genotoxicity assessment of garcinia achachairu rusby (clusiaceae) extract in mammalian cells in vivo

5
Genotoxicity assessment of Garcinia achachairu Rusby (Clusiaceae) extract in mammalian cells in vivo Eduardo de Souza Marques a , Suellen Silva b , Rivaldo Niero b , Se ´ rgio Faloni de Andrade b , Paulo Cesar Pires Rosa c , Fabio Ferreira Perazzo c , Edson Luis Maistro a,d,n a Universidade Estadual Paulista – UNESP – Instituto de Biociˆ encias, Programa de Po ´s-Graduac - ~ ao em Biologia Geral e Aplicada, Botucatu, SP, Brazil b Universidade do Vale do Itajaı ´ – UNIVALI – Programa de Mestrado em Ciˆ encias Farmacˆ euticas, Nu ´cleo de Investigac - ~ oes Quı ´mico-Farmacˆ euticas (NIQFAR), Itajaı ´, SC, Brazil c Universidade Federal de S ~ ao Paulo – UNIFESP – Departamento de Ciˆ encias Exatas e da Terra, Diadema, SP, Brazil d Universidade Estadual Paulista – UNESP – Faculdade de Filosofia e Ciˆ encias, Departamento de Fonoaudiologia, Marı ´lia 17525-900, SP, Brazil. article info Available online 17 May 2012 Keywords: Garcinia achachairu Clusiaceae Comet assay Micronucleus test Guttiferone A abstract Ethnopharmacological relevance: Garcinia achachairu Rusby (Clusiaceae) is popularly known as ‘‘acha- chairu’’, and is used in Bolivian folk medicine for its healing, digestive, and laxative properties, and in the treatment of gastritis, rheumatism and inflammation. Despite its widespread therapeutic use, there is a lack of data regarding its in vivo genotoxic effects. Therefore, in this study, we used the comet assay and the micronucleus test, respectively, to evaluate the possible genotoxic and clastogenic effects of Garcinia achachairu seed extract (GAE) on different cells of mice. Material and methods: The GAE was administered by oral gavage at doses of 500, 1000 and 2000 mg/kg. For the analysis, the comet assay was performed on the leukocytes (collected 4 and 24 h after treatment), liver, bone marrow and testicular cells (collected 24 h after treatment), and the micro- nucleus test (MN) on bone marrow cells. Cytotoxicity was assessed by scoring 200 consecutive polychromatic (PCE) and normochromatic (NCE) erythrocytes (PCE/NCE ratio). Results and conclusion: The results showed that GAE did not induce significant DNA damage in leukocytes (4 h and 24 h samples), liver, bone marrow and testicular cells (24 h samples). GAE also did not show any significant increase in micronucleated polychromatic erythrocytes (MNPCEs) at the three tested doses. The PCE/NCE ratio indicated no cytotoxicity. Under our experimental conditions, the data obtained suggest that a single oral administration of G. achachairu extract does not cause genotoxicity and clastogenicity in different cells of mice. & 2012 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Preparations of medicinal plants are widely used in human therapy. Since in nature, some plants synthesize toxic chemicals, apparently as a primary defense mechanism against bacteria, fungi, insects and other animal predators, in-depth studies on their potential risks to human health are necessary. Garcinia achachairu Rusby (Clusiaceae) is a plant belonging to the genus Garcinia – ex genus Rheedia (Sweeney, 2008), popularly known as ‘‘achachairu’’. This plant is used in Bolivian folk medicine for its healing, digestive, and laxative properties, and for the treatment of gastritis, rheumatism and inflammation (Barbosa and Artiole, 2007). Some species of this genus have different chemical constituents, such as benzophenones and bioflavonoids, with great importance for the pharmaceutical industries due to the wide spectrum of biological activities of these compounds. Its activities include cytophatic inhibition of in vitro HIV infection; free radical scavenging; iNOS and COX-2 expression inhibition in carcinoma of the colon; induction of apoptosis, antiulcer, and trypanocidal properties (Martins et al., 2007). Recently, in our laboratories, we have demonstrated that the seed extract, fractions and a pure compound named Guttiferone A have important antinociceptive activity in different experimental models in mice (Molin et al., 2012). According to the literature, using the comet assay and micro- nucleus test, it is possible to evaluate the potential genotoxicity of many compounds through in vitro and in vivo models (Aquino et al., 2011; Melo-Cavalcante et al., 2011; Ribeiro et al., 2010; Rodrigues et al., 2009). These assays have achieved the status of standard tests in the battery of tests used to assess the safety of novel pharmaceuticals or other chemicals (Candido-Bacani Pde et al., 2011). To our knowledge, there have been no previous studies investigating the genetic toxicity of plants belonging to Contents lists available at SciVerse ScienceDirect journal homepage: www.elsevier.com/locate/jep Journal of Ethnopharmacology 0378-8741/$ - see front matter & 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2012.04.045 n Corresponding author at: Universidade Estadual Paulista – UNESP – Faculdade de Filosofia e Ciˆ encias, Departamento de Fonoaudiologia, Marı ´lia 17525-900, SP, Brazil. Tel.: þ55 1434021324; fax: þ55 1434021302. E-mail address: [email protected] (E.L. Maistro). Journal of Ethnopharmacology 142 (2012) 362–366

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Page 1: Genotoxicity assessment of Garcinia achachairu Rusby (Clusiaceae) extract in mammalian cells in vivo

Journal of Ethnopharmacology 142 (2012) 362–366

Contents lists available at SciVerse ScienceDirect

Journal of Ethnopharmacology

0378-87

http://d

n Corr

de Filos

Brazil. T

E-m

journal homepage: www.elsevier.com/locate/jep

Genotoxicity assessment of Garcinia achachairu Rusby (Clusiaceae) extractin mammalian cells in vivo

Eduardo de Souza Marques a, Suellen Silva b, Rivaldo Niero b, Sergio Faloni de Andrade b,Paulo Cesar Pires Rosa c, Fabio Ferreira Perazzo c, Edson Luis Maistro a,d,n

a Universidade Estadual Paulista – UNESP – Instituto de Biociencias, Programa de Pos-Graduac- ~ao em Biologia Geral e Aplicada, Botucatu, SP, Brazilb Universidade do Vale do Itajaı – UNIVALI – Programa de Mestrado em Ciencias Farmaceuticas, Nucleo de Investigac- ~oes Quımico-Farmaceuticas (NIQFAR), Itajaı, SC, Brazilc Universidade Federal de S~ao Paulo – UNIFESP – Departamento de Ciencias Exatas e da Terra, Diadema, SP, Brazild Universidade Estadual Paulista – UNESP – Faculdade de Filosofia e Ciencias, Departamento de Fonoaudiologia, Marılia 17525-900, SP, Brazil.

a r t i c l e i n f o

Available online 17 May 2012

Keywords:

Garcinia achachairu

Clusiaceae

Comet assay

Micronucleus test

Guttiferone A

41/$ - see front matter & 2012 Elsevier Irelan

x.doi.org/10.1016/j.jep.2012.04.045

esponding author at: Universidade Estadual P

ofia e Ciencias, Departamento de Fonoaudio

el.: þ55 1434021324; fax: þ55 1434021302

ail address: [email protected] (

a b s t r a c t

Ethnopharmacological relevance: Garcinia achachairu Rusby (Clusiaceae) is popularly known as ‘‘acha-

chairu’’, and is used in Bolivian folk medicine for its healing, digestive, and laxative properties, and in

the treatment of gastritis, rheumatism and inflammation. Despite its widespread therapeutic use, there

is a lack of data regarding its in vivo genotoxic effects. Therefore, in this study, we used the comet assay

and the micronucleus test, respectively, to evaluate the possible genotoxic and clastogenic effects of

Garcinia achachairu seed extract (GAE) on different cells of mice.

Material and methods: The GAE was administered by oral gavage at doses of 500, 1000 and 2000 mg/kg.

For the analysis, the comet assay was performed on the leukocytes (collected 4 and 24 h after

treatment), liver, bone marrow and testicular cells (collected 24 h after treatment), and the micro-

nucleus test (MN) on bone marrow cells. Cytotoxicity was assessed by scoring 200 consecutive

polychromatic (PCE) and normochromatic (NCE) erythrocytes (PCE/NCE ratio).

Results and conclusion: The results showed that GAE did not induce significant DNA damage in

leukocytes (4 h and 24 h samples), liver, bone marrow and testicular cells (24 h samples). GAE also

did not show any significant increase in micronucleated polychromatic erythrocytes (MNPCEs) at the

three tested doses. The PCE/NCE ratio indicated no cytotoxicity. Under our experimental conditions, the

data obtained suggest that a single oral administration of G. achachairu extract does not cause

genotoxicity and clastogenicity in different cells of mice.

& 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Preparations of medicinal plants are widely used in humantherapy. Since in nature, some plants synthesize toxic chemicals,apparently as a primary defense mechanism against bacteria,fungi, insects and other animal predators, in-depth studies ontheir potential risks to human health are necessary.

Garcinia achachairu Rusby (Clusiaceae) is a plant belonging tothe genus Garcinia – ex genus Rheedia (Sweeney, 2008), popularlyknown as ‘‘achachairu’’. This plant is used in Bolivian folkmedicine for its healing, digestive, and laxative properties, andfor the treatment of gastritis, rheumatism and inflammation(Barbosa and Artiole, 2007). Some species of this genus havedifferent chemical constituents, such as benzophenones and

d Ltd. All rights reserved.

aulista – UNESP – Faculdade

logia, Marılia 17525-900, SP,

.

E.L. Maistro).

bioflavonoids, with great importance for the pharmaceuticalindustries due to the wide spectrum of biological activities ofthese compounds. Its activities include cytophatic inhibition ofin vitro HIV infection; free radical scavenging; iNOS and COX-2expression inhibition in carcinoma of the colon; induction ofapoptosis, antiulcer, and trypanocidal properties (Martins et al.,2007). Recently, in our laboratories, we have demonstratedthat the seed extract, fractions and a pure compound namedGuttiferone A have important antinociceptive activity in differentexperimental models in mice (Molin et al., 2012).

According to the literature, using the comet assay and micro-nucleus test, it is possible to evaluate the potential genotoxicity ofmany compounds through in vitro and in vivo models (Aquinoet al., 2011; Melo-Cavalcante et al., 2011; Ribeiro et al., 2010;Rodrigues et al., 2009). These assays have achieved the status ofstandard tests in the battery of tests used to assess the safety ofnovel pharmaceuticals or other chemicals (Candido-Bacani Pdeet al., 2011). To our knowledge, there have been no previousstudies investigating the genetic toxicity of plants belonging to

Page 2: Genotoxicity assessment of Garcinia achachairu Rusby (Clusiaceae) extract in mammalian cells in vivo

E.S. Marques et al. / Journal of Ethnopharmacology 142 (2012) 362–366 363

the Garcinia genus. Therefore, the present study was undertakento investigate the genotoxic and clastogenic potential of Garcinia

achachairu seeds extract (GAE) on different cells of mice using thecomet and micronucleus assays, respectively.

2. Material and methods

2.1. Plant material

The material (leaves, seeds and branches) of G. achachairu

were collected separately in Camboriu, Santa Catarina, in March2007 and identified by Dr. Oscar B. Iza (Department of Botany,University of Vale do Itajaı). A voucher specimen was deposited atthe Barbosa Rodrigues Herbarium (Itajaı-SC) under number HBR52637. In this work, only the extract prepared from the seedswas used.

2.2. Extract preparation

Air-dried and powdered seeds (250 g) of G. achachairu wereextracted at room temperature by maceration with methanol(2�1000 mL) for seven days. The extracted material was filteredand concentrated under reduced pressure by rotatory evaporator,yielding 9.01 g (3.6%) of crude methanol seed extract.

2.3. Phytochemical characterization: chromatographic and tandem

mass spectrometric system

The LC system used consisted of a Shimadzu HPLC System(Perkin-Elmer, USA) with an autoinjector for solvent delivery andsample introduction. An Esquire-3000 triple quadrupole LC-MS/MS mass spectrometer with an electrospray ionization source(ESI) was used as a detector. Separation was performed on a2.1�100 mm C18 column packed with 5 mm particles (Spheri-5RP-18 Brownlee, Perkin-Elmer) connected to the outlet of a2.1�15 mm, 5 mm, guard column (Spheri-5 RP18 Brownlee,Perkin-Elmer). The mobile phase consisted of acetonitrile: H2O(9:1 v/v; 30%; solvent A) and 0.5% AcOH in MeOH (70%; solvent B)and was delivered at a flow rate of 0.4 mL/min at room tempera-ture. Analyst software (version 1.4, Applied Biosystems/MDSSCIEX, Toronto, Canada) was used for the control of equipment,acquisition and data analysis. Scans were performed in negativemode and declustering potential was optimized.

2.4. Chemicals

Doxorubicin (DXR, Oncodoxs, Meizler) was used as theDNA damage agent in the comet and micronucleus assays,and was prepared by dissolving it in sterile water. The othermain chemicals were obtained from the following suppliers:normal melting point (NMP) agarose (Cat. no. 15510–019:Invitrogen) low melting point (LMP) agarose (Cat. no. 15517–014:Invitrogen), N-lauroyl sarcosine sodim salt (L-5125: Sigma) andethylenediaminetetraacetic acid (EDTA) (Merck). GAE was dissolvedin 1% DMSO (Dimethyl sulfoxide).

2.5. Animals and dosing

Experiments were carried out on 10-week-old male albinoSwiss mice (Mus musculus), weighing 25–30 g. The animals wereacquired from the biotermium of the Universidade EstadualPaulista (UNESP), Botucatu, S~ao Paulo state, Brazil, and kept inpolyethylene boxes, in a climatecontrolled environment (2574 1C,5575% relative humidity) with a 12 h light–dark cycle (7:00 a.m.to 7:00 p.m.). Food (NUVILAB CR1NUVITAL) and water were

available ad libitum. The mice were divided into five experimentalgroups of six animals each. GAE was diluted in 1% DMSO andadministered in a single dose of 0.5 mL by gavage at concentrationsof 500, 1000 and 2000 mg/kg body weight, chosen on the basis ofour acute toxicity studies in mice, and following the limit doserecommended by OECD 420 (2001) for acute treatments intoxicology assays. Up to 2000 mg/kg dose, there was not any signof toxicity in animals. The negative control group received 1%DMSO by gavage. The positive control group received an intraper-itoneal injection of doxorubicin at 80 mg/kg body weight. Theanimals used in this study were sacrificed by cervical dislocationwithout anesthesia to avoid possible alterations in the DNAdamage analysis. The Animal Bioethics Committee of the Faculdadede Medicina de Marılia (CEP/FAMEMA, Marılia, S~ao Paulo state,Brazil) approved the present study on 26 February 2010 (protocolnumber 780/09), in accordance with federal government legisla-tions on animal care.

2.6. Comet assay

The comet assay (Single Cell Gell Electrophoresis –SCGE) wascarried out by the method described by Speit and Hartmann(1999), which is based on the original work of Singh et al. (1988)and includes modifications introduced by Klaude et al. (1996) aswell as additional modifications. Peripheral blood samples fromthe tail vein were obtained from six Swiss mice of each group, 4 hand 24 h after treatment, before euthanasia. After the animals’sacrifice, liver, bone marrow, and testicle cells samples werewashed in saline solution, in an ice bath. A small portion (about4 mm in diameter) was transferred to a Petri dish containing 1 mlof Hank’s solution (pH 7.5) and then homogenized gently with asmall pair of tweezers and a syringe to avoid clumps of cells. Analiquot of 20 ml was removed from the supernatant of each celltype to determine cell viability. Cell counting was performedusing a hemocytometer. Cell viability was determined by trypanblue dye exclusion. The number of trypan bluenegative cells wasconsidered the number of viable cells, and was greater than 85%.Another equal aliquot of cells from each animal was mixed with120 ml of 0.5% low melting point agarose at 37 1C, and rapidlyspread onto two microscope slides per animal, precoated with1.5% normal melting point agarose. The slides were coverslippedand allowed to gel at 4 1C for 20 min. The coverslips were gentlyremoved and the slides were then immersed in cold, freshlyprepared lysis solution consisting of 89 ml of a stock solution(2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH set to 10.0 with �8 gsolid NaOH, 890 ml of distilled water and 1% sodium laurylsarcosine), plus 1 ml of Triton X100 (Merck) and 10 ml of DMSO(Merck). The slides, which were protected from light, wereallowed to stand at 4 1C for 1 h and then placed in the gel box,positioned at the anode end, and left in a high pH (413)electrophoresis buffer (300 mM NaOH–1 mM EDTA, preparedfrom a stock solution of 10 M NaOH and 200 mM, pH 10.0, EDTA)at 4 1C for 20 min prior to electrophoresis, to allow DNA unwind-ing. The electrophoresis run was carried out in an ice bath (4 1C)for 20 min at 300 mA and 25 V (0.722 V cm�1). The slides werethen submerged in a neutralization buffer (0.4 M Tris–HCl, pH7.5) for 15 min, dried at room temperature and fixed in 100%ethanol for 10 min. The slides were dried and stored overnight orlonger, before staining. For the staining process, the slides werebriefly rinsed in distilled water, covered with 30 ml of 1�ethidium bromide staining solution prepared from a 10� stock(200 mg ml�1) and coverslipped. The material was evaluatedimmediately at 400� magnification, using a fluorescence micro-scope (Olympus BX 50) with a 515–560 nm excitation filter and a590 nm barrier filter. Only individual nucleoids were scored.The extent and distribution of DNA damage indicated by the SCGE

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E.S. Marques et al. / Journal of Ethnopharmacology 142 (2012) 362–366364

assay was evaluated by examining at least 100 randomly selectedand non-overlapping cells (50 cells per coded slide) per animal ina blind analysis (six mice per group). These cells were scoredvisually, according to tail size, into the following four classes:class 0, no tail; class 1, tail shorter than the diameter of the head(nucleus); class 2, tail length 1–2 times the diameter of the head;and class 3, tail length more than twice the diameter of the head.Comets with no heads, with nearly all the DNA in the tail or witha very wide tail were excluded from the evaluation because theyprobably represented dead cells (Hartmann and Speit, 1997).The total score for 100 comets, which ranged from 0 (all undamaged)to 300 (all maximally damaged), was obtained by multiplying thenumber of cells in each class by the damage class.

Table 1Identification of chromatogram peaks and retention time of the major compounds

found in the G. achachairu seeds extract (GAE).

2.7. Bone marrow micronucleus test

The assay was carried out following standard protocols, asrecommended by Schmid (1975) and Krishna and Hayashi (2000).The same six male mice from each group as those used in thecomet assay were also used to this assay. The bone marrow fromone femur was flushed out using 2 ml of saline (0.9% NaCl) andcentrifuged for 7 min. The supernatant was discarded and smearswere made on slides. The slides were coded for a ‘blind’ analysis,fixed with methanol and stained with Giemsa (Gollapudi andKamra, 1979). For the analysis of the micronucleated cells, 2000polychromatic erythrocytes (PCE) per animal were scored, todetermine the clastogenic and/or aneugenic property of theGAE. To detect possible cytotoxic effects, the PCE/NCE (normo-chromatic erythrocytes) ratio in 200 erythrocytes/animal wascalculated (Gollapudi and McFadden, 1995). The cells wereblindly scored using a light microscope at 1000� magnification.The mean number of micronucleated polychromatic erythrocytes(MNPCEs) in individual mice was used as the experimental unit,with variability (standard deviation) based on differencesbetween animals within the same group.

Compound Retention time Mass (m/z) Identification

01 4.1–4.3 609.0 Guttiferone N

02 37.9–38.4 633.3

03 39.2–39.5 618.0 Garcinol

04 39.8–40.2 617.2 Isogarcinol

05 42.1–42.6 599.5 Guttiferone M

06 42.6–42.7 617.5 Camboginol

07 42.8–43.0 389.7

08 43.8–44.0 255.7

09 44.0–44.2 417.2

10 44.8–45.2 601.5 Guttiferone A

11 45.3–45.6 835.2

12 46.2–46.7 601.5 Xanthochymol

2.8. Statistical analysis

After verifying for normal distribution (normality test KSperformed). The data obtained from the comet assay weresubmitted to analysis of variance (ANOVA) and the Tukey–Kramer multiple comparison test (Sokal and Rohlf, 1995) andthe data obtained from the micronucleus assay were submitted tothe analysis of variance test (ANOVA) with linear regression,using the GraphPad Prisms software (version 5.02) in both cases.The results were considered statistically significant at Po0.05.

Fig. 1. Chromatogram (LCMS) of G.

3. Results and discussion

The therapeutic use of natural products, including medicinalplants, has become increasingly prevalent. Many pharmacognos-tical and pharmacological investigations have been performed toidentify lead compounds for drug development (Newman et al.,2003). Due to the biological activities of these compounds,evaluation of its genotoxic/mutagenic potential is essential (Bastet al., 2002; Rodeiro et al., 2006; Santos et al., 2007; Tice et al.,2000).

The phytochemical profile of the Garcinia achachairu seedsextract (GAE) has presented 12 compounds, which were able toidentified 7 compounds (Fig. 1, Table 1). The major compound ofthe extract by the area is the compound 10, identified asGuttiferone A. Our results extend previous phytochemical char-acterization of the G. achachairu seeds extract, where the presenceof benzophenones, xanthones and bioflavonoids as main consti-tuents also were observed, being the benzophenone Guttiferone Athe major compound (Molin et al., 2012).

The present study evaluates the genotoxic property of GAE.The comet assay and micronucleus test are effective tests, in thiscontext. The alkaline version of the comet assay was used in ourstudy. This assay measures low levels of DNA damage, such assingle and double strand breaks, alkali-labile sites, and DNA–DNAand DNA–protein crosslinks (Tice et al., 2000). Considering thatorgan-specific genotoxicity closely parallels organ specific carci-nogenicity (Tsuda et al., 2000), we analyzed mice cells fromdifferent organs. Our results obtained by SCGE (comet) assay arepresented in Fig. 2, which shows the DNA damage (according totail size) in leukocytes from peripheral blood cells (collected4 and 24 h after treatment), and liver, bone marrow, and testi-cular cells (collected 24 h after GAE treatment). The cell viability

achachairu seeds extract (GAE).

Page 4: Genotoxicity assessment of Garcinia achachairu Rusby (Clusiaceae) extract in mammalian cells in vivo

Fig. 2. Genotoxicity of Garcinia achachairu seeds extract (GAE) in different cells of mice by comet assay. npo0.001 (ANOVA/Tukey post-test) when compared to negative

control. Data are expressed as the mean values obtained from six mice per group (n¼6); Score¼DNA damage index.

Table 2Number of micronucleated polychromatic erythrocytes (MNPCEs) observed in the bone marrow cells of male (M) Swiss mice treated with Garcinia achachairu seeds extract

(GAE), and respective controls.

Treatments Number of MNPCE per animal MNPCE Mean7SD PCE/NCE Mean7SD

M1 M2 M3 M4 M5 M6

Control (DMSO 1%) 4 3 4 5 3 4 3.8370.68 1.2570.16Doxorubicin (80 mg/kg) 23 18 20 17 17 22 19.572.36* 1.3870.24GAE (500 mg/kg) 4 5 4 4 3 4 4.0070.57 1.4070.23GAE (1000 mg/kg) 3 3 3 4 3 3 3.1670.37 1.3370.20GAE (2000 mg/kg) 5 4 3 3 4 3 3.6670.74 1.2470.14

Two thousand cells per animal were analyzed. SD¼standard deviation of the mean.n Significantly different from control (po0.001).

E.S. Marques et al. / Journal of Ethnopharmacology 142 (2012) 362–366 365

for all the cells was greater than 85% using Trypan blue staining,which confirms the absence of cytotoxicity observed by the PCE/NCE ratio in the MN test. No death, morbidity or distinctiveclinical signs were observed in the treated animals following GAEtreatment. As expected, doxorubicin, the positive control, induceda significant increase in DNA migration in leukocytes (po0.001)when compared to the negative control, indicating the validity ofthe species selected, and of the study design in the detection ofgenotoxic effects. Our historical laboratory positive control usingdoxorubicin confirmed the results obtained. In all the analyzedcells, no increases in DNA damage (p40.05) were found betweenthe negative control and experimental groups treated with threedoses of GAE. When cells were exposed to three concentrations ofthe test compound, the majority of cells examined on slides didnot show any DNA damage (class 0), with very few nucleoidspresenting class 1 DNA damage. These findings suggest nogenotoxic effects of the GAE on the cells analyzed.

The second cytogenetic assay performed in the present studywas the micronucleus test. This in vivo assay is the primary test in

a battery of genotoxicity tests recommended by the regulatoryagencies worldwide. The assay measures clastogenicity (chromo-some breakage) and aneugenicity (chromosome lagging due tomitotic apparatus dysfunction), and estimating the ratio of poly-chromatic erythrocytes (PCEs) to normochromatic erythrocytes(NCEs) is useful in evaluating any perturbations in hematopoiesisas a result of animal treatment (Krishna and Hayashi, 2000;Gollapudi and McFadden, 1995). Our results for the micronucleusassay and PCE/NCE ratio determined after single gavage admin-istration of three different doses of GAE in Swiss mice are shownin Table 2. There were no statistically significant differences(p40.05) in the frequency of micronucleated polychromaticerythrocytes (MNPCEs) between the control and the groupstreated with the three doses of GAE, indicating absence of theclastogenic/aneugenic effects of this extract. As expected, theanimals treated with doxorubicin showed a high frequency ofMNPCE in bone marrow cells when compared with the control(po0.001). The estimated ratio of PCE–NCE in bone marrowpreparations showed no statistically significant alterations in

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E.S. Marques et al. / Journal of Ethnopharmacology 142 (2012) 362–366366

hematopoiesis as a result of GAE treatment, indicating no cyto-toxic effects. The results obtained in the micronucleus test areconsistent with those observed by the comet assay.

Our literature review found no previous studies involving theevaluation of the genotoxic potential of the Garcinia or Rheedia

extracts. Nevertheless, several species of this genus showed thepresence of benzophenones, xanthones and bioflavonoids as mainconstituents (Deachathai et al., 2006; Lannang et al., 2010;Panthong et al., 2006), and, for some of these compounds, thereare genotoxic and antigenotoxic studies. Almanza et al. (2011)reported that the benzophenone acuminophenone A, and thexanthones formoxanthone C and macluraxanthone isolated fromRheedia acuminate showed no mutagenicity on several Salmonella

typhimurium strains, but on the other hand, these compoundspromoted a strong reduction of mutagenic effect induced byhydrogen peroxide.

In the present study we observed the absence of genotoxicityof the G. achachairu seeds extract. Our results are in agreementwith the above mentioned study that analyzed some of the maincompounds found in the Garcinia extract.

In conclusion, the present results demonstrate that under ourexperimental conditions, Garcinia achachairu seeds extract is notgenotoxic or clastogenic even considering the high doses tested.These results represent a positive step forward in determining thesafe use of these plants in traditional and folk medicine. In theethnopharmacological context, the lack of toxicity and genotoxi-city of this extract is important for the whole population, sincethe rate of cancer, as well as diseases caused by genotoxic agents,is increasing worldwide.

Acknowledgments

E.S. Marques thanks CAPES for a Master’s scholarship andPatrıcia C. Martins Mello for her technical assistance.

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