4.bastos et al.journal of ethnopharmacology

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Journal of Ethnopharmacology 103 (2006) 241245

Antinociceptive effect of the aqueous extract obtained from rootsofPhysalis angulataL. on mice

G.N.T. Bastos a, A.R.S. Santos c, V.M.M. Ferreira a, A.M.R. Costa a,C.I. Bispo a, A.J.A. Silveira b, J.L.M. Do Nascimento a,

a Departamento de Fisiologia, Centro de Ci encias Biologicas, Universidade Federal do Para, Belem 66075-900, BrazilbDepartamento de Qumica, Centro de Ci encias Exatas e Naturais, Universidade Federal do Para, Belem 66075-900, Brazil

cDepartamento de Ciencias Fisiologicas, Centro de Ciencias Biologicas, Universidade Federal de Santa Catarina, Florianopolis 88040-900, Brazil

Received 22 June 2004; received in revised form 5 August 2005; accepted 9 August 2005

Available online 19 September 2005

Abstract

In this study, we attempted to identify the possible antinociceptive action of aqueous extract (AE) obtained from roots ofPhysalis angulata,

known in Brazil as Camapu, used to treat various pain-related physiological conditions. The AE ofPhysalis angulata(1030 mg/kg) given by

i.p. or p.o. route, 0.5 and 1 h prior, produced significant inhibition of abdominal constrictions caused by acetic acid, with ID50 values of 18.5

(17.419.8) and 21.5 (18.924.4) mg/kg and inhibitions of 83 8 and 66 5%, respectively. The AE (1060 mg/kg, i.p.) also caused significant

inhibition of the late-phase of formalin-induced pain, with an ID 50 value of 20.8 (18.423.4) mg/kg and inhibition of 100%. Treatment of mice

with AE (60 mg/kg, i.p.) or with morphine (10 mg/kg, i.p.) produced a significant increase of the reaction time in the hot-plate test. These results

demonstrate, for the first time, that the AE of Physalis angulata produce marked antinociception against the acetic acid-induced visceral pain

and inflammatory pain responses induced by formalin in mice. The mechanism by which the AE produces antinociception still remains unclear.

However, pharmacological and chemical studies are continuing in order to characterize the mechanism(s) responsible for the antinociceptive action

and also to identify the active principles present in Physalis angulata. Moreover, the antinociceptive action demonstrated in the present study

supports, at least partly, the ethnomedical uses of this plant.

2005 Elsevier Ireland Ltd. All rights reserved.

Keywords: Physalis angulata; Antinociception; Formalin test; Writhing test; Hot-plate test

1. Introduction

Physalis angulata L. belongs to the Solanaceae family and

includes about 120 species with herbal characteristicsand peren-

nial habits (Correa, 1962; Kissmann and Groth, 1995). It is

distributed throughout the tropical and subtropical regions of the

world (Kissmann and Groth, 1995; Santos et al., 2003). Extracts

or infusions from this plant have been used in various countries

in popular medicine as a treatment for a variety of illnesses,

such as malaria, asthma, hepatitis, dermatitis and rheumatism

(Chiang et al., 1992a; Lin et al., 1992; Santos et al., 2003; Soares

et al., 2003). In Brazil,Physalis angulatais popularly known as

Camapu, Bucho de Ra, Jua de Capote or Mata-Fome

(Branch and Silva, 1983), and its juice is considered to be seda-

Corresponding author. Tel.: +55 91 2111545; fax: +55 91 2111601.

E-mail address:[email protected] (J.L.M. Do Nascimento).

tive and depurative against rheumatism and earache. The leaves

are sometimes used against inflammations of the bladder, spleen

and liver. The whole plant cooked is recommended in baths for

inflammatory processes, such as rheumatism (Lorenzi, 1982).

It has been demonstrated that some of the extracts or active

principles obtained fromPhysalis angulatahave a broad spec-

trum of biological activities, including antibacterial, molluscici-

dal, antiprotozoal, anticancer, cytotoxic and immunomodulatory

activities (Kasteleinand Camargo,1990;Lee et al., 1991; Chiang

etal.,1992a,b;Linetal.,1992;C aceres et al.,1995;Freiburghaus

et al., 1996; Pietro et al., 2000; Ismail and Alam, 2001; Januario

et al., 2002; Santos et al., 2003; Soares et al., 2003 ).

Phytochemical studies of Physalis angulata have demon-

strated the presence of steroids, known as physalins (D, I, G, K,

B, F, E), physagulins (E, F and G), with anolides and flavonoids

(Row et al., 1978, 1980; Lee et al., 1991; Chiang et al., 1992a,b;

Shingu et al., 1992; Ismail and Alam, 2001). In the present study,

we have attempted to investigate the antinociceptive action of

0378-8741/$ see front matter 2005 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.jep.2005.08.008


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242 G.N.T. Bastos et al. / Journal of Ethnopharmacology 103 (2006) 241245

the aqueous extract obtained from roots of Physalis angulata

on chemical and thermal models of nociception in mice for the

purpose of validating its ethnomedical use.

2. Material and methods

2.1. Preparation of the aqueous extract (AE) of Physalis

angulata

The plant was collected in Para State, Brazil, during the

year 2000 and classified by Dr. Ricardo Seichas (Department

of Botany, Museu Emlio Goeldi). A voucher specimen (ref.

653) was deposited in the Joao Murca Pires herbarium of the

Paras Emlio Goeldi Museum (Belem, PA, Brazil). After col-

lecting the material, roots ofPhysalis angulatawere separated

for extraction. The roots weighing 150 g were cleaned in a water

stream, extracted with 700 ml of Milli-Q water, andconcentrated

at a final volume of 14%. The decoct was cooled and stored in

a freezer at 20 C for subsequent lyophilization, producing

2.712g of the extract.

2.2. Animals

Swiss male mice (3035 g) were obtained from the Evan-

dro Chagas Animal Resources Centre, Belem, Para, Brazil.

They were randomly assigned to groups of 10 animals and

maintained in plastic boxes, with food and water ad libitum,

under a 12 h light/12 h dark cycle. The room temperature was

maintained at 22 1 C. The animals were acclimatized to the

laboratory for at least 2 h before the experiments that were

carried out between 8:00 and 13:00 h in order to avoid cir-

cadian influence. All experiments reported in this study werecarried out in accordance with current guidelines for the care

of laboratory animals and ethical guidelines for investiga-

tion of experimental pain in conscious animals. All efforts

were made to minimize the number of animals used and their

suffering.

2.3. Abdominal constriction by intraperitoneal injection of

acetic acid

Abdominal contraction, induced by i.p. injection of

acetic acid 1%, consisted of a contraction of the abdominal

muscle together with a stretching of the hind limbs ( Tonos

et al., 1999). The animals were pre-treated intraperitoneally(i.p.) with morphine (10 mg/kg) and aspirin (100 mg/kg),

used as positive control, or with the AE of Physalis angulata

(10, 20 or 30 mg/kg) 0.5 h before, or orally with the AE of

Physalis angulata (10, 20 or 30 mg/kg) 1 h before, acetic

acid injection. The control groups received the same volume,

0.9% of NaCl (10 ml/kg). After challenge, pairs of mice

were placed in separate boxes and the number of abdominal

constrictions was counted every 5 min over a 1 h period.

Antinociceptive activity was expressed as the reduction in the

number of abdominal constrictions, i.e. the difference between

control animals (NaCl) and animals pre-treated with AE or

morphine.

2.4. Formalin-induced licking

The procedure used was similar to that described

previously (Santos et al., 1998). Twenty microliters of 2.5%

formalin solution (0.92% formaldehyde) was injected intra-

plantarly (i.pl.) under the ventral surface of the right hindpaw.

The animals were placed individually in clear plexiglass cages

(33 cm23cm 21.5 cm) and observed from 0 to 30 min fol-

lowing formalin injection. The amount of time spent licking the

injected paw was timed with a chronometer and was considered

as indicative of nociception. The initial nociceptive response

normally peaked 5 min afterformalin injection (early-phase) and

1530min after formalin injection (late-phase), representing the

tonic and inflammatory pain responses, respectively (Hunskaar

and Hole, 1987). The animals were pre-treated intraperitoneally

with the AE ofPhysalis angulata (10, 20, 30 or 60 mg/kg), or

with morphine (10 mg/kg) or indomethacin (10 mg/kg) which

were used as positive controls,0.5 h beforehand. Thecontrol ani-

mals received the same volume of vehicle (10 ml/kg, i.p.) used to

dilute these drugs. Following intraplantar injection of formalin,each animalwas immediately placedinto a clear plexiglass cage,

and the time it spent licking the injected paw was determined.

2.5. Hot-plate test

The hot-plate test was used to measure the response laten-

cies according to the method described previously (Santos

et al., 1998).In the experiments the hot-plate was maintained at

551 C. Before beginning the experiments, the basal reaction

time response of all animals was taken. The animals were pre-

treated with saline (10 ml/kg, i.p.), morphine (10 mg/kg, i.p.) or

AEofPhysalis angulata (30 and 60 mg/kg, i.p.) and 0, 0.25, 0.5,1, 1.5, 2 and 2.5 h later, theywereput on the heated surface of the

plate at 551 C. The time necessary for the initial response to

the painful stimulus (elevation of the paws, licking or jumping)

was taken as defining the response. In order to minimize damage

to the animals paws, the cut-off time was 30 s.

2.6. Drugs

The drugs used were: formalin and acetic acid (Merck, Sao

Paulo, Brazil), indomethacin and aspirin (Sigma Chemical Co.,

St. Louis, MO, USA),morphine hydrochloride (Cristalia-Brazil,

Sao Paulo, Brazil). All substances used were dissolved in saline

solution, with the exception of indomethacin and aspirin thatwere dissolved in 5% NaHCO3 and Tween 80 plus 0.9% NaCl

solution, respectively. The final concentration of Tween 80 did

not exceed 5% and did not cause any effect per se.

2.7. Statistical analysis

The results are presented as meanS.E.M., except the ID50values (i.e. the doses of aqueous extract of Physalis angulata

necessary to reduce response by 50% relative to control value)

which are reported as geometric means accompanied by their

respective 95% confidence limits. The ID50values were calcu-

lated from at least three dosages of AE, determined by linear


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G.N.T. Bastos et al. / Journal of Ethnopharmacology 103 (2006) 241245 243

regression from individual experiments using appropriate soft-

ware (GraphPad software, San Diego, CA). The statistical sig-

nificance of differences between groups was obtained by means

of analyses of variance followed by NewmannKeuls multiple

comparison test.P-values less than 0.05 (P < 0.05) were consid-

ered to be significant.

3. Results

The results inFig. 1A and B show that the AE ofPhysalis

angulata, given by i.p. or p.o. (100 mg/kg) routes 0.5 or 1 h

beforehand, caused a dose-related inhibition of acetic acid-

induced visceral nociceptive response in all of the analyzed peri-

ods. The calculated mean ID50values for these effects were 18.5

(17.419.8) and 21.5 (18.924.4) mg/kg, and inhibitions were

83 8 and 665% for AE given by i.p. and p.o. routes, respec-

tively. The pre-treated animals using both morphine (10 mg/kg,

i.p. 0.5 h beforehand) and aspirin (100 mg/kg, i.p. 0.5 h before-

hand) were used as positive control, which produced significant

inhibition of acetic acid-induced visceral nociceptive responsein all of the analyzed periods (Fig. 1A). The maximal inhibition

of acetic acid-induced pain produced by morphine and aspirin,

in the doses used, were 100 and 82 5%, respectively (Fig. 1A).

The AE ofPhysalis angulata, administered intraperitoneally

(1060 mg/kg), produced marked and dose-related inhibition

against the inflammatory (late-phase) pain, but not against neu-

rogenic (early-phase) pain, caused by intraplantar injection of

formalin in mice (Fig. 2A and B). The calculated mean ID50value for the late-phase was 20.8 (18.43.4) mg/kg, and inhibi-

tion was 100%. Similarly, indomethacin (10 mg/kg, i.p.) caused

significant inhibition (767%) of the late-phase, but not the

Fig. 3. Effect of aqueous extract ofPhysalis angulata ((), 30 mg/kg; (),

60mg/kg) or morphine((), 10 mg/kg), given intraperitoneally, on the hot-plate

test in mice. Each point represents the meanS.E.M. of 10 animals. The points

marked by the open square indicate the control values (animals injected with

saline, 10 ml/kg) and the asterisks denote the significance levels in comparison

with control groups, *** P < 0.001. In some cases, the error bars of the mean are

hidden within the symbols.

early-phase, of formalin-induced nociception (Fig. 2Aand B).

In contrast, the treatment of animals with morphine (10mg/kg,

i.p.), given 0.5 h prior, produced marked inhibition of both the

neurogenic pain (early-phase, 784%) and inflammatory pain

(late-phase, 100%) of the formalin test in mice (Fig. 2A and B).

The results in Fig. 3 show that the treatment of animals

with morphine (10 mg/kg, i.p.) caused a marked increase in

the latency of the animals in all analyzed periods according

to assessment in the hot-plate test, under conditions where the

AE ofPhysalis angulata(60 mg/kg, i.p.) produced a significant

Fig. 1. Effect of aqueous extract ofPhysalis angulata [(), 10 mg/kg; (), 20 mg/kg; (), 30 mg/kg, given intraperitoneally (panel A) and orally (panel B)],

morphine ((), 10 mg/kg, i.p.) or aspirin ((), 100 mg/kg, i.p.) against acetic acid-induced visceral pain in mice. Each point represents the meanS.E.M. of 10animals. The points marked by the open square indicate the control values (animals injected with saline, 10 ml/kg) and the asterisks denote the significance levels in

comparison with control groups,* P < 0.05, ** P < 0.01, *** P < 0.001. In some cases, the error bars of the mean are hidden within the symbols.

Fig. 2. Effect of aqueous extract ofPhysalis angulata, morphine or indomethacin given intraperitoneally, against the early-phase (05 min, panel A) or late-phase

(1530 min, panel B) of formalin-induced nociception in mice. Each column represents the mean S.E.M. of 10 animals. The column C indicates the control values

(animals injected with saline, 10 ml/kg) and the asterisks denote the significance levels in comparison with control groups,***

P < 0.001.


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G.N.T. Bastos et al. / Journal of Ethnopharmacology 103 (2006) 241245 245

after administration of the AE. The data indicates that the AE of

Physalis angulatapresent a low acute toxicity. However, these

findings are preliminary and further studies are required to clar-

ify this point.

In summary, the results of the present study demonstrate for

the first time that the AE of Physalis angulata produce dose-

related antinociceptive action in chemical (acetic acid-induced

visceral pain or formalin-inducednociception) and thermal (hot-

plate test) models of nociception in mice. The mechanism by

which the AE produces antinociception still remains unclear,

but pharmacological and chemical studies are continuing so as

to characterize the mechanism(s) responsible for the antinoci-

ceptive action and also to identify the active principles present

in Physalis angulata. Furthermore, the antinociceptive action

demonstrated in the present study supports, at least partly, the

ethnomedical uses of this plant.

Acknowledgements

This study was supported by grants from the NationalCouncil of Scientific and Technological Development (CNPq),

SECTAN-FUNTEC-Para and PROPESP- UFPa, Brazil. The

author is grateful to Dr. Reinaldo de Amorim Carvalho, Instituto

de Pesquisa Evandro Chagas, for the experimental animals used

in this research.

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