
Industrial Crops & Products 125 (2018) 236–240
W.S. de Almeida et al.
prepared by dissolving 10.000 μg of the product in 1 mL−1 of dimethylsulfoxide (DMSO). This initial solution was then diluted in sterile
distilled water to a concentration of 1024 μg mL−1. MIC of the LLEO
was determined by microdilution in BHI with microbial suspensions of
105 CFU mL−1 and with LLEO at concentrations 8, 16, 32, 64, 128, 256,
512 μg mL−1. Microplates were incubated at 37 °C for 24 h. MIC was
defined as the lowest concentration of the drug in which no microbial
growth is observed. All experiments were done in triplicate. The control
group used in this study consisted of culture medium and inoculum
without the addition of essential oil. The control of the inoculum and
the control of the sterility of the culture medium were made.
Antifungal assays were performed by the microdilution method in
SDB double concentrated with a yeast suspension of 105 CFU mL−1 and
LLEO solutions ranging from 8 to 512 μg mL−1. Microplates were incubated at 37 °C for 48 h.
antimicrobial activity of the essential oil of Lippia lasiocalycina was the
objective of the present study.
2. Experimental
2.1. Plant material
The plant material was collected at 9:00 am in the Gilbués city,
Piauí State, Brazil, in April 2016. The region presents subhumid tropical
climate with high temperatures and low rainfall index being located in
the semi-arid region of northeastern Brazil.
Plant material was made available by the Brazilian Agricultural
Research Company - EMBRAPA Meio-Norte (-5.036410, -42.797898).
Voucher specimen was deposited in the Embrapa Herbarium Genetic
Resources and Biotechnology - CEN under the number CEN92437.
2.2. Essential oil extraction
2.6. Determination of the minimum microbicide concentration
Lippia lasiocalycina essential oil (LLEO) was obtained from fresh
leaves of the plant by hydrodistillation using Clevenger type distillation
apparatus for approximately 3 h. The essential oils yields ranged from
0.40 to 0.52%. At the end of the process, the resulting oil was collected,
dried with sodium sulphate, weighed, and stored under refrigeration.
The oil was solubilized in H2CCl for gas chromatography and mass
spectrometry analysis.
Inhibition of the bacterial or fungal growth was confirmed transferring an aliquot of 10 μl from each well of the MIC test microplates to
a Petri dish containing BHIA (or SDA) and checking cell viability after
incubation at 37 °C for 24 to 48 h. Minimal microbicide concentration
(MMC) was defined as the lowest concentration of the drug in which no
microbial growth was observed.
2.3. Chromatography conditions
3. Results and discussion
For the characterization of the chemical composition of the volatile
compounds of LLEO, a gas chromatograph, coupled to the mass spectrometer (GC–MS) was used. A Shimadzu® Chromatograph, model
CGMS-QP2010 SE equipped with AOC-5000 automatic injector and
SLB-5 ms column (30 m x 0.25 mm x 0.25 μm) was used. The conditions
for the CG-MS analysis were as follows: Helium as carrier gas at a flow
rate of 1 mL min−1, a temperature of 250 °C in the injector; a temperature program, starting at 60 °C (3 min), at a rate of 3 °C / min until
reach 240 °C (for 10 min); the detector temperature was 250 °C.
Previously the essential oil was diluted into dichloromethane (1:10)
and so 1 μL was injected. The MS conditions were triple quadrupole
type of ion detector operating by electronic impact (70 eV, 45–450 Da).
Identification of the essential oil components was performed by
comparing their GC–MS retention indices. The spectra were considered
coincident if the similarity index was equal to or greater than 90%. The
Kovats index was estimated by comparison between some known
compounds in the chromatogram and the compatible Kovats indices of
the database records (WILEY, NIST, PHEROBASE).
In the last years we have seen a change in the habits of life of the
population, a change that includes a greater concern with the quality of
the food ingested as well as to the safety of the industrialized products.
At the same time, the industry has been looking for options to reduce
the amount of synthetic chemicals in its products, signaling a global
trend towards adherence to more natural means of conservation and
food production (Machado et al., 2011). In this scenario, essential oils
appear as a promising alternative. One of the main factors that make
them interesting for the food industry is the fact that these oils are
mostly used routinely in cooking or in folk medicine, making their
acceptability much higher when compared with synthetic additives
(Pereira et al., 2006).
The volatile compounds of L. lasiocalycina essential oil were detected for the first time by GC–MS. Representing 90.09% of the essential
oil fourteen constituents were identified of which the major ones, piperitenone oxide (57.55%) and limonene (20.69%), accounted for
78.24% (Table 1).
The chemical composition of species of the genus Lippia has been
extensively studied. Gonçalves et al. (2015) and Guimarães et al. (2014)
2.4. Microbial strains
Table 1
Constituents of Lippia lasiocalycina essential oil analyzed by GC–MS.
Evaluation of the antimicrobial activity of EOLL was performed
against standard strains Staphylococcus aureus ATCC 25923, Escherichia
coli ATCC 25922 and Candida albicans ATCC 10231, as well as, against a
multidrug-resistant S. aureus strain (SA-1199B). Bacterial strains were
maintained on Brain Heart Infusion Agar (BHIA, Himedia, India) slant
at 4 °C, and prior to assay the cells were grown overnight at 37 °C in
Brain Heart Infusion (BHI, Himedia, India). The yeast strain was
maintained on Sabouraud Dextrose Agar (SDA, Himedia, India) slant at
4 °C and prior to assay the cells were grown for 24 h at 37 °C in
Sabouraud Dextrose Broth (SDB, Himedia, India).
2.5. Determination of the minimum inhibitory concentration
The determination of the minimum inhibitory concentration (MIC)
was performed according to the microdilution method (CLSI, 2003). In
this assay, 96-well microplates (12 columns and 8 lines) with flat
bottom were used. A stock solution of the test product was previously
RTa
Compound
KIb
Area
3.677
7.121
8.816
10.195
10.399
10.986
11.538
19.849
21.998
22.354
24.199
25.283
31.175
31.597
Methylbenzene
α-pinene
Myrcene
p-Cimene
Limonene
Trans-β-ocimene
γ-Terpinene
Carvone
m-Thymol
Tridecane
Piperitenone
Piperitenone oxide
Bicyclogermacrene
β-Bisabolene
770
939
991
1026
1031
1050
1062
1242
1290
1299
1342
1365
1494
1509
0.31
1.17
0.20
0.47
20.69
1.01
0.18
0.26
0.83
1.03
3.04
57.55
2.67
0.68
a
b
237
Retention time.
Kovats index.
Industrial Crops & Products 125 (2018) 236–240
W.S. de Almeida et al.
found carvacrol and 1,8-cineol as major components for Lippia sidoides.
Costa et al. (2005) when analyzing the same species found thymol and
α-phellandrene. Nogueira et al. (2007), in a study carried out in Paraná,
Brazil, found citral and trans-dihydrocarvone while Silva et al. (2006)
identified citral as the main component of Lippia alba. Previous researches verified that essential oil from aerial parts of L. origanoides
collected in different Brazilian states showed carvacrol and thymol as
major chemical components (Sarrazin et al., 2015a; Queiroz et al.,
2014; Borges et al., 2012).
A variation of the essential oil composition is influenced by several
factors, such as place of cultivation, soil type, climate, temperature,
luminosity and collection time (Alencar-Filho et al., 2017; Bitu et al.,
2015; Sarrazin et al., 2015b). The chemical profile may vary both between species and interspecies (Lima et al., 2016; Morais, 2009). In
addition, some constituents appear as typical of certain species while
others are considered as an exception.
Piperitenone oxide (PO) is a natural oxygenated monoterpene, also
known as rotundifolone, used in different products such as detergents,
creams, and lotions. In the food industry, it is usually used as a flavoring
agent (Sotto et al., 2017). The growing market demand for exempt food
products or with a reduction in the amount of synthetic chemicals used
in their production and conservation has increased the industry's interest in alternatives that have better consumer acceptance (Galo et al.,
2018). Thus, the presence of PO in high concentration in LLEO beckons
to a new vegetable source of this substance commonly used in food
flavoring, appearing as a more natural option to its synthetic analogues.
Besides that, several pharmacological activities have been evidenced
for PO including antiviral activity against herpesvirus type 1 (Civitelli
et al., 2014), repellent action against Aedes aegypti (Tripathi et al.,
2004), thevasodilatory effect in hypertensive rats (Lan et al., 2017),
anticancer potential (Nakamura et al., 2014) and antiparasitic action
(Teles et al., 2011). Piperitenone oxide is not a common occurring
compound in the genus Lippia being. It is most common in different
species of mint such as Mentha spicata and Mentha rotundifolia. Some
Lippia species may exhibit piperitenone oxide as a minor component
(Bozovic et al., 2015), but their presence as a major constituent is not a
common finding.
Among the species of the genus Lippia the PO was recently identified
in Lippia pedunculosa at a concentration higher than 70% (Nascimento
et al., 2017). The presence of PO as the major constituent of the LLEO
(57.55%) is an unprecedented discovery, indicating that it is a good
source of this compound and signalizing for a range of possible technological applications for the LLEO.
Terpenes are considered the secondary metabolites most produced
by plants. This class of substances is often used in the pharmaceutical
and solvent industries. (Wang et al., 2016). They have a great structural
diversity and can exert crucial functions for plant development, being
usually involved in defense mechanisms (Srividya et al., 2015; Viegas
Júnior, 2003). Limonene, the second main compound found in the
LLEO, is a monocyclic monoterpene present in more than 300 plant
species. It is present in two possible conformations, S-(-)-limonene and
R-(+)-limonene, and both are directly related to inhibition of growth of
microorganisms, especially fungi (Duetz et al., 2003). In citrus essential
oils the presence of the R-(+)-limonene isomer is more common, while
S-(-)-limonene is more common herbs and some plant varieties as in
species of the Mentha genus (Maróstica Júnior and Pastore, 2007). In
the essential oil market, limonene is one of the most exported from
Brazil to European Union countries with the main purposes being the
use of cleaning products. From 2005 to 2008, 427 t of limonene were
exported, generating millions of dollars of revenue (Bizzo et al., 2009).
Natural products from plants are considered as having a good inhibitory activity if they present MICs ≤100 μg/mL−1, a moderate inhibitory activity if they present MICs ranging from 100 to 500 μg/
mL−1, a weak inhibitory activity if they present MICs ranging from 500
to 1000 μg/mL−1, and no inhibitory activity if they present
MICs > 1000 μg/mL−1 (Holetz et al., 2002). According to these
Table 2
MIC and MMC of Lippia lasiocalycina essential oil on microbial strains.
Strains
MICa (μg/mL−1)
S. aureus ATCC
25923
S. aureus
SA1199-B
E. coli ATCC
25922
C. albicans
ATCC
10231
≥1024
–
–
No activity
≥1024
–
–
No activity
≥1024
–
–
No activity
512
512
1
Fungicide
a
b
MMCb
(μg/mL−1)
MMC/MIC
Inhibitory
Effect
Minimum inhibitory concentration.
Minimum microbicide concentration.
criteria, the results obtained from assays to evaluate the antimicrobial
activity (Table 2) showed that LLEO was inactive against both Grampositive and Gram-negative bacterial strains.
On the other hand, the oil was active against the yeast strain tested
(Table 2), and although this activity, it is considered as weak, its inhibitory effect was fungicide. This result indicates that LLEO is a source
of phytochemicals that could be used in the prevention or treatment of
mycoses in human and animal hosts, as well as, they could be used for
prevention of food contamination by fungal species. The antifungal
activity of the LLEO could be attributed to the presence of hydrophobic
monoterpenes, such as PO and limonene, as well as, other minor
components which are able to cause lipid partitioning of cell membrane
and increasing its permeability to essential electrolytes (Bakkali et al.,
2008; Duetz et al., 2003; Oumzil et al., 2002).
Candida spp. are commensal yeasts commonly found in mucous
membranes and epithelial tissues, however species of the genus Candida
can lead to opportunistic fungal infections, being Candida albicans the
most involved (Gullo et al., 2016; Castro and Lima, 2011). The low host
immunity combined with the versatility of surviving in the most different environments and conditions is directly related to the transition
from a harmless microorganism to an infectious one (Cassone, 2015; Li
et al., 2015). Yeasts have several virulence factors such as the ability to
adhere to tissues, the possibility of converting unicellular yeasts into
filamentous forms, a hydrophobic cell wall and the production of some
extracellular enzymes that help in the expression of their pathogenicity
(Ishida et al., 2006; Calderoni and Fonzi, 2001).
Several drugs are used in the treatment of mycotic infections.
Antiseptics such as iodine tincture and gentian violet are examples of
some substances very employed as antimycotics. The development of
pharmacological research has enabled the discovery and use of more
modern antifungal drugs such as ketoconazole, miconazole, amphotericin B, and clotrimazole (Lima et al., 2006). However, extensive use
has led to resistance to treatment and difficulties in combating opportunistic fungal infections (Barbosa et al., 2016). Protein mutations that
reduce the ability of drugs to interact and bind to the cell wall and the
active efflux of drugs out of the cell by means of membrane-expressed
transporters, and pumps are some mechanisms developed by Candida
albicans to circumvent the action of major antifungal agents (Schneider
and Morschhauser, 2015; Shafreen et al., 2014).
Given the increasing occurrence of microorganisms that express
resistance to drug therapies, alternatives should be sought to enable the
treatment of these conditions efficiently. The use of plant products,
such as essential oils, has been identified as one of the most promising
sources, both for the discovery of new drugs and as an aid to existing
ones (Guimarães et al., 2017; Oliveira et al., 2016; Lucena et al., 2015).
The fact that they are a complex mixture of substances allied to action
at multiple target sites makes the development of resistance to essential
oils an unlikely event (Sales et al., 2014). In addition, the synergistic
effect between essential oils and synthetic drugs can inhibit efflux
pumps (Barreto et al., 2014) and eliminate plasmids that are involved in
238
Industrial Crops & Products 125 (2018) 236–240
W.S. de Almeida et al.
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4. Conclusion
The essential oil of L. lasiocalycina presented a high concentration of
piperitenone oxide and fungicide activity on strains of Candida albicans
being a good source for future research aimed at the development of
new drugs and as a source of obtaining flavorings used in foods.
Funding
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-prof it sectors.
Declarations of interest
None.
Acknowledgments
We acknowledge the Coordination for the Improvement of Higher
Education Personnel (CAPES), Laboratory of Geochemistry Analysis
(LAGO) of the Federal University of Piaui, and EMBRAPA Meio-Norte.
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