Journal of Ethnopharmacology 76 (2001) 81 86

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Short communication
Antinociceptive and antiedematogenic effects of the aqueous
extract of Hyptis pectinata leaves in experimental animals
M.D. Bispo, R.H.V. Mourao, E.M. Franzotti, K.B.R. Bomfim,
M. de F. Arrigoni-Blank, M.P.N. Moreno, M. Marchioro, A.R. Antoniolli *
Laboratorio de Farmacologia/Bioqumica, Departamento de Fisiologia, CCBS. Uni6ersidade Federal de Sergipe CEP. 49100 -000,
Sao Cristo6ao-SE, Brazil
Received 30 November 1999; received in revised form 11 October 2000; accepted 28 December 2000

Abstract

The aqueous leaf extract of Hyptis pectinata (L.) Poit (Lamiaceae), popularly known in Brazil as sambaicata or canudinho,
was tested for its antinociceptive effects using the abdominal writhing, hot plate and formalin test models, and for its
aniedematogenic effects using the carrageenin and arachidonic acid-induced rat paw edema. The aqueous extract of Hyptis
pectinata administered orally at doses of 100, 200 and 400 mg/kg had a significant antinociceptive effect in the test of acetic
acid-induced abdominal writhing, with 43, 51 and 54% reduction of writhes, respectively, compared to the control. An increase
in hot-plate latency of 47 and 37.5% was also observed in animals receiving doses of 200 and 400 mg/kg, p.o. when placed on a
hot plate. In the formalin test, doses of 200 and 400 mg/kg, p.o. had no significant effect during the first phase of the test (05
min), while the dose of 200 mg/kg, p.o. reduced the nociceptive effect by 70% during the second phase (20 25 min). At the dose
of 600 mg/kg, p.o., the aqueous extract inhibited carrageenin-induced rat paw edema by 34.1%, and the dose of 300 mg/kg
administered intraperitoneally inhibited the rat paw edema induced by subplantar injection of arachidonic acid by 32.8%. These
results suggest that the aqueous extract from the Hyptis pectinata leaves produces antiedematogenic and antinociceptive effects.
The antinocipetion observed with the hot-plate test probably involves the participation of the opioid system. 2001 Elsevier
Science Ireland Ltd. All rights reserved.

Keywords: Antinociceptive and antiedematogenic activity; Aqueous extract; Hyptis pectinata

1. Introduction ders and fever (Martinez, 1989), and bacterial infections

Hyptis pectinata (L.) Poit (Lamiaceae), popularly
known in Brazil as sambacaita or canudinho, is a
herbaceous plant with opposing crossed, whole and
aromatic leaves. Its flowers are small, clustered into
axillary inflorescences, hermaphrodite, pentamer,
strongly zygomorphous, and bilabiate. The plant is
recommended in folk medicine for various conditions,
among them rhinopharyngitis, nasal congestion, and
certain skin diseases (Malan et al., 1988), gastric disor-
* Corresponding author. Tel.: + 55-79-212-66444.
E-mail address: aroberto@ufs.br (A.R. Antoniolli).
(Rojas et al., 1992). In the state of Sergipe, Hyptis
pectinata is extensively used by the local population for
the treatment of inflammation, bacterial infections,
pain, and cancer.
The essential oil of Hyptis pectinata contains 33
compounds, with a quantitative predominance of
monoterpenes (95.8%). Its main constituents are p-
cymene, thymol and b-terpinene, which, taken together,
correspond to more than 68% of the total. The antisep-
tic property of the oil of this plant is probably favored
by the large amount of thymol (Malan et al., 1988).
Although some investigations of the various activities
of Hyptis pectinata have been performed, no references
were detected concerning pharmacological studies of

0378-8741/01/$ - see front matter 2001 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 1 7 2 - 6
82 M.D. Bispo et al. / Journal of Ethnopharmacology 76 (2001) 8186
the antinociceptive and antiedematogenic effects of the
aqueous extract of this plant. Since Hyptis pectinata is
extensively used for these purposes in the state of
Sergipe, the objective of the present study was to
evaluate the antinociceptive and antiedematogenic ef-
fects on various animal models of the aqueous extract
of Hyptis pectinata leaves collected outside the blos-
soming period, as well as its acute toxicity for mice.
2. Materials and methods
2.1. Plant material
Hyptis pectinata leaves were collected outside the
blossoming period from the Live Pharmacy of Aracaju,
Brazil. A voucher specimen was deposited at the Uni-
versity of Sergipe herbarium under the number ASE
2626.The leaves were dried in an oven with air renewal
and circulation (model MA-037) at 37C until complete
dehydration.
2.2. Preparation of the aqueous extract
Dried Hyptis pectinata leaves were triturated in a
blender until a finely granulated powder was obtained.
The extract was obtained from this powder by adding
distilled water (3:10, w/v) under constant shaking for 4
h at 35C, followed by filtration (pH=6.0). The yield
of the aqueous extract was 8.3%.The filtrate was
lyophilized and stored at 5C. At the time of use, the
extract was re-suspended in distilled water at the de-
sired concentrations.
2.3. Animals
Wistar rats of both sexes (180 240 g) were used for
the antiedematogenic studies, and Swiss mice (2030 g)
were used for the antinociceptive studies. The animals
were divided into groups of eight and kept in plastic
cages at a temperature of 25 28C, with free access to
ration (Purina) and water. The animals submitted to
oral administration of the extract or drugs were fasted
for 12 h before the experiment.
2.4. Acute toxicity (LD50)
Mice of both sexes were divided into five groups of
10 animals each. The control group received only vehi-
cle (water), and the remaining groups received increas-
ing doses of up to 5 g/kg of the aqueous extract of
Hyptis pectinata by the oral route. The animals were
observed for 48 h, and mortality was recorded for each
group at the end of this period (Dietrich, 1983). Animal
weight was monitored for 14 days after treatment.
2.5. Acetic acid-induced writhing test
The response to intraperitoneal injection of 0.6%
acetic acid, i.e. contraction of the abdominal muscle
and elongation of the hind limbs, was induced by the
method of Koster et al. (1959). Swiss mice (20 30 g,
n= 8) of both sexes were pretreated orally with the
aqueous extract (100, 200 and 400 mg/kg), morphine
(2.5 mg/kg, i.p.), and indomethacin (10 mg/kg, p.o.).
The extract and indomethacin were administered 60
min before and morphine 30 min before intraperitoneal
injection of 0.6% acetic acid (0.1 ml/10 g), and 10 min
later, mouse pairs were placed in transparent cages, and
the number of abdominal writhes was counted over a
period of 20 min. The antinociceptive activity was
evaluated in terms of percentage inhibition of writhes.
2.6. Hot-plate test
The hot-plate test was used to measure latency by the
method of Eddy and Leimback (1953), with small
modifications. Mice (2030 g) were divided into groups
of eight animals each. The temperature of the plate
(Ugo Basile, model DS 37) was fixed at 55+ 0.5C. The
reaction time (jumps, licking one of the hind legs or one
of the forelegs, tapping) was measured 0, 15, 30 and 60
min after oral administration of the extract (200 and
400 mg/kg), morphine (10 mg/kg, i.p.) or distilled water
(control group). When 30 s elapsed without the animal
presenting any of the above behaviors, it was removed
from the plate to avoid tissue damage. The possible
participation of the opioid system in the antinociceptive
effect was analyzed with the extract. For the analysis of
this mechanism, we also used the latency time in mice,
with some modifications. The animals were pretreated
intraperitoneally with naloxone (5 mg/kg) 15 min be-
fore oral administration of the extract (200 mg/kg) or
intraperitoneal administration of morphine (10 mg/kg).
The control animals received distilled water (0.1 ml/10
g) and were studied for latency time over a period of 0,
15, 30 and 60 min.
2.7. Formalin test
The formalin test was applied according to the
method of Dubuisson and Dennis (1977), modified by
Hunskaar and Hole (1987). Mice of both sexes (2530
g, n=8) were pretreated with the extract (200 and 400
mg/kg, p.o.), or with morphine (7.5 mg/kg, i.p.) 60 or
30 min after intraplantar injection of 1% formalin (20
ml) into the right hind paw of the animal. The reaction
to pain was then observed, and the time the animal
spent licking or biting its paw was measured with a
chronometer during the first phase (05 min, neuro-
genic pain) and during the second phase (2025 min,
tonic pain).
 M.D. Bispo et al. / Journal of Ethnopharmacology 76 (2001) 8186
2.8. Carrageenin-induced edema
A volume of 0.1 ml of 1% carrageenin (CG, an
edematogenic agent) diluted in 0.9% saline was injected
into the subplantar region of the right hind paw of the
rat (Winter et al., 1962). Paw volume was measured
immediately after CG injection (time 0) and at intervals
of 1, 2, 3, and 4 h using a plethysmometer (model 7150,
Ugo Basile, Varese, Italy). The aqueous extract at
different concentrations (200, 400 and 600 mg/kg) and
indomethacin (10 mg/kg) were administered p.o. 1 h
before CG injection.
2.9. Arachidonic acid-induced edema
A volume of 0.1 ml of 0.5% arachidonic acid diluted
in 0.2 M carbonate buffer, pH 8.43 8.56, was injected
into the subplantar region of the right paw of the rat
(Di Martino et al., 1987). The paw volume was mea-
sured immediately after the injection (time 0) and over
a period of 2 h at 15 min intervals using a plethys-
mometer. The aqueous extract at the dose of 300 mg/kg
and norhydroguaiaretic acid (NDGA, 100 mg/kg) were
administered, i.p., 30 min before the injection of the
edematogenic agent.
Control groups receiving no drugs were used in the
edema model. The data obtained for the various groups
are reported as means+S.E.M. Percentage edema inhi-
bition was calculated using the following formula (Ker-
haro and Adams, 1974): % inhibition=A B 100,
where A is the mean for the control group, and B is the
mean for the treated group.
2.10. Statistical analysis
The results are reported as means+S.E.M. The dif-
ferences were estimated by ANOVA followed by the
Student t-test, with the level of significance set at
P B0.05.
Table 1
Antinociceptive effects of Hyptis pectinata extract (p.o), indomethacin (p.o) and morphine (i.p) on acetic acid-induced writhing in mice
Treatment Dose (mg/kg)
Control 25.509 1.81
Indomethacin 10
Morphine 2.5
Extract 100
Extract 200
Extract 400
a
PB0.001 vs. control (n= 8 animals).
83
3. Results and discussion
The results obtained in the present study demonstrate
that the aqueous extract of Hyptis pectinata had anti-
nociceptive and antiedematogenic effects on the various
models tested. Increasing doses of the aqueous extract
of Hyptis pectinata up to 5 g/kg administered to mice
p.o. were not lethal, so that it was not possible to
determine the LD50. However, if a dose of 5 g/kg was
not lethal, this is an indication of the low toxicity of the
extract (Dietrich, 1983). Animal weight was monitored
for a period of 14 days, with no alterations (results not
shown).
The antinociceptive effect of the aqueous extract of
Hyptis pectinata was evaluated using different pain
stimuli such as heat and chemical agents (acetic acid
and formalin). The extract had a significant effect in
both models. The data concerning the antinociceptive
effects of the aqueous extract of Hyptis pectinata, in-
domethacin and morphine on the abdominal writhes of
mice induced by 0.6% acetic acid are summarized in
Table 1. Indomethacin (10 mg/kg, p.o.), morphine (2.5
mg/kg, i.p.) and oral administration of the aqueous
extract (100, 200 and 400 mg/kg) had a significant effect
on the number of writhes induced by acetic acid, with
47, 66, 43, 51 and 54% inhibition, respectively, com-
pared to control animals (treated with water). The
results indicate that the dose of 200 mg/kg of the
aqueous extract has a maximal effect on the number of
abdominal writhes, since no significant difference was
observed between the doses of 200 and 400 mg/kg.
Collier et al. (1968) postulated that acetic acid acts
indirectly by inducing the release of endogenous media-
tors, which stimulate the nociceptive neurons that are
sensitive to non-steroidal anti-inflammatory drugs and
narcotics. The abdominal writhes induced by acetic acid
are considered to be a less selective antinociceptive
model.
Number of constriction (mean 9 S.E.M.) Percentage inhibition
13.59 1.93a 47.0
8.5 92.90a 66.6
14.591.38a 43.0
12.5 9 1.14a 51.0
11.0 9 0.73a 54.0
84 M.D. Bispo et al. / Journal of Ethnopharmacology 76 (2001) 8186

Table 2
Antinociceptive effects of Hyptis pectinata extract (EA-p.o), morphine (i.p) on heat-induced pain in mice (hot-plate test)a

Treatment (mg/kg) Latency (s) mean9 S.E.M. Percentage inhibition

0 min 15 min 30 min 60 min

Control 8.89 9 0.55 8.749 0.65 8.50 9 0.43 8.86 9 0.61

Morphine 10 11.60 9 0.62b 14.169 0.76c 14.60 90.96c 14.56 91.43b 56.9
Morphine+naloxone 10 7.409 0.54 10.319 1.18 10.71 90.83a 10.87 9 1.18 12.28
EA 100 9.459 0.78 9.139 0.69 6.58 90.54 7.36 9 0.43 7.04
EA 200 10.659 1.34 12.2791.38a 13.78 92.68a 14.73 9 1.92b 47.04
EA 200+naloxone 5.6390.69b 7.079 1.40 7.61 9 1.20 5.99 9 0.81b 24.82
EA 400 8.139 0.55 12.63 9 1.84a 13.55 92.95a 13.80 92.60a 37.55

a
PB0.05.
b
PB0.01.
c
PB0.001 vs. control (n= 8 animals).

Table 3
Antinociceptive effects of Hyptis pectinata extract (p.o) and morphine (i.p) on formalin test in micea

Treatment (mg/kg) First phase (05 min) Inhibition (%) Second phase (2025 min) Percentage inhibition

Control (formalin) 52.1993.62 28.69 94.96

Morphine 7,5 9.879 2.61** 81.08 0 ** 100
Extract 200 41.8094.06 19.90 8.40 92.99* 70
Extract 400 46.63 9 3.56 10.65 20.00 97.29 30.28

a
Each value is the mean 9S.E.M. from eight animals Statistical significance: *PB0.01, **PB0.001 vs. control.

Animals treated with the aqueous extract (100, 200
and 400 mg/kg, p.o.) and morphine (10 mg/kg) in the
hot-plate test presented a longer latency time than the
control group, with the dose of 200 mg/kg provoking
the longest latency (47% inhibition), whereas the doses
of 100 and 400 mg/kg caused inhibition of 7 and 37.5%,
respectively (Table 2). Although the hot-plate test is
commonly used for assays of narcotic analgesics, other
drugs such as sedatives and muscle relaxants or psy-
chomimetic drugs act centrally (Vaz et al., 1996). No
significant latency was observed at time 0 with any dose
of the aqueous extract, whereas morphine (10 mg/kg),
used as a reference drug, had an antinociceptive effect
at all times tested (0, 15, 30 and 60 min) compared to
the control. The results demonstrate that the dose of
200 mg/kg of the aqueous extract of Hyptis pectinata
presented the best antinociceptive effect in both the
abdominal writhing and hot-plate tests.
To determine the possible mechanism of action of the
aqueous extract of Hyptis pectinata to produce analge-
sia, we used naloxone, a non-selective antagonist of
opioid receptors. In some experimental models, nalox-
one apparently acts by antagonizing the action of en-
dogenous opioids involved in pain or stress (Faden,
1988). The data presented in Table 2 indicate that
naloxone (5 mg/kg, i.p.) markedly reversed the anti-
nociceptive effect of the aqueous extract (200 mg/kg)
and of morphine (10 mg/kg) in the model of heat-in-
duced pain (hot plate). These results suggest that the
antinociceptive effect of the aqueous extract of Hyptis
pectinata may involve opioid receptors.
In the formalin test, pretreatment of mice with the
aqueous extract at the doses of 200 and 200 mg/kg had
no significant effect during the first phase of the test
(05 min), whereas during the second phase (20 25
min), the dose of 200 mg/kg significantly reduced the
nociceptive effect by 70% (Table 3). The formalin test
can also be used to elucidate the mechanisms of pain
and analgesia (Tjolsen et al., 1992). Centrally acting
drugs such as narcotics inhibit the two phases equally
(Shibata et al., 1989), but drugs with peripheral action
such as aspirin, oxyphenobutazone, hydrocortisone and
dexamethasone inhibit the second phase only (Hun-
skaar and Hole, 1987; Rosland et al., 1990). Inhibition
of only the second phase of the formalin test is a typical
characteristic of cyclooxygenase inhibitors (Rosland et
al., 1990).
The extract of Hyptis pectinata administered orally at
the dose of 600 mg/kg exhibited a significant antiede-
matogenic activity (34.1% inhibition) during the first 4
h on the rat paw edema induced by carrageenin, a
classical model of acute inflammation used in the study
of non-steroidal anti-inflammatory drugs involving var-
ious types of chemical mediators of inflammation such
as histamine, serotonin, bradykinin and prostaglandins
(Di Rosa, 1974; Vinger et al., 1987). Doses of 200 and
400 mg/kg did not show any significant differences
(Table 4). Another model of inflammation used is the
 M.D. Bispo et al. / Journal of Ethnopharmacology 76 (2001) 8186 85

Table 4
Anti-inflammatory effects of Hyptis pectinata extract (p.o) and indomethacin on carrageenin-induced edema in rats

Treatment (mg/kg) Variation of edema (ml) 9 S.E.M. Percentage inhibition

Time (h)

1 2 3 4

Control 0.21 9 0.023 0.36 90.027 0.49 90.023 0.35 90,022

Indomethacin 10 0.1090.015b 0.24 90.022a 0.33 9 0.026a 0.23 9 0.026a 36.17
Extract 200 0.199 0.023 0.349 0.034 0.45 9 0.037 0.34 9 0.034 6.38
Extract 400 0.18 90.020 0.359 0.017 0.45 9 0.017 0.35 9 0.034 5.67
Extract 600 0.1090.015b 0.239 0.021a 0.35 9 0.031a 0.25 9 0.031a 34.10

a
PB0.01
b
PB0.001 vs. control (n=8 animals). Percentage inhibition: total edema response.

Table 5 Acknowledgements
Anti-inflammatory effects of intraperitoneal administration of Hyptis
pectinata extract (p.o) and NDGA in arachidonic acid-induced edema
in ratsa The authors are grateful to the Living Pharmacy
of Aracaju, Brazil, for constantly supplying the plant.
Time (min) Edema (ml) (mean 9S.E.M.) Research supported by Banco do Nordeste, CNPq
and PIBIC/UFS/CNPq.
Control NDGA (100 EA (300
mg/kg) mg/kg)

15 0.250 90.032 0.052 90.011d 0.1649 0.022b References

30 0.370 90.046 0.0909 0.016 d 0.231 9 0.025b
45 0.441 9 0.041 0.1759 0.013d 0.2909 0.030b Collier, H.O.J., Dinnen, L.C., Johnson, C.A., Schneider, C., 1968.
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