SUPPLEMENTARY MATERIAL
Antioxidant and orofacial antinociceptive activities of the stem bark
aqueous extract of Anadenanthera colubrina (Velloso) Brenan (Fabaceae)
N.P. Damascenaa, M.T.S. Souzaa, A.F. Almeidab, R.S. Cunhab, N.P.
Damascenac, R.L. Curvelloa, A.C.B. Limaa, E.C.V. Almeidaa, C.C.S. Santosa,
A.S. Diasa, M.S. Paixãob, L.M.A. Souzab, L.J. Quintans Júniora, C.S. Estevama,
B.S. Araujoa*
a
Department of Physiology, Federal University of Sergipe, São Cristovão, SE, Brazil
b
Department of Odontology, Federal University of Sergipe, Aracaju, SE, Brazil
c
Department of Medicine, Federal University of Sergipe, Aracaju, SE, Brazil
*Brancilene Santos de Araujo, Universidade Federal de Sergipe, Departamento de Fisiologia,
Laboratório de Bioquímica de Produtos Naturais, Campus São Cristovão, CEP 49100-000, Aracaju,
SE, Brasil. Phone/FAX: +55 79 2105 6647. E-mail: bsa@ufs.br
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Antioxidant and orofacial antinociceptive activities of the stem bark aqueous
extract of Anadenanthera colubrina (Velloso) Brenan (Fabaceae)
The antinociceptive and antioxidant activities of the aqueous extract from
Anadenanthera colubrina stem bark of (AEAC) were investigated. AEAC (30
µg/ml) reduced 94.8% of 2,2-diphenyl-1-picrylhydrazyl radical and
prevented 64% (200 µg/mL) of lipid peroxidation caused by 2,2′-azobis(2-
methylpropionamidine) dihydrochloride induced-peroxyl radicals. AEAC
treatment (200 and 400 mg/kg) significantly (p < 0.001) reduced mice orofacial
nociception in the first (61.4% and 62.6%, respectively) and second (48.9% and
61.9%, respectively) phases of the formalin test. Nociception caused by
glutamate was significantly (p < 0.001) reduced by up to 79% at 400 mg/kg,
while up 56-60% of the nociceptive behavior induced by capsaicin was
significantly inhibited by AEAC (100-400 mg/kg). Mice treated with AEAC did
not show changes in motor performance in the Rota-rod apparatus. It seems that
AEAC is of pharmacological importance in treating pain due to its
antinociceptive effects, which were shown to be mediated by central and
peripheral mechanisms.
Keywords: Anadenanthera colubrina; Fabaceae; orofacial pain; glutamate;
capsaicin; formalin
Experimental
Plant material
Anadenanthera colubrina stem barks were collected in the district of Saúde, Capela, Sergipe,
Brazil, in October 12, 2010 (10°30′45.0864″ W, 37°3′27.2802″ S). The plant was identified by Ana
Paula do Nascimento Prata, botanist of the Department of Biology (DB), Federal University of Sergipe
(FUS), Brazil. A voucher specimen was deposited in the DB/FUS herbarium under the registration
number ASE 18826. To prepare the aqueous extract of A. colubrina (AEAC), the stem barks
(200 g) were dried at room temperature, infused in distilled water (500 mL) for 1h at room
temperature, which was filtered and then lyophilized. The crude extract (AEAC, 3.5 g) obtained
after lyophilization was kept at -20 ºC until further use.
Scavenging and anti-lipoperoxidative activities of AEAC
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AEAC potential for scavenging free radical was evaluated by the 2,2-diphenyl-1-picrylhydrazyl
(DPPH) radical method (Sousa et al. 2007). The results were expressed as inhibition percentage
(IP), while the antioxidant amount necessary to decrease the DPPH concentration by 50% (IC50)
was calculated by plotting the percentage of DPPH remanescent (%DPPHREM) after 60 min
versus extract concentrations. The antioxidant activity index (AAI) was calculated according to
Scherer & Godoy (2009) with modifications, considering the authors actually used DPPH initial
concentration instead of its final concentration to calculate AAI. Thus, AAI = [DPPH initial
concentration (µg/mL)]/[IC50 (µg/mL)], where antioxidant activity is considered weak when AAI
value is less than 0.5, moderate when AAI value is between 0.5 and 1.0, strong when AAI is
between 1.0 and 2.0, and very strong when AAI value is greater than 2.0 (Scherer & Godoy
2009).
The inhibition of lipid peroxidation by AEAC was monitored by measuring the production
of malonaldehyde (MDA), a thiobarbituric acid-reactive substances (TBARS), using egg yolk as
lipid source and 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH) and ferrous
sulphate (FeSO4) as peroxidation inducers (Lapenna et al. 2001). MDA formation was measured
by reading samples absorbance at 532 nm. Results were expressed as inhibition percentage.
Animals
Male Swiss mice (Mus musculus), aging 60-90 days and weighing 28-32g each, obtained from
the Central Animal Facility of FUS, were randomly kept in cages under controlled temperature
(22 ± 3°C) with a 12h light/dark cycle (light from 06h to 18h). Animals had free access to food
and water. The Ethics Committee on Animal Research of UFS (CEPA/UFS) approved the
protocols and experimental procedures under registration number 61/11.
Formalin test
Orofacial nociception was induced in mice by injecting 2% formalin (20 L, s.c.) in the right
upper lip (perinasal area) (Luccarini et al. 2006). Mice groups (n=8, per group) were
systematically pretreated with vehicle (saline, p.o., negative control) and AEAC (100, 200 and
400 mg/kg, p.o.) 1 hour before formalin injection, while morphine (MOR) (5 mg/kg, i.p.,
positive control) was administered 0.5 hour before the pain agent was used. Nociception was
quantified by measuring the time (seconds) that mice spent scratching their faces in the injected
area with its hind or front legs or expressing the flint behavior.
Capsaicin test
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Capsaicin nociception was induced as described by Pelisier et al. (2002). Capsaicin (20 L,
2.5 g, s.c.) was dissolved in a mixture of ethanol, dimethyl sulphoxide and distilled water
(1:1:8), and injected in the perinasal area. The effect of AEAC (100, 200 and 400 mg/kg, p.o),
MOR (5 mg/kg, i.p.) and vehicle on the face scratching behavior was observed during 42 min.
Glutamate test
Glutamate nocicetpion was induced according to Beirith et al. (2002) with modifications.
Glutamate (40 µL, 25 mM, s.c.) was injected in mice perinasal area and the animals were
observed individually for 15 min. The groups of animals were treated with AEAC, MOR and
vehicle as previously described for the formalin and capsaicin tests.
Motor activity evaluation
The influence of AEAC on mice motor activity was evaluated through the Rota-rod apparatus
(AVS®, Brazil). Mice that were able to remain on the top of the apparatus for more than 180
seconds (at 7 rpm) were chosen 24 h prior to the test. The selected animals were divided into five
groups (n=8) and treated with vehicle, AEAC (100, 200 and 400 mg/kg, p.o.) and diazepam (1.5
mg/kg, i.p.). After 30, 60 and 120 min of treatment, each animal was put in the apparatus and the
time it spent over the platform was counted up to 180 seconds (Dunham & Miya 1957).
Statistical analysis
Results obtained were expressed as mean  standard deviation (SD). The data were evaluated by
one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test, using GraphPad
Prism version 5.0 for Windows. Differences were considered significant when p < 0.05.
References
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Luccarini P, Childeric A, Gaydier A, Voisin D, Dallel R. 2006. The orofacial formalin test in the
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