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Evaluation of the Mutagenicity and Genotoxicity of

Arrabidaea chica Verlot (Bignoneaceae), an Amazon
Plant with Medicinal Properties
a a a
Vinícius Carlotto dos Santos , Thais Basso Longo , Ana Letícia Hilário Garcia , Marc
b c c
François Richter , Temenouga Nikolova Guecheva , João Antonio Pêgas Henriques ,
d a
Alexandre de Barros Falcão Ferraz & Jaqueline Nascimento Picada
a
Universidade Luterana do Brasil (ULBRA), Laboratório de Genética Toxicológica, Canoas,
RS, Brazil
b
Universidade Estadual do Rio Grande do Sul (UERGS), Pró-Reitoria de Pesquisa e Pós-
Graduação, Porto Alegre, RS, Brazil
c
Instituto de Educação para Pesquisa, Desenvolvimento e Inovação Tecnológica–ROYAL
Unidade GENOTOX–ROYAL/Centro de Biotecnologia, Universidade Federal do Rio Grande do
Sul (UFRGS), Porto Alegre, RS, Brazil
d
Universidade Luterana do Brasil (ULBRA), Laboratório de Fitoquímica, Canoas, RS, Brazil
Published online: 04 Apr 2013.

To cite this article: Vincius Carlotto dos Santos , Thais Basso Longo , Ana Letcia Hilrio Garcia , Marc Franois Richter ,
Temenouga Nikolova Guecheva , Joo Antonio Pgas Henriques , Alexandre de Barros Falco Ferraz & Jaqueline Nascimento
Picada (2013) Evaluation of the Mutagenicity and Genotoxicity of Arrabidaea chica Verlot (Bignoneaceae), an Amazon Plant
with Medicinal Properties, Journal of Toxicology and Environmental Health, Part A: Current Issues, 76:6, 381-390, DOI:
10.1080/15287394.2012.761947

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 Journal of Toxicology and Environmental Health, Part A, 76:381–390, 2013
Copyright © Taylor & Francis Group, LLC
ISSN: 1528-7394 print / 1087-2620 online
DOI: 10.1080/15287394.2012.761947

EVALUATION OF THE MUTAGENICITY AND GENOTOXICITY OF Arrabidaea chica

VERLOT (BIGNONEACEAE), AN AMAZON PLANT WITH MEDICINAL PROPERTIES

Vinícius Carlotto dos Santos1, Thais Basso Longo1, Ana Letícia Hilário Garcia1,
Marc François Richter2, Temenouga Nikolova Guecheva3, João Antonio Pêgas Henriques3,
Alexandre de Barros Falcão Ferraz4, Jaqueline Nascimento Picada1
1
Universidade Luterana do Brasil (ULBRA), Laboratório de Genética Toxicológica, Canoas, RS,
Brazil
2
Universidade Estadual do Rio Grande do Sul (UERGS), Pró-Reitoria de Pesquisa e
Pós-Graduação, Porto Alegre, RS, Brazil
3
Instituto de Educação para Pesquisa, Desenvolvimento e Inovação Tecnológica–ROYAL Unidade
GENOTOX–ROYAL/Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul
(UFRGS), Porto Alegre, RS, Brazil
Downloaded by [University of Sydney] at 16:11 01 August 2013

4
Universidade Luterana do Brasil (ULBRA), Laboratório de Fitoquímica, Canoas, RS, Brazil

Arrabidaea chica Verlot (Bignoniaceae) is an important folk medicine plant native to the
Amazon region and used to treat anemia, hemorrhage, inflammation, intestinal colic, hepati-
tis, and skin affections. Although studies showed its therapeutic properties, little knowledge
regarding genotoxic properties of this plant is available. The aim of this study was to deter-
mine the potential mutagenic and genotoxic/antigenotoxic effects of an A. chica chloroformic
fraction (Ac-CF) obtained from leaves containing bioactive metabolites. The mutagenic
effects were evaluated using the Salmonella mutagenicity assay, with TA98, TA97a, TA100,
TA102, and TA1535 strains, with and without metabolic activation. In vivo mutagenic and
genotoxic/antigenotoxic effects were investigated using the micronucleus (MN) test in bone
marrow and alkaline comet assay in blood and liver after administration of 100, 500, or
1000 mg/kg Ac-CF in CF-1 mice by gavage (once a day for 3 d). In vitro antioxidant poten-
tial was evaluated using DPPH and xanthine/hypoxanthine assays. Ac-CF was not mutagenic
in any of the Salmonella typhimurium strains tested and showed negative responses for
mutagenicity and genotoxicity in mice. Further, Ac-CF displayed antigenotoxic effects by
decreasing the oxidative DNA damage induced by hydrogen peroxide by greater than 50%
in blood and liver. The antioxidant action detected in the in vitro assays demonstrated IC50 of
0.838 mg/ml in the xanthine/hypoxanthine assay and IC50 of 28.17 µg/ml in the DPPH assay.
In conclusion, Ac-CF did not induce mutagenic and genotoxic effects and was able to protect
DNA against oxidative damage in vivo, suggesting that this fraction may not pose genetic risks,
although further toxicology assays are necessary.

Arrabidaea chica is a shrub native to
South America, commonly known as “Crajiru,”
“Carajuru,” or “Pariri.” This plant belongs to the
Bignoneaceae family, which comprises approx-
imately 120 genera and 800 species. Leaves
of A. chica are used in folk medicine as
antidiarrheic and antianemic agents, and are
also indicated to treat intestinal colic, hepati-
tis, ictericia, leukemia, albuminuria, and skin
infections (Jorge et al., 2008). Antifungal and
trypanocidal actions of A. chica were reported
(Barbosa et al., 2008; Höfling et al., 2010), in

Received 17 November 2012; accepted 20 December 2012.

This work was supported by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico); FAPERGS (Fundação de
Amparo à Pesquisa do Estado do Rio Grande do Sul); and GENOTOX-ROYAL, Brazil.
Address correspondence to Jaqueline Nascimento Picada, Laboratório de Genética Toxicológica, ULBRA: Avenida Farroupilha 8001,
Bairro São José, CEP 92425-900, Canoas–RS, Brazil. E-mail: jnpicada@cpovo.net

381
 382
addition to anti-inflammatory (Oliveira et al.,
2009) and wound healing properties (Jorge
et al., 2008).
Native Indian tribes in the Brazilian territory
have used leaves of this plant as dye in body
painting in rituals, to protect the skin against
sunlight, and to repel insects. In folk medicine,
the plant is used as an infusion of fresh or
dried leaves taken continuously as a drink for
1 to 3 d, replacing any usual beverage con-
sumed daily, and at times is also used to cleanse
skin wounds (Barbosa et al., 2008; Jorge et al.,
2008). The chemical constituents of a red dye
present in the leaves of A. chica were examined
Downloaded by [University of Sydney] at 16:11 01 August 2013
and some desoxyanthocyanidins were iden-
tified (Scogin, 1980), the most important of
which are carajurin and carajurone (Figure 1)
(Zorn et al., 2001; Devia et al., 2002).
Other species of the genera Arrabidaea
have also been studied. Arrabidaea triplinervia
was tested against the trypomastigotes form of
Trypanosoma cruzi, and found to promote total
elimination of these parasites in blood (Leite
et al., 2006). González et al. (2000), using the
species A. bilabiata, and Alcerito et al. (2002),
using A. brachypoda, showed that these species
possess antifungal properties. Recently, anti-
inflammatory and antinociceptive effects of A.
brachypoda were demonstrated in rodents (Da
Rocha et al., 2011).
Arrabidaea chica is considered by the
Brazilian health authorities as one of the
71 most important medicinal plants used in
the folk medicine, especially in the Amazon
region (RENISUS, 2009). However, only a
few studies examined its safety. The aim
of the present study was to determine the
FIGURE 1. Desoxyanthocyanidins identified in Arrabidaea chica:
Carajurin (R = CH3 , R1 = H) (6,7-dihydroxy-5,4 -dimethoxy-
flavone); Carajurone (6,7,4 -trihydroxy-5-methoxy-flanone)
(R, R1 = H) (Zorn et al., 2001; Devia et al., 2002).
V. C. DOS SANTOS ET AL.
genotoxic/antigenotoxic and mutagenic prop-
erties of the chloroformic fraction obtained
from leaves of A. chica, which presents a char-
acteristic red color due to the presence of
desoxyanthocyanidins.
MATERIALS AND METHODS
Plant Material
Aerial parts of A. chica were collected
in Tabaí, RS, Brazil. Voucher specimens were
identified by Dr. Sergio Bordignon. An exsicata
n. 3173 was deposited in the Herbarium at
Lutheran University of Brazil (HERULBRA).
Preparation of Chloroform Fraction
The aerial parts of the plant were air-
dried at room temperature for 7 d. The
aqueous extract was prepared by infusion
(1/10 plant/solvent) of A. chica leaves (850 g).
The infusion stood at room temperature for
30 min. After cooling and filtration, the aque-
ous extract was partitioned into five 50-ml
fractions using chloroform, yielding the A. chica
chloroformic fraction (Ac-CF) (5.95 g). After
that, the Ac-CF was evaporated in a rotary
evaporator at 40◦ C until dry.
Phytochemical Screening
The plant extracts were subjected to qual-
itative chemical screening for the identifica-
tion of the major classes of active chem-
ical constituents. The phytochemical profile
of A. chica leaves was determined accord-
ing to methodology described by Harborne
(1998). The method consists of colorimet-
ric reactions for detection of flavonoids,
tannins, anthraquinones, alkaloids, saponins,
coumarins, and cardiac glycosides. The con-
firmatory thin-layer chromatography analyses
were performed following traditional proce-
dures (Wagner and Bladt, 1996).
Salmonella/Microsome Mutagenicity
Assay
Salmonella typhimurium strains TA98,
TA97a, TA100, TA102, and TA1535 were
 MUTAGENICITY AND GENOTOXICITY OF Arrabidaea chica
kindly provided by B. M. Ames (University of
California, Berkeley). Mutagenicity was assayed
according to the pre-incubation procedure.
The S9 metabolic activation mixture (S9 mix)
was prepared according to Maron and Ames
(1983). Briefly, 100 µl test bacterial cultures
(1–2 × 109 cells/ml) was incubated at 37◦ C
with different amounts of Ac-CF (5, 10, 50,
100, or 250 µg/plate) in the presence or
absence of S9 mix for 20 min, without shaking.
Subsequently, 2 ml soft agar (0.6% agar, 0.5%
NaCl, 50 µM histidine, 50 µM biotin, pH
7.4, 42◦ C) were added and poured imme-
diately onto a plate of minimal agar (1.5%
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agar, Vogel-Bonner E medium, containing
2% glucose). Aflatoxin B1 (1 µg/plate) was
used as positive control for all strains in the
presence of metabolic activation (with S9 mix).
In the absence of metabolic activation, 4-
nitroquinoline oxide (4-NQO, 0.5 µg/plate)
was used for TA97a, TA98, and TA102 strains,
and sodium azide (1 µg/plate) for TA100 and
TA1535 strains. Plates were incubated in
the dark at 37◦ C for 48 h before counting
the revertant colonies. Assays were repeated
threefold and the plating for each dose was in
triplicate.
Pharmacological Procedure
Thirty-six CF-1 male mice were housed in
cages with food and water available ad libitum
under a 12-h light/dark cycle and a constant
temperature of 23 ± 3◦ C. All experimental pro-
cedures were carried out in accordance with
the Guide for the Care and Use of Laboratory
Animals of the National Institutes of Health
(NIH). This study was approved by an internal
ethics committee on animal experimentation of
the Lutheran University of Brazil under protocol
number 2009-014A.
Mice were divided in 5 groups of n = 6
animals, which were given an oral gavage of
vehicle (saline solution NaCl 0.9% with 5%
dimethyl sulfoxide) or Ac-CF (100, 500, or
1000 mg/kg) in a volume of 10 ml/kg body
weight, once a day for 3 d. Peripheral blood
samples were collected at three periods (3 h
and 24 h after the first administration and on
383
d 4) for the comet assay in order to evaluate
possible DNA damage and repair. All animals
were euthanized on d 4. Liver tissue was dis-
sected for the comet assay, and bone marrow
was obtained from femurs for the micronucleus
(MN) test. A positive control group with n =
6 animals (treated with cyclophosphamide at
25 mg/kg) was included for the MN test.
Comet Assay
The alkaline comet assay was carried out
as described by Tice et al. (2000). Pieces of
liver were placed in 0.5 ml cold phosphate-
buffered saline (PBS) and finely minced in order
to obtain a cell suspension; blood samples were
placed in 15 µl anticoagulant (heparin sodium
25,000 IU, Liquemine). The suspension of cells
from liver and from peripheral blood (5 µl) was
embedded in 95 µl 0.75% low-melting-point
agarose (Gibco BRL). The mixture (cell/agarose)
was spread onto a fully frosted microscope
slide coated with a 300-µl layer of normal
melting agarose (1%) (Gibco BRL). After solidi-
fication, slides were transferred to either PBS or
0.25 mM freshly prepared hydrogen peroxide
(H2 O2 ) solution (ex vivo treatment) for 5 min
at 4◦ C as described by Rodrigues et al. (2009).
Slides were washed 3 times with PBS and then
placed in lysis buffer (2.5 M NaCl, 100 mM
ethylenediamine tetraacetic acid [EDTA], and
10 mM Tris, freshly added 1% Triton X-100
[Sigma] and 10% dimethyl sulfoxide [DMSO],
pH 10) for 48 h at 4◦ C. Subsequently, the
slides were incubated in freshly made alkaline
buffer (300 mM NaOH and 1 mM EDTA, pH
> 13) for 20 min, at 4◦ C. An electric current
of 300 mA and 25 V (0.9 V/cm) was applied
for 15 min to induce DNA electrophoresis. The
slides were then neutralized (0.4 M Tris, pH
7.5), stained with silver, and analyzed using a
microscope. Images of 100 randomly selected
cells from each animal (50 cells from each of
2 replicate slides) were analyzed. Cells were
also scored visually according to tail size into
five classes, ranging from undamaged (0), to
maximally damaged (4), resulting in a single
DNA damage score for each animal, and con-
sequently each group studied. Therefore, the
 384
damage index (DI) may range from 0 (com-
pletely undamaged, 100 cells × 0) to 400 (with
maximum damage, 100 × 4). The damage
frequency (DF) was calculated based on num-
ber of cells with tail versus those with no tails
(Rodrigues et al., 2009). The percent reduction
in DI was calculated using the equation: R% =
[DI H2 O2 – DI Ac-CF with H2 O2 ]/[DI H2 O2 –
DI vehicle] × 100.
Micronucleus (MN) Test
The MN test was performed according
to the U.S. Environmental Protection Agency
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Gene-Tox Program (Mavournin et al., 1990).
Bone marrow extracted from both femurs
of mice was suspended in fetal calf serum
and smeared on clean glass slides. Slides
were air-dried, fixed in methanol, stained
in 10% Giemsa, and coded for a blind
analysis. To avoid false negative results and
obtain a measure of toxicity on bone marrow,
the polychromatic erythrocytes:normocromatic
erythrocytes (PCE:NCE) ratio was scored in
1000 cells. The incidence of MN was observed
in 2000 PCE for each animal.
Hypoxanthine/Xanthine Oxidase Assay
The method employed to assay the
hydroxyl radical scavenging ability of Ac-CF
was based on the method developed by
Owen et al. (1996). Briefly, Ac-CF was dis-
solved in the assay buffer (hypoxanthine, Fe(III),
EDTA, and salicylic acid) to a concentration of
2 mg/ml and diluted appropriately (in tripli-
cate) in assay buffer up to a final volume of
1 ml, giving a range of 0.1–2 mg/ml. A 5-ml
aliquot of xanthine oxidase dissolved in 3.2
M (NH4 )2 SO4 was added to initiate the reac-
tion. The samples were incubated for 3 h at
37◦ C, and the reaction was stopped afterwa-
rd by adding 10 ml HCl. A 30-ml aliquot
of reaction mixture was analyzed by high-
performance liquid chromatography (HPLC)
under chromatographic conditions as described
by Owen et al. (2003). Chromatographic
analysis was conducted using a gradient
based on methanol/water/acetic acid with
V. C. DOS SANTOS ET AL.
a µBondaPak C18 reverse-phase column
(Waters) and detection at 325 nm. The HPLC
equipment had a 2695 separation module
(Waters) and an ultraviolet (UV) detector 2487
(Waters). Hydroxylation of salicylic acid and
hypoxanthine was monitored at A = 325 and
A = 278 nm, respectively. The amount of dihy-
droxyphenols (2,5-dihydroxibenzoic acid and
2,3-dihydroxibenzoic acid; 2,5-DHBA and 2,3-
DHBA) produced by hydroxyl radical (OH•)
attack on salicylic acid was determined from
standard curves prepared with the respective
pure dihydroxyphenols. Assays were repeated
at least twice.
DPPH–Scavenging Assay
Scavenging of DPPH free radical was mea-
sured according to the method described by
Yamaguchi et al. (1998) with modifications
in which the Ac-FC was added to Tris-HCl
(100 mM) buffer, pH 7, containing 250 mM
DPPH dissolved in methanol. At least 6 dif-
ferent dilutions were tested and allowed to
stand for 20 min in the dark, before absorbance
was measured at 517 nm using a Shimadzu
spectrophotometer model UV-1602PC (Kyoto,
Japan). The experiment was conducted in trip-
licate. Antioxidant activity (AOA) was expressed
as IC50 (inhibitory concentration in micrograms
per milliliter of samples or positive controls nec-
essary to reduce the absorbance of DPPH by
50% compared to negative control).
Data Analysis
Data from Salmonella/microsome mutage-
nicity assay were analyzed by the Salmonella
statistic assay (Environmental Monitoring
System Laboratory, U.S. EPA, Software Version
2.3, April 1988). A test substance was consid-
ered mutagenic when significant concentration
response and analysis of variance (ANOVA)
variance were observed, and the increase in
mean number of revertants on test plates was
at least twofold higher than negative control
plates (or at least threefold higher for the
TA1535 strain). Comet assay and MN test data
are expressed as means ± SD, and statistical
 MUTAGENICITY AND GENOTOXICITY OF Arrabidaea chica
significance was determined by one-way anal-
ysis of variance (ANOVA) complemented by
Dunnett’s test. In all comparisons, p < .05 was
considered as indicating statistical significance.
RESULTS
Phytochemical analyses of A. chica indi-
cated the presence of saponins, alkaloids,
flavonoids, and phenolic compounds. Other
secondary metabolites such as anthraquinones,
cardiac glycosides, cumarins, and tannins
were not detected (data not shown). In the
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mutagenicity assay, the concentration range
between 5 and 250 µg Ac-CF/plate was
not cytotoxic in the presence or absence of
S9 metabolic activation (Table 1). No marked
mutagenic effects were observed on the
frameshift mutation-detecting strains TA98
(DNA target –C–G–C–G–C–G–C–G) and TA97a
(DNA target -C-C-C-C-C-C-; +1 cytosine),
in the absence or in the presence of
S9 metabolic activation. In addition, no sig-
nificant mutagenicity was seen in the strains
detecting base pair substitutions: TA1535 and
the corresponding isogenic strain TA100, which
detects base pair substitutions of a leucine
[GAG] by proline [GGG]. Negative results were
also observed in TA102 strain, which is sensi-
tive to oxidative and alkylating mutagens that
detect transversions or transitions in TAA DNA
sequences.
Table 2 shows the genotoxic and antigeno-
toxic effects of Ac-CF in peripheral blood.
No marked genotoxic effects were observed
after treatment with any of concentrations in
blood samples collected 3 and 24 h after the
first administration of Ac-CF. In order to eval-
uate the antigenotoxic activity, the blood cells
were challenged ex vivo with H2 O2 . The val-
ues of DI and DF from blood collected at
3 h were similar for all groups. However, 24 h
after the first administration a decrease in DI
and DF in all treated groups was noted in
comparison with vehicle control, indicating an
antigenotoxic effect. The percent reduction in
DI reached 74.2 % in the group treated with
Ac-CF 1000 mg/kg.
385
After repetitive doses administered for 3 d,
Ac-CF did not induce DNA damage in blood
or liver tissues (Table 3). In the ex vivo treat-
ments with H2 O2 , blood samples from the
group treated with the highest dose of Ac-
CF showed significantly lower DI compared
to vehicle control. Percentage reduction in
DI reached 56.6%. In liver, a fall in DI was
observed after treatment with all doses.
As shown in Table 4, the PCE/NCE ratio was
similar across all groups, indicating no apparent
toxicity in bone marrow of animals. There was
no marked change in MN frequency in Ac-CF-
treated groups compared with vehicle control.
The positive control group (cyclophosphamide)
showed a significant increase in MN frequency
in PCE in agreement with the historical values
reported in our laboratory.
The in vitro antioxidant activity of the Ac-
CF was determined by monitoring the pro-
duction of hydroxyl benzoic acids (DHBA) as
a product of the attack by hydroxyl radicals
on salicylic acid in the hypoxanthine/xanthine
oxidase assay. The reduction in total oxi-
dation products as a function of the con-
centration of Ac-CF added to the assay
resulted in a concentration-dependent in vitro
antioxidant capacity. The calculated IC50 value
was 0.84 mg/ml. Trolox (vitamin E) was used
as positive control and displayed an IC50 of
0.34 mg/ml.
In addition, the free radical scavenging
effect of Ac-CF, as well as that of ascorbic
acid and rutin, both used as positive controls,
was tested using the DPPH free radical scav-
enging assay (Yamaguchi et al., 1998). It was
observed that the scavenging effect of Ac-CF
is a function of concentration (Table 5). The
free radical scavenging activity of Ac-CF was
93.8% at a concentration of 1000 µg/ml, while
it was 86.1% at a concentration of 100 µg/ml.
The IC50 value for Ac-CF was 28.17 µg/ml.
In comparison, the results of the free radical
scavenging effect of ascorbic acid (IC50 = 4.03
µg/ml) and rutin (IC50 = 17.45 µg /ml) were
99.6% and 95.4%, respectively, for the con-
centration of 1000 µg/ml, and 99.4%, and
89.4%, respectively, for the concentration of
100 µg/ml.
 Downloaded by [University of Sydney] at 16:11 01 August 2013

TABLE 1. Induction of his+ Revertants (Rev) in S. typhimurium strains by A. chica Chloroformic Fraction With and Without Metabolic Activation (S9 Mix)

Salmonella typhimurium strains

TA98 TA97a TA100 TA1535 TA102
Concentration
Substance (µg/plate) Rev/platea MIb Rev/platea MIb Rev/platea MIb Rev/platea MIb Rev/platea MIb

Without metabolic activation (−S9)

NCc — 25.3 ± 8.3 — 93.0 ± 9.9 — 103.7 ± 15.2 — 9.0 ± 3.0 — 214.7 ± 20.4 —
Ac-CFd 5 28.0 ± 8.0 1.11 79.3 ± 4.5 0.85 112.0 ± 21.8 1.08 6.7 ± 2.5 0.74 253.7 ± 21.5 1.18
10 31.7 ± 5.0 1.25 86.0 ± 14.2 0.92 112.7 ± 19.8 1.09 10.0 ± 0.0 1.11 264.7 ± 12.7 1.23
50 21.0 ± 5.0 0.83 92.3 ± 2.5 0.99 106.7 ± 7.6 1.03 9.7 ± 2.5 1.08 234.3 ± 21.0 1.09
100 26.0 ± 8.7 1.03 76.0 ± 17.1 0.82 97.7 ± 1.2 0.94 6.7 ± 2.9 0.74 215.0 ± 29.1 1.00
250 27.7 ± 8.6 1.10 94.0 ± 14.7 1.01 97.0 ± 6.2 0.94 5.7 ± 1.2 0.63 220.7 ± 1.2 1.03
PCe 0.5 (4NQO)1 192.0 ± 15.0 7.6 289.7 ± 32.7 3.12 272.0 ± 24.3 2.62 536.3 ± 28.7 59.6 2412.7 ± 390.2 11.2
(NaN3 )

386
With metabolic activation (+ S9)
NCc — 22.3 ± 7.7 — 110.0 ± 7.2 — 120.7 ± 16.0 — 7.3 ± 1.5 — 269.0 ± 29.5 —
Ac-CFd 5 23.7 ± 8.1 1.06 105.7 ± 2.9 0.96 117.0 ± 14.2 0.98 8.3 ± 0.6 1.13 293.7 ± 17.2 1.09
10 24.7 ± 4.2 1.11 107.0 ± 10.0 0.97 105.0 ± 8.2 0.88 6.7 ± 0.6 0.91 265.0 ± 35.8 0.99
50 20.0 ± 5.6 0.90 98.7 ± 12.4 0.89 110.3 ± 15.3 0.92 7.3 ± 1.5 1.00 254.3 ± 37.6 0.94
100 24.7 ± 2.5 1.11 97.0 ± 6.1 0.88 115.7 ± 21.5 0.96 7.7 ± 0.6 1.05 288.7 ± 19.9 1.07
250 22.7 ± 5.9 1.01 99.0 ± 11.0 0.90 107.3 ± 21.1 0.89 9.3 ± 2.1 1.27 312.3 ± 4.7 1.16
PCe 1 (AFB1 ) 273.0 ± 75.5 12.2 394.7 ± 115.7 3.59 252.0 ± 15.6 2.10 130.7 ± 64.2 17.8 1647.0 ± 18.1 6.12

a Number of revertants/plate: mean of three independent experiments ± SD.

b MI: mutagenic index (number of his+ induced in the sample/number of spontaneous his+ in the negative control).
c NC: negative control 50% DMSO (10 µl) used as a solvent for the extract.
d Ac-CF: A. chica chloroformic fraction.
e PC: positive control (−S9) sodium azide to TA100 and TA1535; 4-NQO to TA97a, TA98 and TA102; (+ S9) aflatoxin B .
1
Note: MI results are in bold to confirm the reversion properties of the strains by the mutagens.
 MUTAGENICITY AND GENOTOXICITY OF Arrabidaea chica 387

TABLE 2. Genotoxic Activity in Blood of Mice Treated With a Single Dose of Vehicle or A. chica Chloroformic Fraction (Ac-CF)
(100, 500, or 1000 mg/kg)

Vehicle 100 mg/kg 500 mg/kg 1000 mg/kg

Without H2 O2
3h DIa 13.8 ± 8.0 16.6 ± 4.1 20.1 ± 11.7 19.8 ± 5.2
DFb 23.8 ± 13.3 13.5 ± 2.9 15.3 ± 9.6 14.2 ± 4.6
With H2 O2
DI 87.0 ± 27.2 118.8 ± 49.3 82.5 ± 23.8 96.5 ± 38.5
DF 53.0 ± 18.0 47.8 ± 21.5 42.2 ± 22.0 54.2 ± 21.4
Without H2 O2
24 h DI 15.9 ± 10.7 17.7 ± 11.4 13.6 ± 8.2 21.6 ± 13.2
DF 12.5 ± 8.3 13.7 ± 8.9 11.7 ± 7.6 16.9 ± 8.7
With H2 O2
DI (R%c ) 112.5 ± 17.8 52.0 ± 7.9∗∗ (62.6%) 43.3 ± 27.2∗∗ (71.6%) 40.8 ± 22.1∗∗∗ (74.2%)
DF 64.0 ± 13.6 33.0 ± 2.6∗ 20.0 ± 6.0∗∗ 25.6 ± 15.8∗∗
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Note. Significance indicate by ∗ p < .05; ∗∗ p < .01; ∗∗∗ p < .001, in terms of statistically significant difference from the vehicle group
(ANOVA, Dunett’s test).
a DI: damage index: can range from 0 (completely undamaged, 100 cells × 0) to 400 (with maximum damage 100 cells × 4).
b DF: damage frequency; was calculated based on number of cells with tail versus those with no tail.
c R% (% DI reduction) = [DI H O – DI Ac-CF with H O ]/[DI H O – DI vehicle] × 100.
2 2 2 2 2 2

TABLE 3. Genotoxic Activity in Blood and Liver of Mice With Vehicle or A. chica Chloroformic Fraction (Ac-CF) (100, 500, or
1000 mg/kg) by 3 Consecutive Days and Sacrificed 24 h After the Last Administration

Vehicle 100 mg/kg 500 mg/kg 1000 mg/kg

Blood Without H2 O2
DIa 4.2 ± 3.0 4.7 ± 3.8 6.0 ± 4.9 6.6 ± 5.7
DFb 3.8 ± 2.5 4.0 ± 2.3 5.7 ± 4.8 5.6 ± 4.9
With H2 O2
DI (R%c ) 75.7 ± 26.9 56.2 ± 17.3 48.6 ± 8.3 35.2 ± 12.2∗ (56.6%)
DF 33.0 ± 13.9 35.2 ± 7.3 35.0 ± 13.0 28.0 ± 13.6
Liver Without H2 O2
DI 20.0 ± 15.8 20.2 ± 18.9 29.6 ± 19.8 33.3 ± 23.4
DF 17.4 ± 13.4 17.7 ± 17.8 23.6 ± 16.0 27.7 ± 16.5
With H2 O2
DI (R%) 106.0 ± 16.4 61.9 ± 22.7∗∗ (51.3%) 67.0 ± 3.6∗ (45.3%) 64.2 ± 10.8∗ (48.6%)
DF 77.0 ± 15.8 57.9 ± 26.6 40.0 ± 9.8 51.8 ± 11.3

Note. Significance indicate by ∗ p < .05; ∗∗ p < .01; ∗∗∗ p < .001, in terms of statistically significant difference from the vehicle group
(ANOVA, Dunett’s test).
a DI: damage index: can range from 0 (completely undamaged, 100 cells × 0) to 400 (with maximum damage 100 cells × 4).
b DF: damage frequency; was calculated based on number of cells with tail versus those with no tail.
c R% (% DI reduction) = [DI H O – DI Ac-CF with H O ]/[DI H O – DI vehicle] × 100.
2 2 2 2 2 2

DISCUSSION drugs (Mortelmans and Zeiger, 2000). Ac-CF

was not mutagenic in any of the Salmonella
Considering the lack of studies regard-
typhimurium strains, in the presence or absence
ing toxicity of A. chica, despite its use in
of metabolic activation (Table 1), suggesting
folk medicine, this investigation determined
absence of phytochemicals inducing frameshift
the possible genotoxic and mutagenic effects
mutation, base pair substitutions, oxidative
of a fraction of this plant by means of in
damage, or DNA alkylations, directly or after
vitro and in vivo assays. Ac-CF was tested
metabolism. Hence, Ac-CF showed negative
in Salmonella mutagenicity assay, a short-term
results for gene (point) mutations.
bacterial reverse mutation assay used glob-
In order to evaluate in vivo genotoxicity,
ally as an initial screen to determine the
Ac-CF was administrated in mice reaching
mutagenic potential of new chemicals and
 388 V. C. DOS SANTOS ET AL.

TABLE 4. Mutagenic Activity in Bone Marrow of Mice Treated and liver samples after challenge of cells with
With Vehicle or A. chica Chloroformic Fraction (Ac-CF) (100,
500, or 1000 mg/kg) by 3 Consecutive Days and Sacrificed
H2 O2 , determining possible protective effects
24 h After the Last Administration against DNA oxidative damage. H2 O2 acts as
a precursor of the hydroxyl radical, which
MNPCEa in Ratio
2000 PCE, PCE/NCEb ,
then ultimately attacks cellular DNA, promot-
Group mean ± SD mean ± SD ing genotoxic effects (Brozmanová et al., 2001).
Ac-CF showed antigenotoxic effects, inhibiting
Vehicle 10.1 ± 4.1 1.6 ± 0.6
Ac-CF 3 × 100 mg/kg 11.8 ± 5.3 2.3 ± 1.1 DNA damage induced by H2 O2 in blood and
Ac-CF 3 × 500 mg/kg 12.2 ± 3.5 1.7 ± 1.0 liver (Tables 2 and 3). In a previous study, 7-
Ac-CF 3 × 1,000 mg/kg 14.2 ± 7.4 1.6 ± 0.6 d treatment with hydroethanolic extract of A.
Cyclophosphamide 25 mg/kg 24.3 ± 5.0∗∗ 1.6 ± 0.4
chica protected liver against carbon tetrachlo-
Note. Significance indicated by ∗∗ p < .01 as significant ride (CCl4 )-induced hepatotoxicity (Lima de
difference from vehicle group (ANOVA, Dunett’s test). Medeiros et al., 2011). In this case, Ac-CF pro-
a MNPCE: micronucleus in polychromatic erythrocytes.
b Ratio PCE/NCE: ratio polychromatic erythrocytes/normo-
tected cells predominantly 24 h after admin-
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chromatic erytrocytes. istration, reaching modulation over 50%, sug-

1000 mg/kg as the highest dose, the solubil-
ity limit of this fraction. At this dosage, no
mice presented signs of toxicity during the
experiments. The lack of toxicity was later
confirmed by similar polychromatic erythro-
cytes:normochromatic erythrocytes (PCE:NCE)
ratios of treated groups in comparison to nega-
tive control (Table 4).
The comet assay was used to detect recent
DNA damage, such as single- and double-
strand breaks, alkali-labile sites, and DNA–DNA
and DNA–protein cross-links. Blood samples
were collected at several periods in order to
evaluate possible DNA repair, as recommended
by Tice et al. (2000). Ac-CF was not able to
induce DNA damage in blood collected 3 or
24 h after the first administration (Table 2).
The same was observed 24 h after the third
administration (Table 3). Further, no marked
genotoxic effect was observed in liver. At the
same time, the antigenotoxicity was evaluated
by ex vivo comet assay using the same blood
gesting an antigenotoxic effect by (1) direct pro-
tective actions through scavenging free radicals
generated by H2 O2 or (2) adaptive responses,
via induction of antioxidant enzymatic systems,
which may detoxify H2 O2 . Indeed, Ac-CF
showed in vitro antioxidant properties scaveng-
ing the free radical produced in the DPPH assay
(IC50 of 28.17 µg/ml) (Table 5). The antioxidant
effect noted in our study is in agreement with
previous findings where the crude extract of
A. chica showed antioxidant activity in the
DPPH assay (Jorge et al., 2008). The Ac-CF
antioxidant potential was 7-fold lower than that
of ascorbic acid and only 1.6-fold lower than
rutin. The in vitro antioxidant activity was also
determined by monitoring production of DHBA
as a product of the attack by hydroxyl radical
on salicylic acid in the hypoxanthine-xanthine
oxidase assay. The antioxidant activity of Ac-
CF (IC50 0.84 mg/ml) was 2.5-fold lower than
Trolox (IC50 of 0.34 mg/ml), used as positive
control.
It is known that anthrocyanidins and
flavonoids possess antioxidant properties

TABLE 5. Inhibition of DPPH, IC50 Values for the DPPH Assay of the A. chica Chloroformic Fraction (Ac-CF)

Inhibition (%)
Sample 1 µg/ml 10 µg/ml 100 µg/ml 1000 µg/ml IC50 (µg/mL)a

Ascorbic acid 9.7 98.1 99.4 99.6 4.03 ± 0.16

Rutin 5.9 37.8 89.4 95.4 17.45 ± 0.87
Ac-CF 4.1 25.3 86.1 93.8 28.17 ± 1.94

Note. Results were based on the values measured at 20 min. Ascorbic acid and rutin were used as positive controls.
a Mean ± standard deviation of three determinations.
 MUTAGENICITY AND GENOTOXICITY OF Arrabidaea chica
in vitro (Pool-Zobel et al., 1999; Wang
et al., 1999; Azevedo et al., 2007). The
phytochemical screening of A. chica showed
the presence of flavonoids, alkaloids, saponins,
and phenolic compounds. These findings
are in accordance with a study reporting the
identification of flavonoids, saponins, and
alkaloids in this plant (Alves et al., 2010).
Recently, protective effects against DNA oxida-
tive damage induced by H2 O2 from acerola
fruit (Malpighia glabra L.) were attributed to
vitamin C and other antioxidant compounds
(Nunes et al., 2011). In the present study,
the antigenotoxic effects produced by Ac-CF
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may be due to scavenger action of flavonoids
and phenolic compounds. Further, Ac-CF may
reinforce antioxidant defenses by inducing
endogenous adaptive responses. The total
flavonoid content was 1.82%, determined by
spectroscopy. Moreover, Ac-CF also contains
desoxyanthocyanidins, which are substances
that belong to the phenolic compounds class,
like carajurin and carajurone, to which the anti-
inflammatory and wound healing properties of
this plant are attributed.
The MN test was performed at the
end of the experimental procedures to
detect clastogenic/aneugenic activities, which
lead to increasing MN frequency and sug-
gests mutagenic effects at chromosomal level
(Mavournin et al., 1990). As shown in Table 4,
Ac-CF did not alter MN frequency in bone
marrow, suggesting that this plant does not pro-
duce mutagenic effects. This is the one of the
first studies to evaluate the safety of this plant.
Studies using repeated oral doses for 28 d in
rodents need to be conducted to establish the
long-term safe use of this A. chica fraction.
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