Toxicology Letters 151 (2004) 481–487

Genotoxic potentiality of aqueous extract prepared from

Chrysobalanus icaco L. leaves
S.C. Ferreira-Machado a , M.P. Rodrigues a , A.P.M. Nunes a ,
F.J.S. Dantas a , J.C.P. De Mattos a , C.R. Silva a , E.G. Moura b ,
R.J.A.C. Bezerra a , A. Caldeira-de-Araujo a,∗
a Departamento de Biofı́sica e Biometria, Universidade do Estado do Rio de Janeiro, Instituto de Biologia Roberto Alcantara Gomes,
Av. 28 de setembro, 87, Rio de Janeiro 20551-030, Brazil
b Departamento de Ciências Fisiológicas, Universidade do Estado do Rio de Janeiro, Instituto de Biologia Roberto Alcantara Gomes,
Av. 28 de setembro, 87, Rio de Janeiro 20551-030, Brazil

Received 23 March 2004; received in revised form 23 March 2004; accepted 24 March 2004

Available online 24 April 2004

Abstract

Plants have been related to our lives, being used as medicine, regardless of scientific evidence of side effects. This work analyses
the toxicological effects of Chrysobalanus icaco L. aqueous extract, used in different pathologies. It was studied through: (i)
alteration of plasmid pUC 9.1 topology; (ii) survival of bacterial strains submitted, or not, to previous treatment with SnCl2 ; (iii)
transformation efficiency of E. coli strain by the treatment with the plasmid pUC 9.1. In (i), the treatment of the plasmid resulted
in DNA single-strand breaks (SSB). A decrease of the lethal effect induced by SnCl2 in presence of the extract was found, while
no C. icaco bacterial survival reduction was observed. The transformation efficiency of the plasmid was also reduced. Results
suggest that the extract could present a potential genotoxic effect, as demonstrated either by the induction of SSB in plasmid or
in transformation efficiency experiments. Finally, it presents an antioxidant action.
© 2004 Published by Elsevier Ireland Ltd.

Keywords: Chrysobalanus icaco; Plasmid pUC 9.1; Escherichia coli; Genotoxic potentiality

1. Introduction side effects. Various practices of traditional medicine

have been developed in different cultures and regions,
During the last decade, plants have been employed based on indigenous experiences, philosophy and
in the prevention and treatment of diseases. Their use personal attitudes without a parallel development
has considerably increased among populations, as they of international standards and appropriate methods
are believed to be beneficial and having few significant for evaluating traditional medicine (Rates, 2001;
Ankli et al., 2002; Steenkamp, 2003). The concept
∗ Corresponding author. Tel.: +55-21-25876391; that these products are “natural” is neither ade-
fax: +55-21-22543532. quate nor sufficient to assure safety and efficacy.
E-mail address: adriano@uerj.br (A. Caldeira-de-Araujo). In addition, the amount of available information on

0378-4274/$ – see front matter © 2004 Published by Elsevier Ireland Ltd.

doi:10.1016/j.toxlet.2004.03.014
482 S.C. Ferreira-Machado et al. / Toxicology Letters 151 (2004) 481–487

the toxicity and therapeutic properties in the hu- C. icaco aqueous extract, as it is frequently used by
man organism about several medicinal herbs is still the population. This research was performed in three
quite limited. Most of the information does not experimental models: (i) agarose gel electrophoresis
have scientific support. Medicinal herbs have their analysis of structural conformation changes of plasmid
use as medicines, based only on traditional folk pUC 9.1, previously treated with different concentra-
use, which has been passed on from generation to tions of the extract; (ii) survival of Escherichia coli
generation (Beyerstein, 2001; Talalay, 2001; Ernst, (E. coli) strains treated with aqueous extract, alone or
2002). simultaneously with stannous chloride; and (iii) the
Recently, interest has increased on the research of capacity of the treated plasmids in transforming com-
antioxidant properties of several plants against free petent E. coli AB1157 (wild-type) cells.
radicals or reactive oxygen species (ROS), to pre-
vent cellular injuries in the human organism. ROS
are generated from both endogenous and exogenous 2. Material and methods
sources and may be involved in the aetiologies of
several human diseases. ROS, such as hydroxyl rad- 2.1. Plant material and reagents
icals (OH• ), produced in the cells, are capable of
damaging important cellular targets, such as mem- C. icaco was collected at a sand bank area in Cabo
branes and deoxyribonucleic acid (DNA) (Reiniger Frio, Rio de Janeiro state, Brazil, from October to
et al., 1998; Nakagawa and Yokozawa, 2002; Auddy November 2002, between 9 and 10 a.m., the leaves
et al., 2003; Lee et al., 2003; Sroka and Cisowski, being processed all at once to obtain all the extract
2003). Dantas et al. (2002) related the toxic effects used during this work. The plant was classified and
of stannous chloride, a powerful reducing agent used deposited in the Herbarium Bradeanum, at the Rio
for packing canned food, in dental amalgams and for de Janeiro State University under registration num-
preparing 99m Technetium-radiopharmaceuticals, on ber 85422. Decoctions were prepared in the traditional
the generation of ROS in K562 cell line. way: 10 g of leaves were boiled in 1 L of water on
Emphasis is given, in this paper, to the poorly slow heat for 5 min and cooled at room temperature
studied medicinal plant species Chrysobalanus (25 ◦ C). Then, the supernatant was filtered with a pa-
icaco Linnaeus (C. icaco), also known as abajeru, per filter. Finally, the filtered was lyophilized, being
coco-plum and many other common names. This stored at −20 ◦ C, until further use. The dry extract
is a medium-sized shrub, native to coastal areas of obtained was suspended either in Mili-Q water (Milli-
the American continent, belonging to the Chrysobal- pore Corp., Bedford, MA, USA), in order to proceed
anaceae family (Dahlgren, 1980). Consumption of with DNA electrophoresis and bacterial transforma-
C. icaco as a tea prepared with various parts of the tion experiments, or solved in 0.9% NaCl solution in
plant has been thoroughly used, in folk medicine, for order to run the bacterial inactivation assays. After-
the control of several pathologies such as leucorrhea, wards, it was sterilized by filtration through 0.22 ␮m
haemorrhages and chronic diarrhea. The species is Millipore membrane.
also known to bring forth diuretic, hypoglycemic and Stannous chloride (SnCl2 ·2H2 O) obtained from
antiangiogenic effects (Costa, 1977; Vargas-Simon Sigma Chemicals Co., USA, was employed as
et al., 1997; Paulo et al., 2000). In the literature, there positive control to induce DNA strand breaks
is at least one publication showing the induction of (Caldeira-de-Araújo et al., 1996; Dantas et al.,
apoptosis by triterpenoids obtained from methanol 1999; De Mattos et al., 2000). The SnCl2 solution
extract of C. icaco leaves (Fernandes et al., 2003). (200 ␮g/mL) was freshly prepared in Mili-Q water
The abusive use of the extract of C. icaco leaves and before each experiment.
the poor knowledge of its therapeutic and side effects
emphasize the need for a deeper study, including the 2.2. Bacterial strains and plasmid
evaluation of its toxicity in several biological systems.
The purpose of this work was to examine the relative DNA repair wild-type E. coli K12 AB1157
antioxidant activity and the genotoxic potential from (Howard-Flanders and Theriot, 1966), PQ35 (rfa)
 S.C. Ferreira-Machado et al. / Toxicology Letters 151 (2004) 481–487 483

and PQ37 (rfa and uvrA) (Quillardet and Hofnung, the control (not treated). A significance level of 5%
1985) strains were used. E. coli DH5␣F’Iq (rec−) was adopted to compare data.
(Hannanah, 1983) strain hosted plasmid pUC 9.1. The data collected by densitometry provided us with
This plasmid, carrying an ampicillin resistance gene, the null events percentage (no breaks = p(0; µ)) for
was obtained through alkaline lysis method, as de- each one of the aqueous extract concentrations tested.
scribed in Sambrook et al. (1989). Plasmid samples Thus, the mean values of breaks per genome for each
were further purified from high molecular weight one of the concentrations, using Poisson distribution
RNA contaminants, performing LiCl precipitation were obtained as follows (Remington and Shor, 1985):
(2.5 M final concentration), while the residual RNA
contaminants were digested by RNAse (20 ␮g/mL) µ = −ln p(0; µ)
treatment for 30 min at room temperature (25 ◦ C).
2.5. Bacterial inactivation
2.3. DNA treatment
E. coli AB1157, PQ35 and PQ37 exponentially
To evaluate the action of C. icaco aqueous ex- growing cultures in LB medium (Miller, 1992) were
tract in DNA topology, plasmid pUC 9.1 (200 ng per centrifuged, washed twice in 0.9% NaCl solution
tube) was incubated either with increasing concen- and suspended in the same solution. Samples of this
trations of the extract (0.7, 1.75, 3.5 and 7 mg/mL) suspension were incubated with the aqueous extract
or with SnCl2 solution 200 ␮g/mL (positive control) (0.7, 3.5 and 7 mg/mL), or SnCl2 (25 ␮g/mL) or
in 10 mM Tris–HCl buffer at pH 7.4. In all cases, both agents, for 60 min at 37 ◦ C. At 20 min intervals,
the reaction mixture was incubated at room temper- aliquots were diluted and spread onto glass Petri
ature for 40 min. After incubation, each sample was dishes containing solidified LB medium (1.5% agar).
mixed with loading buffer (0.25% xylene cyanol FF; Colonies were counted after overnight incubation at
0.25% bromophenol blue; 30% glycerol in water), 37 ◦ C, and the survival fractions (N/No) determined
and applied in 0.8% agarose horizontal gel elec- as the mean of three isolated experiments. Standard
trophoresis chamber in Tris acetate–EDTA buffer at deviations did not exceed 10%.
pH 8.0 and run at 6 V/cm. Here, the electrophoresis
assay was performed to separate different structural 2.6. Bacterial transformation
conformations of plant extract-treated plasmid DNA:
form I supercoiled (SC) native conformation, form II Competent bacteria (E. coli AB1157), sensitive to
open circle (OC), resulting from single-strand breaks, ampicillin, were obtained as described by Sambrook
and form III linear (L), resulting from double-strand et al. (1989). The transformation efficiency of plasmid
breaks. The gel was stained with ethidium bromide DNA, previously treated with different aqueous ex-
(0.5 ␮g/mL) and the DNA bands were visualized tract C. icaco concentrations (0.7, 3.5 and 7 mg/mL),
by fluorescence under an ultraviolet (UV) transil- was then analysed. Following, 200 ␮L of competent
luminator system. The assay was repeated, at least bacterial suspensions (∼109 cells/mL) were mixed
five times, the best result being digitalized (Kodak with pretreated plasmid DNA (5 ng) and incubated
Digital Science 1d, EDAS 120), and the bands quan- on ice for 30 min. Next, the tubes were placed in
tified through the Gel-Pro Analyzer 3.0 computer water-bath at 42 ◦ C for 2 min and immediately in-
program. cubated on ice for 2 min. Afterwards, 500 ␮L of
pre-warmed LB (37 ◦ C) were added. The samples
2.4. Statistical analysis of DNA strand breaks were then incubated for 1 h at 37 ◦ C and aliquots
were withdrawn and plated onto LB-agar, supple-
ANOVA followed by the Student Newman Keuls mented with 60 ␮g ampicillin/mL. Plates were incu-
multiple comparison test through the statistical pro- bated at 37 ◦ C for 24 h and the number of colonies
gram InStat version 3.01 (GraphPad Software, San was counted. A mean value from each C. icaco ex-
Diego, CA, USA) was used to compare the results ob- tract concentration employed was obtained, and the
tained from the different extract concentrations against transformation efficiency was expressed as the per-
484 S.C. Ferreira-Machado et al. / Toxicology Letters 151 (2004) 481–487

Fig. 1. A percentage of 0.8 agarose gel electrophoresis and densitometric analysis of plasmid pUC 9.1 (200 ng) treated with increasing
concentrations of C. icaco or with SnCl2 as a positive control. Aliquots of pUC 9.1 plasmid DNA (200 ng) were incubated with different
concentrations of C. icaco extract for 40 min at room temperature (25 ◦ C). Each sample was mixed with loading buffer and submitted to 0.8%
agarose gel electrophoresis. (A) The experiment was performed, at least, five times and the best photo is presented here. (B) Densitometric
measures were obtained from the gel, through Gel Pro Analyser computer program. Lanes: (1) pUC 9.1; (2) SnCl2 (200 ␮g/mL); (3)
0.7 mg/mL; (4) 1.75 mg/mL; (5) 3.5 mg/mL and (6) 7 mg/mL of the extract plant.

centage of the maximal value (100%), attributed to 0.7–7.0 mg/mL. This result could classify the C. icaco
the control (not treated). This experiment was carried extract as a weak strand break inducer. This genotoxic
out in triplicate and the results presented are the mean effect was confirmed by densitometry analysis. The
average. The standard deviations did not exceed 15% open circle and supercoiled forms were analysed and
of the respective mean values. Data were analysed the result suggests that, regarding the control, there
by using ANOVA, followed by the Student Newman was a significative (P < 0.001) increase in the num-
Keuls multiple comparison test through the statisti- ber of lesions when plasmid DNA was treated with C.
cal program InStat version 3.01 (GraphPad Software, icaco, without be observed the linear form (III).
San Diego, CA, USA). To evaluate the transformation
efficiency significance, a significant level of 5% was 0,45
adopted to compare these data. 0,4
0,35
SSB/Genome

0,3
0,25
3. Results and discussion 0,2
0,15
0,1
The conformational changes in pretreated plasmid
0,05
DNA were analysed through agarose gel electrophore- 0
sis. Our results showed that plasmid DNA had their 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 5,5 6 6,5 7 7,5 8

[mg/mL] C. icaco extract

supercoiled form (I) changed to relaxed open circular
form (II) after treatment with increasing concentra- Fig. 2. Number of single-strand breaks/genome in plasmid DNA
tions of extract plant (from 0.7 to 7 mg/mL) (Fig. 1). treated with C. icaco extract. Analysis of densitometric data per-
Thus, this extract was able to induce single-strand formed by the Gel Pro Analyser computer program, gave the null
events percentage (no breaks) for each one of the C. icaco extract
breaks, as can be seen in Fig. 2. On the other hand,
concentrations tested. Using the Poisson distribution, the mean
a dose–response pattern was not obtained (Fig. 2), value of breaks for each one of the concentrations, was obtained as
because there was no significative difference (P > follows µ = −ln p(0; µ). Number of single-strand breaks/genome
0.05) in the number of breaks induced at the interval values are the means of five isolated experiments.
 S.C. Ferreira-Machado et al. / Toxicology Letters 151 (2004) 481–487 485
Survival Fraction

10 10
1
0,1
Survival Fraction

1 0,01
0,001
0,0001
0,1 0,00001
0 20 40 60 0 30 60
treatment time (min) Time (min)

Fig. 3. Effect of C. icaco extract on the survival of E. coli AB1157. Fig. 5. Effect of C. icaco extract on the inactivation induced
Exponential growing cells, suspended in 0.9% NaCl solution, were by stannous chloride on E. coli PQ37 (rfa and uvrA). Expo-
treated with C. icaco for different incubation times. The survival nential growth cells suspended in 0.9% were treated with SnCl2
fractions were determined: (䉬) 0.9% NaCl; (䊐) 0.7 mg/mL C. (25 ␮g/mL) for different incubation times in the presence or ab-
icaco extract; ( ) 3.5 mg/mL C. icaco extract; (䊊) 7 mg/mL C. sence of the extract (7 mg/mL). The survival fraction were deter-
icaco extract. mined: (䉬) 0.9% NaCl; (䊐) SnCl2 ; ( ) C. icaco extract; (䊊)
SnCl2 + C. icaco extract.

The inactivation induced in different DNA repair

proficient E. coli strains by various concentrations of was used to transform wild-type E. coli AB1157 com-
the aqueous extract was also evaluated. As it can be petent cells (ampR), as described in Section 2. Thus,
observed (Figs. 3–5), there was no important decrease the genotoxic ability of this extract was observed in
in the survival fractions, either in the wild-type or in this assay (Fig. 6). In fact, there was a decrease in
the repair mutant tested (uvrA). Also, an important the transformation efficiency of plasmid DNA treated
decrease of the survival levels was not observed in with the extract, when compared to the control and
other repair mutant E. coli strains (AB2463, recA; among them (P < 0.05). In this system, the ability of
BW9091, xthA; BW544, xthA, nfo; BW535, nth, nfo, a transformed wild-type bacterial cell to grow in se-
xthA; GY5022, envA; and GY5027, envA, uvrB) (data lective media is a result of its capacity to repair plas-
not shown). mid DNA induced lesions. Thus, transformed bacteria
These results could initially suggest the absence of were incubated for 1 h in LB medium, without ampi-
toxicity of this extract, but we cannot discard the fact cillin. So, bacteria were allowed to recover the plasmid
that the substances, present in extract composition, DNA from the lesions to further encode ␤-lactamase
did not enter in sufficient quantity into the bacterial plasmid gene that would enable bacteria to replicate
cells to cause damage to the genetic material. To elu- in solid LB containing ampicillin medium (Hannanah,
cidate this point, 5 ng of the treated plasmid fraction
120
Transformation

100
efficiency %

10
Survival Fraction

80
1
60
(N/No)

0,1
0,01 40
0,001 20
0,0001 0
0 30 60 1 2 3 4 5
Treatment Time (min) Treatment

Fig. 4. Effect of C. icaco extract on the inactivation induced by Fig. 6. Transformation efficiency of plasmid pUC 9.1 onto
stannous chloride on E. coli PQ35 (rfa). Exponential growth cells wild-type E. coli AB1157. Aliquots of plasmid pUC 9.1 treated
suspended in 0.9% were treated with stannous chloride (25 ␮g/mL) with increasing concentrations of C. icaco extract plant and SnCl2
for different incubation times in the presence or absence of the solution 200 ␮g/mL (positive control) were mixed to the competent
extract (7 mg/mL). The survival fraction was determined: (䉬) 0.9% bacterial suspension and submitted to transformation. Values are
NaCl; (䊐) SnCl2 ; ( ) C. icaco extract; (䊊) SnCl2 + C. icaco the mean of three isolated experiments: (1) pUC 9.1; (2) SnCl2 ; (3)
extract. 0.7 mg/mL; (4) 3.5 mg/mL and (5) 7.0 mg/mL of C. icaco extract.
486 S.C. Ferreira-Machado et al. / Toxicology Letters 151 (2004) 481–487

1983). From these results, we could suggest that the radicals generated by the SnCl2 . Similarly, Melo et al.
decrease in transformation efficiency could be due to (2001) demonstrated that the lethal effect of stannous
the inability of the bacteria to repair plasmid DNA chloride was reduced by the extract of Cymbopogon
whenever augmented concentrations of C. icaco aque- citratus, Maytenus ilicifolia and Baccharis genistel-
ous extract are employed. On the other hand, hidden loides. Thus, these data indicate that DNA is an im-
lesions should also be generated, but could not be de- portant target to the C. icaco extract effects, while the
tected to the extent of the methodology employed in protective action induced against the metal lethal ef-
this work. This could explain why a dose response pat- fect is probably due to unspecific activities such as:
tern in the transformation efficiency experiments was scavenging of stannous ions prior to it uptake in E.
observed (Fig. 6), but not in the electrophoresis as- coli.
says, in which there was only the DNA being injured Finally, the results presented in this work ratify the
(Figs. 1–2). However, these results may reflect differ- need for further toxicological studies of substances
ent transformation efficiencies, due to the difficulty of taken for granted as innocuous, which have been used
the damaged plasmids enter into the cell, rather than by the population without medical prescriptions.
decreasing in repair ability of lesioned plasmids.
Natural products have been used as antioxidants
against reactive oxygen species generated by our own Acknowledgements
metabolism. To test whether C. icaco had an antioxi-
dant effect, the strains PQ35 (rfa) and PQ37 (rfa and This research was supported by grants from Capes,
uvrA) were incubated with SnCl2 (25 mg/mL) in the UERJ, CNPq and FAPERJ.
presence, or not, of the extract (0.7 mg/mL). The strain
PQ35, when compared to the deficient in the excision
repair (PQ37), presented a lower inactivation, when References
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