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Original Article
Biosci. J., Uberlândia, v. 33, n. 6, p. 1608-1616, Nov./Dec. 2017
SEASONAL EFFECT IN ESSENTIAL OIL COMPOSITION AND
ANTIOXIDANT ACTIVITY OF Plectranthus amboinicus LEAVES
EFEITO SAZONAL NA COMPOSIÇÃO E ATIVIDADE ANTIOXIDANTE DO ÓLEO
ESSENCIAL DE MALVARISCO (Plectranthus amboinicus)
Rita de Cássia de Freitas BEZERRA¹; Francisco Barros de OLIVEIRA NETO²;
Francisco Felipe Maia da SILVA³; Luciana Medeiros BERTINI
4
;
Leonardo Alcântara ALVES
4
1. Mestranda do Programa de Pós-graduação em Energias Renováveis – IFCE, Instituto Federal de Educação, Ciência e Tecnologia do
Ceará, Macaranaú, CE, Brasil; 2. Graduado em Licenciatura Plena em Química, Instituto Federal de Educação, Ciência e Tecnologia do
Rio Grande do Norte - IFRN, Apodi, RN, Brasil; 3. Docente do curso de Licenciatura Plena em Química – IFRN, Apodi, RB, Brasil; 4.
Docentes do Programa de Pós-graduação em Ensino (POSENSINO), em associação ampla entre a Universidade do Estadual do Rio
Grande do Norte – UERN, Universidade Federal do Semi-Árido – UFERSA e IFRN, Apodi, RN, Brasil. leonardo.alcantara@ifrn.edu.br
ABSTRACT: In view of the increasing search for natural products with biological activities, such as essential
oils are of high quality because of their therapeutic and economic importance and represent the second class of natural
compounds with the largest number of active constituents and production through Plants minimizes the environment and
sanitary impacts . The objective of the present work is to assess the seasonal effect on the composition and antioxidant
activity of essential oil in leaves of Plectranthus amboinicus (Lour.) Spreng using hydro and steam distillation techniques.
With regarding to the extraction methodology of essential oils from the P. amboinicus, the hydrodistillation technique is
the most efficient in obtaining the volatile product with the best features for the determination of antioxidant activity. The
analyses of the essential oil indicated a similarity in their composition, being observed the Carvacrol as major compound
in almost all analysis, except in January 2015 using steam distillation extraction. Regarding antioxidant activity and
seasonality, it was verified that in October 2014 obtained the best value of inhibitory concentration, with IC50 = 124.97
ppm. However, it was possible to obtain the essential oil from the P. amboinicus leaves through hydro and steam
distillation methodologies, but also noted the seasonality influence on it’s the context of antioxidant capacity.
KEYWORDS: Antioxidant activity. P. amboinicus. Extraction methods. Essential oil. Seasonal
INTRODUCTION
It is known that the use of plants for
therapeutic purposes began thousands of years ago
and, currently, this usage has been intensifying,
even with advances in medicinal drug. This is
because these natural medicines are bringing
positive results to the population that seeks in these
products an alternative healing and disease
prevention.
Inserted in this context, Brazil gains
prominence because it presents a large and diverse
flora, besides having the largest equatorial and
tropical rain forest on the planet, making it a good
location for the exploitation of natural products. The
Natural Products Chemistry (NPC) is, within the
chemistry, the oldest area and that groups the largest
number of researchers (PINTO et al., 2002).
However, it is important the development of
research that seek isolation, characterization and
identification of these compounds present in plants
with medicinal properties and pharmacological
activities. Thus, it is noticed the importance of
multidisciplinary research that aggregate their
chemical, microbiological, pharmacological, and
biotechnological knowledges (FOGLIO et al.,
2006).
Among these studies, it can be cited the
determination of the chemical composition from the
essential oils and volatile compounds obtained from
plants and animals. The aforementioned show great
relevance in industrial, medicinal, and other areas,
because they are potentially useful in phytosanitary
control, resulting in the development of techniques
that seek to reduce the negative effects of oxidants,
radicals, and microorganisms that cause losses in the
food industries and in agriculture (BAKKALI et al.,
2008; MATTEI et al., 2014).
Antioxidants can be defined as those that
inhibit and/or reduce the effects triggered by free
radicals and oxidant compounds in oxidable
substrates. These substances can be enzymatic, such
as superoxide dismutase, catalase, and glutathione
peroxidase, or not enzymatic, such as α-tocopherol
(vitamin E), β-carotene, ascorbate (vitamin C), and
phenolic compounds (HALLIWELL, 2001). It is
remarkable that research with interest in finding
natural antioxidants that may be used in food
products or with pharmaceutical purposes have
increased significantly. This occurs due to the
Received: 04/01/17
Accepted: 05/07/17
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intention to replace the synthetic antioxidants that,
in turn, have shown their limited use due to the
carcinogenic potential (ALMEIDA et al., 2006;
MELO; GUERRA, 2002; SIMÃO, 1985; ZHENG;
WANG, 2001).
In this way, the essential oils are being
increasingly studied as antioxidant agents, in an
attempt to spur the development of techniques that
seek to reduce the negative effects of oxidative
substances (SANTOS, 2004).
Ruberto and Baratta (2000), in their work
with approximately 100 compounds that compose
the essential oils, found that, in general, their
antioxidant activity may be relevant when
associated with phenolic components, followed by
the oxygenated constituents and allyl alcohols
present in their composition. However, many studies
have reported the antioxidant properties of essential
oils, referring to them as potential substitutes for
synthetic antioxidants and, in some cases,
suggesting them to a direct application in food,
farming, pharmaceutical and cosmetic products
(ANDRADE et al., 2012).
In this sense, the genus Plectranthus
(L'Her.), from the family Lamiaceae, is considered
one of the richest in essential oils, having as main
constituents the mono- and sesquiterpenes
(ABDEL-MOGIB et al., 2002). From the 300
species of the genus Plectranthus already identified,
62 are mentioned because they have medicinal,
food, flavoring, antiseptic, and repellent properties,
besides for using as pastures and as ornamental
plants (LUKHOBA; SIMMONDS; PATON, 2006).
Plectranthus amboinicus (Fig. 1), also
known as Indian borage, is a medium-sized herb,
whose leaves are used in the preparation of
homemade syrups for treating cough, sore throat and
bronchitis, and in the treatment of wounds caused
by cutaneous leishmaniasis. The juice of the leaves
is used to treat ovarian and uterine problems,
including cases of cervicitis (LORENZI; MATOS,
2002; COSTA, 2003). Figure 1 shows the plant
species P. amboinicus.
Figure 1. Plant species Plectranthus amboinicus
Thereby, this study aimed to perform
seasonal extraction of the essential oil present in
leaves of P. amboinicus (Lour.) Spreng using hydro
and steam distillation techniques, as well as to
evaluate the influence of the seasonal effect in
determining the composition and antioxidant
activity of the obtained volatile oil.
MATERIAL AND METHODS
Plant Material
The plant material was cultivated and
collected in Instituto Federal de Educação, Ciência e
Tecnologia do Rio Grande do Norte (IFRN), Apodi,
RN, Brazil (05º37’34” S, 37º48’34” W). The leaves
of P. amboinicus were initially ground and then
weighted. Essential oil extraction was performed
from April 2014 to January 2015, being the plant
material always collected in the morning every three
months.
Hydrodistillation Extraction
The essential oil was extracted from 450 g
of the ground plant material, which was placed in a
round-bottom flask containing approximately 1 L of
distilled water and, subsequently, maintained for 2 h
uninterrupted in boiling, using a distiller of
Clevenger-type. The essential oil was separated by
decantation, dried with anhydrous sodium sulfate
(Na2SO4), weighted and collected in amber vials.
The material was stored in a freezer for chemical
analysis.
Steam Distillation Extraction
A total of 1.0 kg of P. amboinicus leaves
were ground and placed in extractor (SILVA et al.,
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2014), containing 10 L of distilled water. The
material was heated for 4 h uninterrupted. The
essential oil was separated by decantation, collected,
dried with anhydrous sodium sulfate (Na2SO4) and
weighted. The material was stored in a freezer for
chemical analysis.
Chemical Analysis Of The Essential Oil
The oil samples were analyzed in a gas
chromatography Shimadzu model KP 5050A
coupled to a mass detector using a DB-1 capillary
column (30 m x 0.25 mm, i.d. x 0.25 µm film
thickness) in the following: carrier gas hydrogen
flow rate 1.7 mL/min and with split mode at ratio
1:27, ion source temperature 280ºC, ionizations
voltage 70 eV, electron impact detection. The
thermal conditions of the column were from 40ºC to
180ºC at 4ºC/min and then 180ºC to 280ºC at
20ºC/min, and held isothermal for 7 min.
The essential oils were analyzed by GC-MS
and individual components was identified
comparing the mass spectrum obtained with the
Wiley library, simulation of the Kovat index
(ALENCAR et al., 1990), as well as visual
comparison with the standard of fragmentation of
spectra of mass described in the literature (ADAMS,
2007) and the database provided with the mass
spectra (SciFinder).
Antioxidant Activity
The evaluation of the antioxidant capacity
of the essential oils from the leaves of P.
amboinicus was performed through the capture
method of DPPH radicals (2,2-diphenyl-1-
picrylhydrazyl) described by Cosmoski et al. (2015)
with alterations. It was weighted 0.01 g of the
volatile oil diluted in 10 mL of methanol. From this,
solutions of 1000, 500, 100, 50, and 10 ppm were
prepared. A rate of 1.0 mL from each solution was
removed and added into test tubes containing 1.0
mL of methanolic solution of DPPH 60µM. The
solutions were left at room temperature and in the
absence of light for 30 minutes. Readings were
performed in spectrophotometer UV TEN-KA T-
2000 at 520 nm wavelength, having as white a
solution containing 1.0 mL methanol and 1.0 mL
DPPH solution. All analyses were performed in
triplicate, as for the essential oil obtained by
hydrodistillation as for steam distillation method.
The inhibition percentage (%In) for each sample
was obtained from the use of the absorbance values:
Where:
% In: Inhibition percentage of the extract.
Abs. DPPH: Absorbance obtained with
sample DPPH/methanol.
Abs. sample: Absorbance obtained from
sample.
RESULTS AND DISCUSSION
After the completion of seasonal extraction
of essential oil from the leaves of P. amboinicus,
using the methods of hydro and steam distillation, it
was possible to obtain the efficiency of each oil,
described in Table 1. Efficiencies were calculated
by the ratio between the weights of obtained oil and
the fresh plant material.
Table 1. Efficiency of the essential oil from the leaves of P. amboinicus obtained in different methods and
periods
Months
Extraction methods /Obtained efficiency (%)
Hydrodistillation Steam distillation
April 2014 0.0512 0.0046
July 2014 0.2371 0.0109
October 2014 0.0386 0.0066
January 2015 0.0117 0.0022
According to Table 1, for the analyzed
methods, hydrodistillation showed the highest
essential oil content. With regard to the month, July
2014 obtained the best efficiency of the essential oil
from the leaves of P. amboinicus in both extraction
methods, 0.2371 % in hydrodistillation and
0.0109% in steam distillation.
In studies conducted by Bandeira et al.
(2011), Oliveira et al. (2011) and Rodrigues et al.
(2013) were obtained 0.19%, 0.43%, and 0.3%,
respectively, using hydrodistillation as extraction
method. According to Reis (2012), the extraction
conditions are determining factors for the efficiency,
chemical composition and quality of essential oil.
% In = Abs. DPPH – Abs. sample x 100%
Abs. DPPH
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Concerning the composition of the essential
oil (Table 2) , the Carvacrol is the major component
in leaves of P. amboinicus during almost the entire
year, especially April 2014, showing a level of
73.4%. Only in January 2015, using the steam
distillation technique, it was not possible to identify
the Carvacrol as major substance. With regard to the
most efficient methodology to obtain the essential
oil from the leaves of P. amboinicus, it is reaffirmed
that the hydrodistillation method proved to be more
effective, since in the referred methodology the
major constituent appeared in higher concentrations
in all extraction periods.
However, from the seasonal extraction of
essential oil from leaves of P. amboinicus, as well
as the characterization and quantification of their
constituents, it was verified that there is a similarity
in their chemical composition, showing some
changes in different periods. In addition, it was
possible to observe that the chemical composition of
the essential oil from the leaves of P. amboinicus
can be characterized both by the extraction method
and by the period in which the same was realized.
The antioxidant capacity of essential oils
obtained by the methods of hydro and steam
distillation were evaluated according to the capture
method of radicals 2,2-diphenyl-1-picrylhydrazyl
(DPPH), proposed by Cosmoski et al. (2015). The
values of IC50 for each essential oil are described in
Table 3.
It was verified that the hydrodistillation
technique resulted in a better antioxidant capacity.
In the referred extraction methodology, in the
months of April 2014 and October 2014, the best
values were obtained of IC50, 132.6 and 125.0 ppm,
respectively. It is noteworthy that these were the
months where the largest concentrations of
Carvacrol were observed. Furthermore, only in the
month of July 2014 was not observed significant
antioxidant activity for the essential oils obtained
from this method. It was noted that in the month of
January 2015 the worst values of inhibitory
concentrations with IC50 = 352.1 ppm in
hydrodistillation and 595.0 ppm in steam
distillation.
It is proved that the harvest season can
influence the efficiency and composition of essential
oil in plants. Several studies report the physiological
reactions of the plant metabolism due to climatic
aspects: time of year and their characteristics
associated with rates of temperature, rainfall and
humidity. It is important to point out that there is a
positive correlation between well-established
intensity of solar radiation and the production of
phenolic compounds. The different plant species are
adapted to a huge variation in the intensity and
quantity of light incidence (GOBBO NETO;
LOPES, 2007).
Andrade et al. (2012), when examining the
antioxidant capacity of the essential oils of
Cymbopogon nardus (citronella grass),
Cinnamomum zeylanicum (true cinnamon), and
Zingiber officinale (ginger), using the same
methodology of this research obtained the results
below. For the citronella grass, the inhibitory
concentration value was 517.4 ppm and significant
antioxidant activity was not observed for the
essential oils of true cinnamon and ginger.
Therefore, from the values found in this
research, it can be affirmed that the essential oil
from the leaves of P. amboinicus presents a good
antioxidant capacity when compared to other oils.
However, the period and the extraction method of
volatile oil are crucial for better inhibitory
concentrations.
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Table 2. Seasonal analysis of the chemical composition of the essential oil of P. amboinicus by steam and hydrodistillation
Steam distillation Hydrodistillation
Months April 2014 July 2014 October 2014 January 2015 April 2014 July 2014 October 2014 January 2015
Compound Content (%) Content (%)
Isoterpinolene 0.3 - - - - - - -
p-Cimene 6.1 - - - - - - -
γ-Terpinene 9.6 4.6 - - 7.1 5.4 3.9 -
Terpinen-4-ol 0.5 - - - 1.4 - - -
Carvacrol 27.3 40.0 34.0 - 73.4 46.8 68.1 48.0
Caryophyllene 26.1 17.2 19.1 - 4.9 4.3 8.1 -
α-Bergamotene 16.3 11.7 16.1 - - 2.4 5.3 -
Humulene 7.1 - - - 1.4 - - -
Caryophyllene oxide 3.8 2.5 7.9 3.5 1.0 1.1 2.6 9.4
Phytol 0.2 - 1.1 - - - - -
β-Myrcene - 0.3 - - 0.5 - - -
α-Terpinene - - - - 1.1 - - -
o-Cymene - 4.2 - - 5.1 6.5 4.9 -
Sec-butyl acetate - 0.2 - 1.0 - - - -
Isobutyl acetate - 6.4 - 30.0 - 18.3 - 15.3
o-xylene - 0.7 - - - 1.8 - -
α-cytral - 1.1 2.4 - - - - -
α-caryophyllene - 4.8 6.2 - - 1.0 2.4 -
β-bisabolene - 0.7 - - - - - -
n-decane - - - - - 0.7 - -
(+)-4-carene - - - - - 0.9 - -
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β-cytral - - 1.2 - - - - -
4-terpineol - - - - - - 2.0 -
Tridecanone - - 2.5 - - - - -
Isopentyl - - - 1.3 - - - -
Toluene - - - 41.6 - - - 20.6
m-xylene - - - 1.3 - - - 0.7
( + ) - Spathulenol - - - 1.9 - - - -
Pentadecane-2-one - - - 1.5 - - - -
1,2-Benzenedicarboxylic
acid
- - - 6.0 - - - -
Cedrol - - - 1.3 - - - -
Total Monoterpenes 43.8 50.2 37.6 - 88.6 59.6 78.9 48.0
Total Sesquiterpenes 53.3 36.9 49.3 6.7 7.3 8.8 18.4 9.4
Total Diterpenes 0.2 - 1.1 - - - - -
Others - 7.3 2.5 82.7 - 20.8 - 36.6
Totalcomposition (%) 97.3 94.4 90.5 89.4 95.9 89.2 97.3 94.0
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Table 3. Antioxidant activity obtained by DPPH method of the essential oils from the leaves of P. amboinicus
Months
Extraction methods / IC50 (ppm)
Hydrodistillation Steam distillation
April 2014 132.6 309.4
July 2014 NI NI
October 2014 125.0 NI
January 2015 352.1 595.0
IC50= inhibiting concentration 50%; NI = not present inhibition
CONCLUSIONS
As regards the extraction methodology of
essential oils from the leaves of P. amboinicus, the
hydrodistillation technique is the most efficient in
obtaining the volatile product by offering better
efficiency and antioxidant activity. However, it is
important to note that the extractions performed by
steam distillation, despite the values found are lower
than those of hydrodistillation, are considered
satisfactory when compared to other works from
literature.
It was also verified that the Carvacrol might
be the main compound that influences the
antioxidant activity of studied oil.
RESUMO: Tendo em vista a crescente busca de produtos naturais com atividades biológicas, os óleos
essenciais apresentam alta qualidade devido à sua importância terapêutica e econômica e representam a segunda classe de
compostos naturais com o maior número de constituintes ativos com produção através das plantas, o que minimiza os
impactos ao meio ambiente. O objetivo do presente trabalho foi avaliar o efeito sazonal na composição e na atividade
antioxidante do óleo essencial presente nas folhas da Plectranthus amboinicus (Lour.) Spreng utilizando as técnicas de
hidrodestilação e arraste a vapor d’água. No que se refere à metodologia de extração dos óleos essenciais do malvarisco,
constatou-se que a técnica de hidrodestilação é a mais eficiente na obtenção do produto volátil com as melhores
características para a determinação da atividade antioxidante. Quanto à sazonalidade, verificou-se que no mês de
Outubro/2014 obteve-se o melhor valor da concentração inibitória com CI50=124,97 ppm. Este trabalho mostra que é a
obtenção do óleo essencial das folhas do malvarisco, utilizando as metodologias de hidrodestilação e arraste a vapor de
água, como também, constatar a influência da sazonalidade na determinação da capacidade antioxidante do mesmo.
PALAVRAS-CHAVE: Atividade antioxidante. P. amboinicus. Métodos de extração. Óleo essencial. Sazonal
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