Journal of Ethnopharmacology 96 (2005) 87–91

Analgesic and anti-inflammatory activity of a crude root extract

of Pfaffia glomerata (Spreng) Pedersen
A.G. Netoa , J.M.L.C. Costaa , C.C. Belatia , A.H.C. Vinhólisa , L.S. Possebomc ,
A.A. Da Silva Filhob , W.R. Cunhaa , J.C.T. Carvalhoc ,
J.K. Bastosb , M.L.A. e Silvaa,∗
a Núcleo de Pesquisa em Ciências Exatas e Tecnológicas da Universidade de Franca, Av. Dr. Armando Salles Oliveira,
201, Parque Universitário, 1404-600 Franca, SP, Brazil
b Faculdade de Ciências Farmacêuticas de Ribeirão Preto da Universidade de São Paulo, São Paulo, Brazil
c Laboratório de Fitofármacos da Universidade de Alfenas-MG, Brazil

Received 23 October 2003; received in revised form 17 August 2004; accepted 23 August 2004
Available online 28 October 2004

Abstract

The anti-inflammatory and antinociceptive effects of the crude hydroalcoholic extract (PE) of Pfaffia glomerata roots was assessed in the
carrageenan-induced rat paw edema at the doses of 100, 200 and 300 mg/kg, using different animal models. An anti-inflammatory dose effect
response correlation of r = 0.997 and Y = 11.67x + 0.02 was found. At the same doses, the extract-inhibited acetic acid-induced writhing in
mice, but no dose response correlation was found. Oral administration of 100 mg/kg of PE and 0.5 mg/kg of dexamethazone inhibited by
29 and 61%, the granulomatous tissue formation (p > 0.05), respectively. These results indicate the potential of this plant extract to treat
chronic inflammation. At the assayed doses no significant activity was found in the hot plate test, as well as in the cell migration-induced by
carrageenan.
© 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords: Pfaffia glomerata; Amaranthaceae; Anti-inflammatory activity; Analgesic activity

1. Introduction 1984; Shiobara et al., 1993a,b). The genus Pfaffia comprises

Several Pfaffia species (Amaranthaceae) are commercial-
ized in Brazil as substitutes for the Asian ginseng (Panax
spp., Araliaceae). They are popularly known as “Brazilian
ginseng” and “paratudo” and have been used as tonic and
aphrodisiac, as well as for the treatment of diabetes and
rheumatism (De Oliveira, 1986). There are many literature
reports for Panax spp., while for Pffafia species there is
still much to be done (Walker, 1994). The main components
isolated so far from Pfaffia spp. roots are stigmasterol,
sitosterol, allantoin, pfaffic acid and their glycosides (nor-
triterpene saponins) (Nishimoto et al., 1984; Nakai et al.,
∗ Corresponding author. Tel.: +55 16 3711 8871; fax: +55 16 3711 8878.
E-mail address: mlasilva@unifran.br (M.L.A. e Silva).
about 22 species distributed in Brazil and, due to its difficult
botanical identification, there is much confusion among the
employed species. De-Paris et al. (2000) reported a study
using six different behavioral animal models for the central
activity, suggesting that the alcoholic extract of Pfaffia
glomerata roots can act as a central nervous system depres-
sant. In the state of Rio de Janeiro, the vernacular name of
Pfaffia glomerata is “dipyrone”, but so far no analgesic or
anti-inflammatory studies have been reported for this specie.
For Pfaffia paniculata, Mazzanti and Braghirolli (1994)
reported the analgesic and anti-inflammatory activity of its
crude extract, bearing low toxicity for rodents.
Therefore, the aim of this study was to carry out a pharma-
cological evaluation of the crude hydroalcoholic root extract
(PE) of Pfaffia glomerata for analgesic and anti-inflammatory
activities.

0378-8741/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2004.08.035
88 A.G. Neto et al. / Journal of Ethnopharmacology 96 (2005) 87–91
2. Material and methods
2.1. Plant material and crude extract preparation
Pfaffia glomerata (Spreng) Pedersen (Amaranthaceae)
was cultivated in Sı́tio Nova Esperança, Ribeirão Corrente,
São Paulo, Brazil, from branches collected in São Gonçalo,
Rio de Janeiro and identified by Dr. João C. Siqueira in the
Botanical Institute of the University of Campinas, São Paulo,
Brazil. Voucher specimens are deposited at the Herbarium of
that Institute under the reference number 24200.
The roots and rhizomes were harvested from 4 years old
individuals and sliced into small pieces, dried in a circulat-
ing air stove and grounded (1600 g). The powder was extract
with aqueous ethanol (7:3, v/v) solution by maceration for 3
weeks at room temperature. Ethanol was removed by distil-
lation under reduced pressure and the remaining solution was
lyophilized and stored under light protection, yielding 19%
(360 g) of crude hydroalcoholic lyophilized extract (PE). For
the pharmacological tests, the lyophilized extract was dis-
solved in saline solution prior to its use.
2.2. Animals
Male Swiss albino mice weighting 20–25 g were used for
the writhing test assay. Male Wistar rats weighting 160–170 g
were used for the hot plate, paw edema, cell migration and the
inhibition of granulomatous tissue assays. The animals were
housed in standard cages at room temperature (25 ± 4 ◦ C)
with food and water ad libitum. Twenty-four hours before
the experiments they were transferred to the laboratory and
were maintained only with water ad libitum.
Animals used in this study were housed and cared for in
accordance with the College of Medicine of Ribeirão Preto of
the University of São Paulo. The experiments were authorized
by the Ethical Committee for Animal Care of the University
of São Paulo (Protocol number 02.1.597.53.1), in accordance
with the Federal Government Legislation on Animal Care.
2.3. Drugs
Morphine sulfate (Innovatec, Brazil), indomethacin
(Prodome, Brazil), kappa carrageenan type III (Iota-Fluka-
Biochemika Co., St. Louis, USA) and dexamethazone (Sigma
Co.), sodium chloride and acetic acid (Merck Co.) were used
in the biological assays.
2.4. Anti-inflammatory activity
The rat paw edema was induced by carrageenan
(100 ␮g/paw), which was injected into the right hind paw
plantar surface to groups of five animals each (Winter et al.,
1962). Saline solution (0.9%, 0.1 mL) was injected into the
left paw as the control reference for the tested paw. The mea-
surement of foot volumes was carried out following the plesti-
mographic method described by Ferreira (1979). It was done
by recording the rat paw volume before the drug injection
and then at 1 h intervals for 5 h.
Acute carrageenan-induced inflammatory reaction in the
peritoneal cavity of rats. Four groups of five animals each re-
ceived PE at 100, 200 and 300 mg/kg by gavage. The standard
drug dexamethazone (0.5 mg/kg) was used as a positive con-
trol. The negative control group received 5% Tween in saline
solution. One hour later, a volume of 3 mL of carrageenan at
100 ␮g/mL in a 0.9% saline sterile solution was injected into
the rat’s peritoneal cavity. Cell migration was quantified 4 h
after the injection of carrageenan, according to the method
described by Souza and Ferreira (1985).
2.5. Granulomatous tissue assay
The assay was performed using groups of five animals
each under aseptic condition and anesthesia using diethyl
ether, and a ventral longitudinal scission was made in each
animal. Then, four rolls of hydrofoils white cotton (Johnson
& Johnson), measuring 5 mm of length and weighting 40 mg
each, were implanted through the diffusion of the subcuta-
neous tissue at four equidistant sites of the incision. The cot-
ton rolls were previously autoclaved and treated with 0.4 mL
of 5% ampicilin (Ariston) aqueous solution, following the
method of Meier et al. (1950) and Niemegeers et al. (1975).
The groups were orally treated with 4 mL/kg of a 5% Tween
solution, 0.5 mg/kg of dexamethazone and 100 mg/kg of PE,
2 h after cotton “pellets” implantation until the 6th day. At
the 7th day, the animals were sacrificed using diethyl ether
and the granulomatous tissue was removed by dissection and
dried at 60 ◦ C. The weight of the granulomatous tissue was
calculated by the difference between the initial and the final
dry weight of the cotton pellets.
2.6. Antinociceptive activity
The writhing test in mice was carried out using the method
of Koster et al. (1959). The writhes were induced by intraperi-
toneal injection of 0.6% acetic acid (v/v) (80 mg/kg). Three
different doses (100, 200 and 300 mg/kg) of PE were admin-
istered orally to groups of five animals each, 30 min before
chemical stimulus. The number of muscular contractions was
counted over a period of 20 min after acetic acid injection.
The data represent the total number of writhes observed dur-
ing 20 min and are expressed as writhing numbers.
The hot plate test in rats was performed by the method of
Woolfe and McDonald (1944), which was adapted for rats.
The evaluated parameters were the latency time for paw lick-
ing and jumping responses on exposure to the hot plate sur-
face, kept at 55 ± 1 ◦ C. The animal was kept on the hot plate
until it lifted one of its hind paws. Three different doses (100,
200 and 300 mg/kg) of Pfaffia glomerata extract were ad-
ministered orally to groups of five animals, 30 min before
the thermal stimulus and the response was determined every
30 min, during 120 min. The data represent the mean reac-
tion time for the animals. Latency time was recorded and the
 A.G. Neto et al. / Journal of Ethnopharmacology 96 (2005) 87–91
results are expressed as hot plate analgesic index (Yaksh et
al., 1976).
2.7. Statistical analysis
Data were statistically analyzed by analysis of variance
(ANOVA) with the level of significance set at p < 0.05. Crit-
ical differences between means were evaluated by Dunnett’s
multiple comparison test and Student’s t-test set at p < 0.05
(Sokal and Rohlf, 1995).
3. Results and discussion
PE at 100, 200 and 300 mg/kg administered 30 min before
the injection of carrageenan inhibited the formation of edema
by 46.3, 56.8 and 63.2%, respectively, 3 h after injection of
the inflammatory stimulus (Fig. 1). This result is quite sim-
ilar to the one observed for indomethacin at 5 mg/kg, which
inhibited the edema by 71.1%. Both results were statistically
significant (Fig. 1, p < 0.05). Moreover, there was a dose re-
sponse correlation for the tested concentrations for the paw
edema test, with r = 0.997 and Y = 11.67x + 0.02. It is known
that the third phase of the edema-induced by carrageenan,
in which the edema reaches its highest volume, is character-
ized by the presence of prostaglandins (PGI2 ) and other com-
pounds of slow reaction (Spector, 1960). Ueno et al. (2000)
found that the injection of carrageenan into the rat paw in-
duces the liberation of bradykinin, which later induces the
biosynthesis of prostaglandin and other autacoids, which are
responsible for the formation of the inflammatory exudate.
Besides, in the carrageenan-induced rat paw edema model,
the production of prostanoids has been through the serum ex-
pression of COX-2 by a positive feedback mechanism (Nantel
et al., 1999). Therefore, it is suggested that the mechanism
of action of CHAE may be related to prostaglandin synthesis
inhibition, as described for the anti-inflammatory mechanism
of indomethacin in the inhibition of the inflammatory process
Fig. 1. Effects of the oral administration of Pfaffia glomerata extract (PE) on
the rat paw edema-induced by intraplantar injection of carrageenan (n = 5)
at 3rd hour. (1) Saline, (2) indomethacin (5 mg/kg), (3) PE at 100 mg/kg, (4)
200 mg/kg and (5) 300 mg/kg. Results are expressed as mean ± S.E.M. of
paw edema volume. Significance relative to control values * p < 0.05.
89
induced by carrageenan (Di Rosa et al., 1971). In addition,
the classification of antinociceptive drugs is usually based
on their mechanism of action either on the central nervous
system or on the peripheral nervous system (Planas et al.,
2000).
Moreover, the cell migration within the injured tissue is
an important step of the inflammatory process. Thus, us-
ing carrageenan as stimulus it was possible to produce an
acute inflammatory response inside of the peritoneal cavity
of rats 4 h later, with a large number of polimorphonuclear
cells in the exudate. Nevertheless, the administration of PE
(100, 200 and 300 mg/kg) did not inhibit the cell migration,
once the obtained result was similar to the one obtained for the
negative control group (0.9% saline solution). On the other
hand, dexamethazone inhibited 80% of cell migration. Ac-
cordingly to Larsen and Henson (1983), the direct participa-
tion of prostaglandins in the chimiotatic response is uncom-
mon to occur. However, leucotrien B4 is a potent chimiotatic
compound for the polimorphonuclear leukocytes and lipoxy-
genasis, an enzyme for the leucotriene synthesis, which is
very sensible to steroidal anti-inflammatory drugs, such as
dexamethazone (Blackwell et al., 1980).
Likewise, the granulomatous tissue formation is related to
the chronic inflammatory process, which is characterized by
several phases (Swingle and Shideman, 1972). In this regard,
the oral treatment with 100 mg/kg of the PE and 0.5 mg/kg
of dexamethazone lead to 29 and 61% reduction of the gran-
ulomatous tissue formation, respectively (p < 0.05).
With respect to the writhing test, the research group
of Deraedt et al. (1980) described the quantification of
prostaglandins by radioimmunoassay in the peritoneal exu-
dates of rats, obtained after intraperitoneal injection of acetic
acid. They found high levels of prostaglandins PGE2␣ and
PGF2␣ during the first 30 min after acetic acid injection.
Nevertheless, it was found that the intraperitoneal admin-
istration of acetic acid induces the liberation not only of
prostaglandins, but also of the sympathetic nervous system
mediators (Hokanson, 1978; Duarte et al., 1988). Thus, the
results obtained for the writhing test using acetic acid are
similar to those obtained for the edematogenic test using car-
rageenan, since Pfaffia glomerata crude hydroalcoholic ex-
tract (PE at 100, 200, 300 mg/kg was effective in inhibiting
the writhings in mice by 69.1, 66.4 and 74.1%, respectively,
in comparison to both the negative control group and the pos-
itive control group-treated with indomethacin, which inhibit
the writhings by 94.1% (Fig. 2). Therefore, anti-inflammatory
substances may also be involved in the peripheral analgesic
activity.
It is known that non-steroidal anti-inflammatory drugs
usually do not increase the pain threshold in normal tissues,
whereas local anesthetics and narcotics do (Ferreira et al.,
1978). However, the hot plate test was undertaken to verify if
PE would have any central analgesic effect. The results for the
group-treated with PE did not differ significantly from those
obtained for the negative control group. On the other hand,
the group-treated with morphine (4 mg/kg, subcutaneously)
90 A.G. Neto et al. / Journal of Ethnopharmacology 96 (2005) 87–91
Fig. 2. Effect of oral administration of PE at 100 mg/kg (3), 200 mg/kg
(4) and 300 mg/kg (5) on the writhing number-induced by intraperitoneal
administration of a 0.6% acetic acid solution in mice (n = 5). Negative control
was treated with 0.9% saline solution (1) and positive control was treated with
indomethacin (5 mg/kg) (2). Results are shown as mean ± S.E.M. inhibition
of writhing movements and a significant difference from the control group
is shown as * p < 0.05.
showed a highly significant result. Hence, it is assumed that
PE has no analgesic effect on the central nervous system that
would contribute to its peripheral analgesic effect. Therefore,
despite of the mild effect observed for the doses of 200 and
300 mg/kg for this test in relation to the control group, it was
not statistically significant, according to the linear correla-
tion test, once this assay evaluates compounds with central
activity like morphine.
4. Conclusion
From these investigations, it may be concluded that PE
showed both anti-inflammatory and analgesic effects, sim-
ilar to those observed for non-steroidal drugs, such as in-
domethacin. It is also suggested that the mechanism of action
of PE might be associated with the inhibition of prostaglandin
synthesis, as observed for most non-steroidal drugs.
It is important to point out that work is in progress to
isolate and to characterize the active compounds present in
hydroalcoholic extract of Pfaffia glomerata roots.
Acknowledgments
We thank “Fundação de Amparo à Pesquisa do Estado de
São Paulo—FAPESP” for the financial support.
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