Yield and Phytochemical Characterization of Essential Oil from Ocimum

selloi B. Obtained by Hydrodistillation and Supercritical Fluid Extraction

L. Aparecida Salgado de Morais1,a, Lin Chau Ming2, M. Ortiz Mayo Marques3 and
M.Â. Almeida Meireles4
1
Embrapa Meio Ambiente; Laboratório de Produtos Naturais, Rod. SP 340, Km 127,5,
Bairro Tanquinho Velho, Jaguariúna, SP, CEP 13820-000, Brazil
2
Faculdade de Ciências Agronômicas, UNESP, Campus Fazenda Experimental Lageado,
Departamento de Horticultura, Caixa Postal 237 - CEP 18610-307, Botucatu, SP, Brazil
3
Centro de Pesquisa e Desenvolvimento de Recursos Genéticos Vegetais, Caixa Postal
28, CEP 13001-970 Campinas, SP, Brazil
4
Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos,
Departamento de Engenharia de Alimentos, Caixa-Postal: 6121, CEP 13083-862,
Campinas, SP, Brazil

Keywords: methyl-chavicol; trans-anethole; SFE; organic treatment; mineral treatment.

Abstract
The yield and chemical composition of essential oils from leaves of Ocimum
selloi B. submitted to organic and mineral fertilization, obtained by hydrodistillation
and supercritical fluid extraction (SFE) were compared. Essential oil was extracted
in a Clevenger-type apparatus for 2 h 30 min and analyzed by GC-MS (Shimadzu,
QP 5050-DB-5 capillary column - 30 m × 0.25 mm × 0.25 μm). Carrier gas was
helium (1.7 ml/min); split ratio: 1:30. Temperature program: 50°C, rising to 180°C
at 5°C/min, 180°C, rising to 280°C at 10°C/min. Injector temperature: 240°C and
detector temperature: 230°C. Identifications of chemical compounds were made by
matching their mass spectra and Kovat’s indices (IK) values with known compounds
reported in the literature. An Applied Separations-apparatus (Speed SFE, model
7071, Allentown, PA, EUA) was used for SFE extractions. They were conducted at
pressure 200 bar and temperature 30°C (20 min in static mode and 40 min in
dynamic mode). The supercritical CO2 flow rate was (6.8±0.7)×10-5 kg-CO2/s. The
essential oil collected was immersed in ethylene glycol bath (5°C). The yield of
essential oils obtained by SFE was larger than hydrodistillation in both fertilization
treatments (279 and 333% for organic and mineral fertilizations, respectively).
There were no differences between the fertilization treatments. The amount of the
volatile components showed by GC-MS chromatogram was highest in the essential
oil obtained by hydrodistillation than SFE. The main volatile constituents of the
essential oils were trans-anethole (Hydrodistillation: organic - 52.4%; mineral -
55.0%/ SFE: Hydrodistillation - 62.8%; mineral - 66.8%) and methyl-chavicol
(Hydrodistillation: organic - 37.3%; mineral - 38.3%/ SFE: organic - 8.4%; mineral
- 4.3%). A reduction of methyl-chavicol relative proportion of essential oil obtained
by SFE was observed. Cys-anethole, α-copaene, trans-cariofilene, germacrene-D,
β-selinene, biciclogermacrene and spathulenol were expressed only in hydrodistilla-
tion. The extraction of essential oil by SFE presented larger yield of essential oil than
hydrodistillation technique, presenting, however, these essential oils, different phyto-
chemical profiles.

INTRODUCTION
Ocimum selloi B. is a native species from Brazil that produces essential oil. It
presents potential use in popular medicine and it is known as “elixir paregórico” in Rio de
Janeiro and Espírito Santo States, as “anis and alfavaquinha” in Minas Gerais State and
“atroveran” in São Paulo State, Brazil. These popular terms are used because their
chemical and pharmacological properties, and its similarity to other species of the
a
lilia@cnpma.embrapa.br

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Acta Hort. 925, ISHS 2011
Ocimum genus. It has been used as anti-diarrhea, anti-spasmodic and anti-inflammatory.
These properties were observed in pre-clinic assays (Vanderlinde et al., 1994).
The extraction of essential oils by supercritical fluid extraction has been used as an
alternative technique to conventional process, because this methodology reduces the
problems with decomposition, due the utilization of lower extraction temperatures; it
produces organic solvents residues free extracts and it permits to increase the yield of
extraction and to obtain total extracts or enriched fractions of active principle, by
changing operational conditions (pressure, temperature and others).
In despite of the potential use of Ocimum selloi as flavorizant and mainly as a
medicinal plant, there are few studies about this species, under phytochemical and
agronomic aspects. So, studies about techniques of cultivation of O. selloi on aerial
phytomass production, yield and chemical composition of essential oil can contribute to
establish agronomical parameters of this species.
Research about supercritical fluid extraction of essential oil will furnish relevant
information that can contribute to increase the yield of essential oil or its main
compounds. It could increase the value of raw material.
The aim of this work was to compare the yield and chemical composition of
essential oils from leaves of Ocimum selloi B. submitted to organic and mineral
fertilization, obtained by hydrodistillation and supercritical fluid extraction (SFE).

MATERIAL AND METHODS

The experiment of growth in the field was conducted at the Experimental Farm
Lageado/Faculdade de Ciências Agronômicas (FCA), UNESP, Botucatu District, SP-
Brazil. Drying of raw material and extraction of essential oils by hydrodistillation was
developed in the Department of Plant Production, Horticulture, FCA, UNESP. Analyses
of chemical composition of essential oils were carried out at the Natural Products
Laboratory of Center for R&D Plant Genetic Resources of Instituto Agronômico de
Campinas (IAC) and the extraction of essential oils by supercritical fluid extraction (SFE)
was accomplished on LASEFI (Laboratory of Supercritical Technology: Extraction,
Fractionation, and Identification of vegetable extracts) that belongs to the Department of
Food Engineering (DEA), College of Food Engineering (FEA), State University of
Campinas (UNICAMP), São Paulo State, Brazil.
Plant samples collected were submitted to organic fertilization (cattle manure,
5000 kg/ha) and mineral fertilization (NPK - ammonium sulfate, 75 kg/ha; single
superphosphate, 50 kg/ha; potassium chloride, 105 kg/ha). For essential oil extraction,
dried leaves (50.0 g) and inflorescences (30.0 g) were submitted to hydrodistillation in a
Clevenger-type apparatus for 2 h 30 mim. Extraction time was optimized through
preliminary tests, observing the volume of essential oil (every 30 min), considering the
optimal time, when no more changes were observed in the total volume. Essential oils
extracted were transferred to transparent glass vials (5 ml) with stopper and screw cap,
pre-weighed on an analytical balance (AND, model HR-120) and kept in a freezer until
the moment of chemical composition analysis. Extractions were performed in triplicate.
The yield of essential oils was expressed as the average value of three replicates and
calculated using the following equation:

% yield=[Average mass of essential oil (g) × 100]/part of plant mass (g) (1)

For the extraction with supercritical carbon dioxide, dried leaves of Ocimum selloi
were triturated in a mill (Tecnal mod. TE-631 /1, Brazil) at 20,500 rpm for 15 s. Particle
size distribution was determined using sieves of 24, 32, 48, 80 and 100 mesh with the aid
of a sieve shaker (Bertel, CATEL, Caileiras, São Paulo State, Brazil). In the extraction
experiments, mixture of particles with size distribution from 32 to 80 mesh were used.
Essential oil extractions from O. selloi were realized on Applied Separations apparatus
(Speed SFE, model 7071, Allentown, PA, EUA), in an extraction column with capacity of
5 ml (Thar Designs, CL 1165, Pittsburgh, PA, EUA, with internal volume of 6 ml).

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Triturated raw material (2.51±0.01 g), composed by a mixture of granulometric
distribution of 32 mesh (26±2%), 48 mesh (52±1%) and 80 mesh (21±2%) was added in
the column extraction and packaged (height: 0.019 m; length: 0.020 m) with the help of a
steel shank (diameter: 0.014 m). Extraction column was connected to the equipment.
Pressure (200 bar) and temperature (30°C) operating conditions were set, keeping the
static period of carbon dioxide (CO2, 99.98% purity, Gama Special Gases, Campinas, São
Paulo State, Brazil), in contact with the particles for 5 min. The flow of CO2 was
(6.8±0.7) × 5.10 kg CO2/s. The essential oil was collected in glass bottle (50 ml)
immersed in a bath of ethylene glycol (5°C) in order to reduce losses of more volatile
components. Extraction time was about 1 h, approximately. After removal of the column
system, cleaning of the line extraction with ethyl acetate (P.A., Lot. 55893, LabSynth, São
Paulo, Brazil) was carried out and the concentrated solution, placed on the rotary
evaporator (Heildoph Instruments GMBH, Laborota 4001, Schwabach, Germany) with
vacuum controller (Heildoph Instruments GMBH, Rotavac/Rotavac Control, Schwabach,
Germany) and stored in a glass flask. Yield of essential oil was determined by weighing
the storage and cleaning flasks (Sartorius, model A200S, ±0.0001 g, Goettingen,
Germany), at the beginning and at the end of extraction and after solvent concentration
(ethyl acetate), respectively.
The essential oil extracted was analyzed by GC-MS (Shimadzu, QP-5000, with
DB - 5 - capillary column: 30 m × 0.25 mm × 0.25 μm). Helium was used as the carrier
gas at the constant flow of 1.7 ml/min. The samples were injected in split mode (split ratio
1:30) and the column temperature was programmed from the initial temperature of 50°C,
rising to 280°C (50°C (5 min)-180°C (5 min); 180-280°C (10°C/min) (Moraes et al.,
2002). The injector temperature was 240°C and detector temperature was 230°C. The
mass spectrometer was operated in the 70 eV. Essential oils were dissolved in ethyl
acetate (5 mg oil/1 ml solvent).
Identifications of chemical compounds were made by matching their mass spectra
with the database of the GC-MS (Nist 62 Libr), literature (Mclafferty and Stauffer, 1989)
and Kovats retention index (Adams, 1995). The data of yield and chemical composition
were submitted to ANOVA, followed of Tukey’s test.

RESULTS AND DISCUSSION

The yield of essential oils obtained by supercritical fluid extraction was higher
than hydrodistillation (Table 1), corresponding to 279 and 333% for organic and mineral
treatments, respectively. Regardless of the treatment used in cultivation (organic and
mineral fertilizers) the chemical composition of essential oils of Ocimum selloi obtained
by two techniques are different (Table 2). Essential oils obtained by hydrodistillation
showed high amount of compounds compared to essential oils obtained by supercritical
fluid extraction. The major compounds observed for two techniques were trans-anethole
(52.4 a 66.8%) and methyl-chavicol (4.3 and 38.3%), with a reduction in the relative
proportion of methyl chavicol extracted by supercritical fluid extraction.
Lower number of compounds, mainly the most volatiles, on essential oils obtained
by supercritical fluid extraction may be due to loss by drag at the moment of gas
decompression. This problem could be minimized by using two alternatives: capture of
volatiles in a glass column with adsorbent material, Porapaq for example, after the
collector flask of essential oil or change in operating conditions (pressure, temperature
and flow of the solvent). In the essential oils extracted supercritical fluid extraction, a
heavier fraction is present, and it is composed by higher molecular weight not identified
substances (28.8 and 28.9%). This factor (heavy fraction) associated with a high yield of
supercritical fluid extraction indicates that this technique may be drawing other classes of
substances not belonging to essential oils. Results like these were noticed by Rodrigues et
al. (2003). Essential oil of fennel obtained by supercritical fluid extraction showed
besides essential oil compounds, including the major component (anethole), fatty acids
and hydrocarbons after fractionation of oil-resin by preparative thin layer chromato-
graphy.

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CONCLUSION
The results obtained by this assay showed that yield of essential oil obtained by
supercritical fluid extraction (SFE) was higher than hydrodistillation, but differences in
the chemical characteristics of essential oils were observed.

Literature Cited
Adams, R.P. 1995. Identification of essential Oil Components by Gas Cromatography/
Mass Spectroscopy, Allured Publ. Corp, Carol Stream.
McLafferty, F.W. and Stauffer, D. 1989. The Wiley/NBS Registry of Mass Spectral Data,
v. 1-6, John Wiley Sons, New York.
Moraes, L.A.S., Facanali, R., Marques, M.O.M., Ming, L.C. and Meireles, M.A.A. 2002.
Phytochemical characterization of essential oil from Ocimum selloi. An. Acad. Bras.
Ciênc. 74:183-186.
Rodrigues, V.M., Rosa, P.T.V.E., Marques, M.O.M., Petenate, A.J. and Meireles, M.A.A.
2003. Supercritical extraction of essential oil from aniseed (Pimpinella anisum L.)
using CO2: solubility, kinetics, and composition data. Journal of Agricultural and Food
Chemistry. Estados Unidos 51:1518-1523.
Vanderlinde, F.A., Costa, E.A. and D’angelo, L.C.A. 1994. Atividades farmacológicas
gerais e atividade antiespasmódica do extrato etanólico de Ocimum selloi Benth,
(elixir paregórico). In: Anais do Simpósio de Plantas Medicinais do Brasil, Fortaleza,
UFCE.

Tables

Table 1. Yield of essential oils of Ocimum selloi leaves extracted by hydrodistillation and
supercritical fluid extraction (200 bar and 30°C).

Yield (%)
Techniques
Organic treatment Mineral treatment
Hydrodistillation 0.83 0.81
Supercritical fluid extraction 2.32 2.70

Table 2. Chemical composition of essential oils of leaves from Ocimum selloi submitted
to organic and mineral treatments, extracted by hydrodistillation and supercritical fluid
extraction (200 bar and 30°C).

%
Hydrodistillation Supercritical fluid extraction
Substances (%)
Organic Mineral Organic Mineral
treatment treatment treatment treatment
Methyl chavicol 37.3 38.3 8.4 4.3
Cys-anethole 1.6 1.7 -- --
Trans-anethole 52.4 55.0 62.8 66.8
α-copaene 1.0 0.5 -- --
Trans-cariofilene 1.7 0.9 -- --
Germacrene D 1.1 0.5 -- --
β-selinene 1.9 1.1 -- --
Bicyclogermacrene 1.8 0.9 -- --
Spathulenol tr 0.4 -- --
Cariophyllene oxide 0.4 0.4 -- --
N.I. 0.8 0.3 28.8 28.9
tr = traces <0.20%; NI = not identified.

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