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Phytomedicine 15 (2008) 334339

www.elsevier.de/phymed

Antinociceptive activity of the volatile oils of Hyptis pectinata L. Poit.

(Lamiaceae) genotypes
M.F. Arrigoni-Blanka,, A.R. Antoniollib, L.C. Caetanoc, D.A. Camposd,
A.F. Blankd, P.B. Alvese
a
Nucleo de Ciencias Biologicas, Campus Prof. Alberto Carvalho, Universidade Federal de Sergipe, Itabaiana,
Av. Vereador Olmpio Grande s/n, 49500-000 Sergipe, Brazil
b
Departamento de Fisiologia, Universidade Federal de Sergipe, Sao Cristovao, Sergipe, Brazil
c
Departamento de Qumica, Universidade Federal de Alagoas, Maceio, Alagoas, Brazil
d
Departamento de Engenharia Agronomica, Universidade Federal de Sergipe, Sao Cristovao, Sergipe, Brazil
e
Departamento de Qumica, Universidade Federal de Sergipe, Sao Cristovao, Sergipe, Brazil

Abstract
Hyptis pectinata L. Poit (Lamiaceae) is known popularly in Brazil as sambacaita or canudinho and is used in
the treatment of inammations, bacterial infections and ache. The antinociceptive activity of the volatile oils of six
genotypes, at doses of 100, 200 and 400 mg/kg body wt., were investigated using abdominal writhe models induced by
acetic acid and hot plate tests. The volatile oils of all the genotypes are composed mainly of sesquiterpenoids. All the
genotypes showed antinociceptive effects in both models used; the SAM002 genotype showed the major inhibitory
effect at dose of 100 mg/kg body wt. These results suggest that the volatile oil of H. pectinata has peripheral (writhe
reduction) and central (time delay of thermal reaction) effects. These observations indicate that H. pectinata may be
useful as an analgesic drug.
r 2007 Elsevier GmbH. All rights reserved.

Keywords: Hyptis pectinata; Acetic-acid-induced abdominal constrictions; Hot-plate test; Acute toxicity; Native medicinal plant

Introduction in traditional medicine (Calixto, 2005). Volatile oils

The use of natural products with therapeutic proper-
ties is as ancient as human civilization (Esquenazi et al.,
2002). Most people living in developing countries are
almost completely dependent on traditional medical
practices for their primary health care needs and higher
plants are known to be the main source for drug therapy
Corresponding author. Tel.: +55 79 3431 2410;
fax: +55 79 2105 6494.
E-mail address: fatima.blank@terra.com.br
(M.F. Arrigoni-Blank).
from a broad spectrum of plant species have shown
antinociceptive, antiinammatory, antimicrobial, anti-
viral, antitumoral and antioxidant activities (Asekun
et al., 1999; Allahverdiyev et al., 2004; Sousa et al., 2004;
Al-Burtamani et al., 2005).
The northeast region of Brazil contains a large
diversity of native plant species that are known for
their medicinal properties and their common use in
popular medicine. With increased use of the land for
agriculture and exploitation of natural resources, many
of these species are in danger of being lost before they
have been studied. They include Hyptis pectinata L.

0944-7113/$ - see front matter r 2007 Elsevier GmbH. All rights reserved.
doi:10.1016/j.phymed.2007.09.009
 ARTICLE IN PRESS
M.F. Arrigoni-Blank et al. / Phytomedicine 15 (2008) 334339 335

Poit. (Lamiaceae), known popularly as sambacaita is
used as tea, decoction and infusion, mainly as a natural
anti-inammatory.
H. pectinata is an odoriferous plant that grows on
wild land or is cultivated near homes in Alagoas and
Sergipe states. Because of its popular use as an anti-
inammatory, studies have been carried out to ascertain
the antinociceptive and antiedematogenic effects of the
aqueous leaf extract (Bispo et al., 2001), and antimicro-
bial activity of the volatile oil of the leaves (Asekun
et al., 1999), validating its popular use.
Other studies have shown that genetic variation
among the same species can alter the concentration of
active principles. Other factors, in addition to genetics,
such as climate, soil, seasonality, time and type of
culture, and fertilizer, may affect the composition of
active principles. Such variations were veried among
species of Hyptis suaveolens that were characterized by
the evaluation of the yield of the volatile oil and its
chemical constituents in different genotypes found in the
Brazilian cerrado (Azevedo et al., 2002). No studies
have been reported to date on Hyptis pectinata
concerning the variations in chemical constituents, yield
and pharmacological activity of its volatile oil.
The objective of this work was therefore to evaluate
the chemical composition and antinociceptive activity of
the volatile oils of six genotypes of H. pectinata from the
Active Germplasm Bank at Universidade Federal de
Sergipe-UFS.
Material and methods
Plant material
Aerial parts of six genotypes of Hyptis pectinata in
different stages of maturation collected from the active
germplasm bank in June 2002 were dried in an oven with
circulating air at 40 1C. The origin of the genotypes is
described in Table 1. Samples of each genotype have
been deposited in the Herbarium of the Universidade
Federal de Sergipe, Sao Cristovao-SE, Brazil.
Volatile oil analysis
Volatile oils of dried leaves from six genotypes were
obtained via hydrodistillation on a Clevenger-type
apparatus (Guenther, 1972) for approximately 3 h.
Samples of oils were analysed by GC/MS in a Shimadzu
QP5050A with a capillary column DB-5 (30 m 
0.25 mm  0.25 mm) connected to an electron impact
detector at 70 eV; helium (ow rate 1 ml/min) was used
as carrier gas with the following program: 80 1C (1 min),
3 1C/min, 180 1C (0 min), 10 1C/min, 300 1C (3 min), type
of injection split 1:100. The retention index calculations
were carried out through co-injections with n-alkanes.
The identication of the constituents of the volatile oils
was based on the retention time index (Adams, 1995)
and comparison with the mass spectral data bank
library NIST21 and NIST107. Concentrations of the
respective constituents were calculated using the area of
the signal in the GC spectra according to the order of
elution.
Analgesic activity
Animals
White Swiss mice of both sexes weighing 2530 g each
were used distributed in groups of 10 (writhing test) and
8 (hot-plate test) animals for treatment. The animals
received Purina ration and water ad libitum throughout
the experiment.
Drugs and treatment
The effects of the volatile oils from the six genotypes
of H. pectinata were tested. Doses of 100, 200 and
400 mg/kg body wt., in propylene glycol as a vehicle,
were administered via subcutaneous injection (s.c.).

Table 1. Geographical coordinates of the Hyptis pectinata genotypes harvested in Sergipe State, Brazil, and kept in the active
germplasm bank of the experimental station Campus Rural da UFS

Genotypes Origin Geographical coordinates Herbarium

(localization)
Latitude (S) Longitude (W) Altitude (m) number

SAM001 Sao Cristovao 101550 25.600 371110 56.400 24 7455

SAM002 Neopolis 101180 20.700 361390 7.200 120 7454
SAM003 Santana do Sao 101160 55.300 361380 33.800 80 7453
Francisco
SAM004 Malhada dos Bois 101210 36.000 361540 27.600 130 7452
SAM005 Sao Francisco 101190 1.400 361530 36.800 55 7451
SAM006 Propria 101140 26.300 361510 11.100 25 7456

Abbreviations: Ssouth; Wwest; mmeter.

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Abdominal writhe tests (acetic acid-induced writhing in
mice)
Volatile oils were injected 30 min before the intraper-
itoneal injection (i.p.) of acetic acid 0.6% (10 ml/kg
body wt.) (Koster et al., 1959). The control animals
received only vehicle and acetic acid. Indomethacin
(10 mg/kg body wt., i.p.) was used as a standard drug.
Ten minutes after administration of acetic acid, the
number of writhes was registered over a period of
20 min.
Hot plate test
The animals were placed on a hot plate (5570.5 1C)
and the thermal stimuli reaction was observed with the
maximum incubation time of 30 s. The reaction time
(licking of paw, jumping, shaking) was measured at 30,
45, 60 and 90 min after administration of the volatile oils
and other drugs. The vehicle (s.c.) and morphine (10 mg/kg
body wt., i.p.) were used as control and standard drugs,
respectively. The possible participation of the opioid
system in the antinociceptive effect was analysed with
the volatile oils. The animals were pretreated intraper-
itoneally with naloxone (5 mg/kg body wt.) 15 min
before subcutaneous administration of the volatile oils
(100 mg/kg body wt.) or equivalent volumes of the
vehicle.
Acute toxicity studies: LD50 values
The method described by Lorke (1983) was employed
in the determination of the LD50 values. Swiss mice
(n 10) of both sexes were fasted overnight and the
volatile oils were administered at the following doses: 0
(vehicle), 1, 2, 3, 4 and 5 g/kg body wt., i.p. Animals
were observed for 48 h after treatment and the nal
LD50 value was calculated as the square root of the
product of the lowest lethal dose and the highest non-
lethal dose, i.e., the geometric mean of the consecutive
doses for which 0% and 100% survival rates were
recorded.
Statistical analysis
All results were reported as means7S.E.M. They
were further analyzed using one-way analysis of
variance (ANOVA) to calculate the signicance of
results.
Results and discussion
Analysis and yield of the volatile oils
The volatile oils of the six genotypes of H. pectinata
averaged 0.5% on a dry weight basis. Their compounds
were identied previously and only the major constitu-
ents are listed in Table 2.
Analgesic effects of volatile oils of H. pectinata
genotypes
The subcutaneous administration of the volatile oils
of the six genotypes signicantly reduced the number of

Table 2. Main compounds of the essential oil of six H. pectinata genotypes from the active germplasm bank of the experimental
station Campus Rural da UFS

Compounda KIb Percentage in genotypes

SAM001 SAM002 SAM003 SAM004 SAM005 SAM006

b-Pinene 981 1.24 2.55 5.88 0.46 1.08 0.85

b-Elemene 1393 3.09 4.47 1.40 4.12 2.17 1.53
Cis-b-Guaiene 1494 0.55 2.98 7.58 0.00 4.85 5.30
g-Cadinene 1516 5.12 1.03 0.56 0.40 1.22 1.38
Cubenol 1617 11.44 0.81 2.01 0.00 1.30 1.22
b-Caryophyllene 1422 12.91 27.10 8.88 45.09 28.15 23.58
Germacrene-D 1483 8.20 9.95 2.37 5.77 5.80 4.77
Caryophyllene oxide 1586 1.98 4.10 2.47 20.92 4.01 9.10
Cubenol 1617 11.44 0.81 2.01 0.00 1.30 1.22
a-Muurolol 1646 25.45 0.67 0.00 0.00 0.00 0.00
Calamusenone 1688 1.85 21.04 40.70 0.00 26.95 23.00
Monoterpene hydrocarbons 2.81 5.48 11.35 0.46 6.07 2.86
Oxygenated monoterpenes 0.37 0.58 3.14 0.00 0.38 0.00
Sesquiterpene hydrocarbons 49.36 62.57 27.40 66.42 53.27 51.65
Oxygenated sesquiterpenes 43.62 29.44 46.02 25.23 32.26 38.70
Essential oil content (%) 0.50 0.50 0.50 0.45 0.57 0.48
a
Compounds are listed in order of elution from a DB-5 column.
b
Kovats indexes.
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writhes induced by acetic acid, as compared to animals
that received only vehicle (Table 3). The inhibitory
effects of the volatile oils ranged from 62.5% to 84.8%.
While all the genotypes inhibited the number of
abdominal writhes, the genotype SAM002 presented
the highest inhibitory effect at a dose of 100 mg/kg body
wt (78.4%) followed by the genotype SAM004 (75%).
However, these effects were not dose-dependent, be-
cause doubling the dose did not signicantly alter the
response.
Using the hot plate test, administration of the volatile
oils of all genotypes delayed the time of reaction as
compared to the control group (Table 4). In this test,
genotype SAM002 showed the best analgesic activity
(62.34% at a dose of 100 mg/kg body wt).
Two different models of nociception were employed
in the present study with the objective of identifying the
possible peripheral and central effects of the volatile oils.
The volatile oils from all six genotypes of H. pectinata
showed analgesic effects in both models used.
This observation indicates that the volatile oil of
H. pectinata has peripheral (reduced writhing) and
central (delayed reaction time) effects. According to
Collier et al. (1968), acetic acid acts indirectly, inducing
the liberation of endogenous mediators that stimulate
the nociceptive neurons which are sensitive to the anti-
inammatory non steroidal drugs (NSAIDs) and to
opioids. In general, our results show that the volatile oil
of H. pectinata induces variable analgesic effects,
depending on the origin of the genotypes. These
observations are indicative of the potential for
H. pectinata to be used as an analgesic drug.
Bearing in mind that the major constituents of the
genotype SAM002 (major inhibitory effect in both
models) are b-caryophyllene (27.1%) and pectinone
(21.04%) and the major constituents of genotype
SAM004 are b-caryophyllene (45.1%) and caryophyl-
lene oxide (20.9%), it appears that the inhibitory effects
of the volatile oils are due mainly to the interaction of all
constituents and not only to the majority compounds,
since previous work has demonstrated that pure
b-caryophyllene does not show antinociceptive effects
at doses of 400 mg/kg body wt (Santos et al., 1998). The
action of the volatile oils is the result of the combined
effect of both their active and inactive compounds, and
the latter might inuence absorption, pharmacokinetics,
and bioavailability of the active compounds (Svoboda
and Deans, 1995).
The administration (i.p.) of the opioid agonist
naxolone (5 mg/kg body wt) completely reversed the
antinociceptive effect of the volatile oils of all genotypes
on hot plate test (Table 5). These results suggest that
opioid receptors are involved in the antinociceptive
action of the volatile oil of H. pectinata.

Table 3. Analgesic effect of the essential oil of Hyptis pectinata genotypes on acetic acid-induced abdominal writhes in mice

Group Dose (mg/kg Number of abdominal Inhibition (%)a

body wt.) constrictions7S.E.M.

Control (vehicle) 33.0074.77

Indomethacin 10 8.0071.24** 75.7
SAM001 100 11.3770.71** 65.5
200 9.0071.94* 72.7
400 9.3771.66* 71.6
SAM002 100 7.2571.33** 78.4
200 7.2570.97** 78.0
400 6.7571.44** 79.5
SAM003 100 12.3871.36** 62.5
200 10.8871.08* 67.1
400 7.0071.00** 78.8
SAM004 100 8.2571.51* 75.0
200 9.8771.38* 70.1
400 10.5070.94** 68.2
SAM005 100 10.2572.51** 68.9
200 10.7571.44** 67.4
400 5.7570.96** 82.6
SAM006 100 10.5072.31** 68.2
200 7.8771.22** 76.2
400 5.0070.89** 84.8

*po0.05, **po0.01 vs. control.

a
Percent inhibition of total writhing test response (n 10).
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338 M.F. Arrigoni-Blank et al. / Phytomedicine 15 (2008) 334339

Table 4. Antinociceptive effect of the essential oil from H. pectinata genotypes in the hot-plate test in mice (n 8)

Group Dose (mg/kg Time after administration (min) Inhibition (%)a

body wt.)
30 45 60 90
Latency time (s) (mean7S.E.M.)

Control 9.1770.70 9.7570.76 9.7070.39 10.9070.89

Morphine 10 14.6371.19** 13.9570.68** 13.8671.05** 12.9470.68 41.39
SAM001 100 14.9971.15** 14.2571.59* 14.8671.76** 14.1872.30 48.85
200 13.9971.08** 15.7371.38** 16.3671.45** 16.1871.97* 58.97
400 15.6870.86** 16.5170.94** 15.9071.18** 17.4671.30** 67.45
SAM002 100 18.7171.47** 15.4371.39** 13.6172.05 15.8171.30** 62.34
200 14.3071.64* 17.6871.97** 15.2171.62** 17.2471.49** 64.59
400 15.6171.44** 15.8171.49** 15.7871.24** 16.7871.42** 63.36
SAM003 100 15.5971.00** 14.6470.90** 15.6870.98** 15.4071.23* 56.62
200 13.2170.59** 15.0970.71** 14.8470.87** 15.8071.11** 50.49
400 14.9071.14** 14.1370.87** 15.6571.23** 15.6371.32* 54.07
SAM004 100 12.5071.01* 14.2571.52* 15.3171.18** 12.5870.92 39.56
200 12.9470.96** 13.1170.75** 13.5571.46* 15.7471.04** 41.29
400 16.4170.83 15.3370.80 15.0370.80 14.3170.46 56.01
SAM005 100 12.7971.23* 14.2370.86** 14.7872.40 13.2071.35 40.48
200 13.6171.06** 12.24 7 1.45 12.5870.67** 12.6171.15 30.36
400 13.8670.93** 14.3171.01** 14.1371.30** 14.5471.06* 45.18
SAM006 100 13.4471.26* 16.1970.98** 16.0671.73** 15.5072.03 56.31
200 13.2971.49* 15.1870.96** 13.3970.83** 14.6071.20* 44.16
400 15.4171.10* 17.5870.98** 16.2870.74** 15.9970.61** 66.63

*po0.05, **po0.01 vs. control.

a
Percent inhibition of total hot-plate response.

Table 5. Effect of naloxone on the antinociceptive effect of the essential oil from H. pectinata genotypes on the hot-plate test in
mice (n 8)

Group/dose (mg/kg body wt.) Time after administration (min) Inhibition (%)a

30 45 60 90
Latency time (s) (mean7S.E.M.)

Control 10.1570.84 10.3070.74 9.5470.75 9.6370.91

Morphine 10 14.2371.09* 14.0570.65* 13.9671.01* 13.9870.71* 41.50
Nal 5 8.9170.54 8.8970.97 8.3970.67 9.0770.74 12.65
Morphine 10+Nal 5 9.4570.68 10.2270.98 9.8570.59 10.1271.01 0
SAM001 100 12.9970.79* 14.3971.40* 13.4970.80** 14.3871.13** 39.45
SAM001 100+Nal 5 8.1170.87 9.5970.68 9.5871.07 9.5071.02 0
SAM002 100 15.4671.30** 14.3171.30* 14.2571.42* 16.1870.91** 53.28
SAM002 100+Nal 5 9.4470.93 10.4471.01 9.0170.57 10.2871.09 0
SAM003 100 13.8070.61** 14.9571.18** 16.0570.96** 16.1471.54** 53.79
SAM003 100+Nal 5 10.3571.18 10.7671.01 10.5370.60 9.4470.62 3.70
SAM004 100 14.1970.41** 13.1370.46** 15.1971.26** 16.2571.23** 48.34
SAM004 100+Nal 5 11.1771.44 10.7670.99 11.4971.21 10.7971.23 14.69
SAM005 100 14.6471.17** 14.9570.91** 15.1171.11** 16.6170.30** 54.80
SAM005 100+Nal 5 9.4370.68 10.7971.09 11.2171.10 11.4571.26 8.25
SAM006 100 13.4170.53** 15.2870.71** 15.5870.93** 15.6871.31** 51.27
SAM006 100+Nal 5 8.5970.84 9.6870.46 8.5170.60 10.7370.68 0

*po0.05, **po0.01 vs. control.

a
Percent inhibition of total hot-plate response.
 ARTICLE IN PRESS
M.F. Arrigoni-Blank et al. / Phytomedicine 15 (2008) 334339
The toxicity of the volatile oils was veried; the LD50
value was estimated at 1.5170.38 g/kg body wt. During
the experiment, animals exhibited low motility, respira-
tory difculty and shaking when treated with higher
doses (4 and 5 g/kg body wt.).
Acknowledgements
The authors wish to thank the Fundacao de Amparo
a Pesquisa de Sergipe-FAP-SE for nancial support for
this research, Coordenacao de Aperfeicoamento de
Pessoal de Nvel Superior-CAPES for the rst authors
fellowship and Conselho Nacional de Desenvolvimen-
to Cientco e Tecnologico-CNPq for the second and
fth authors productivity grants.
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