Journal of Ethnopharmacology 153 (2014) 694–700

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Research Paper

Genotoxic assessment of Rubus imperialis (Rosaceae) extract in vivo and its potential chemoprevention against cyclophosphamide-induced DNA damage Ana Beatriz Costa Rodrigues Alves a, Rafaella Souza dos Santos a, Susana de Santana Calil a, Rivaldo Niero b, Jhonny da Silva Lopes b, Fábio F. Perazzo c, Paulo César Pires Rosa c, Sérgio Faloni Andrade b, Valdir Cechinel-Filho b, Edson Luis Maistro a,n a Universidade Estadual Paulista – UNESP – Faculdade de Filosofia e Ciências, Departamento de Fonoaudiologia, Av. Hygino Muzzi Filho, 737, Caixa Postal 181, Marília, São Paulo 17525-900, Brazil b Programa de Pós-Graduação em Ciências Farmacêuticas, Núcleo de Investigações Químico-Farmacêuticas (NIQFAR), Universidade do Vale do Itajaí – UNIVALI, Itajaí, Santa Catarina, Brazil c Universidade Federal de São Paulo – UNIFESP – Instituto de Ciências Ambientais, Químicas e Farmacêuticas, Departamento de Ciências Exatas e da Terra, Diadema, São Paulo, Brazil

art ic l e i nf o

a b s t r a c t

Article history: Received 12 December 2013 Received in revised form 26 February 2014 Accepted 14 March 2014 Available online 29 March 2014

Ethnopharmacological relevance: Rubus imperialis Cham. Schl. (Rosaceae) is frequently used in traditional medicine as hypoglycemic, antinociceptive and antiviral remedy. Materials and methods: Swiss albino mice were distributed in eight groups for acute treatment with Rubus imperialis extract (24 h). The extract doses selected were 50, 250 and 500 mg/kg b.w. administered by gavage alone or plus to CPA (50 mg/kg b.w.) administered by intraperitoneal injection. Control groups were treated in a similar way. Analyses were performed using the comet assay, on leukocytes (collected 4 and 24 h after treatment) and liver (collected 24 h after treatment), and using the micronucleus test (MN) in bone marrow cells. Cytotoxicity was assessed by scoring 200 consecutive polychromatic (PCE) and normochromatic (NCE) erythrocytes (PCE/NCE ratio). Results and conclusion: The main compounds identified in the Rubus imperialis extract were saponins and steroidal compounds, with niga-ichigoside and tormentic acid being the major compounds. Tested doses of Rubus imperialis extract showed no genotoxic effects on leukocytes from peripheral blood or liver cells by the comet assay. However, the MN test showed an increase in the frequency of micronucleated cells at the two higher doses tested, indicating that this extract has clastogenic/aneugenic effects on bone marrow cells at higher doses. On the other hand, for all cells evaluated, the three tested doses of the Rubus imperialis extract promoted inhibition of DNA damage induced by CPA. Despite the chemoprevention observed, the clastogenicity/aneugenicity observed suggested caution about either continuous or high-dose usage of Rubus imperialis aerial parts extract by humans. & 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Rubus imperialis Rosaceae Comet assay Micronucleus test Antigenotoxic effects

1. Introduction Herbal medicines have been used for centuries for treatment of various ailments. They differ from conventional drugs in that they usually consist of a mixture of compounds. Since little is known about the relative safety of herbal medicines compared to synthetic drugs, its allergic, toxic, and possible mutagenic effects need to be investigated (Ernst, 1998). Rubus species (family Rosaceae) consists of many species cultivated for centuries for their fruits. These and other parts of

n

Corresponding author. Tel.: þ55 1434021324; fax:þ 55 1434021302. E-mail address: [email protected] (E.L. Maistro).

http://dx.doi.org/10.1016/j.jep.2014.03.033 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.

the plants have been used as folk medicinal herbs because of their accumulation of tannins (Patel et al., 2004). Rubus imperialis Cham. Schl. grows abundantly in Southern Brazil, being known as amora-verde, amora-branca, amora-do-mato or amora-brava (Cirilo, 1993). Rubus imperialis is used as a popular remedy due to its hypoglycemic activity (Lemus, 1999). Despite wide folkloric use of Rubus imperialis as a therapeutic agent, there are few reports regarding its pharmacological and phytochemical properties. The antidiabetic action of this plant was evaluated and confirmed in animal model (Lemus, 1999; Novaes et al., 2001; Kanegusuku et al., 2002). Niero et al. (1999) demonstrated that aerial parts of Rubus imperialis presented potent antinociceptive effects when analyzed in both models of nociception in mice—

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writing and formalin tests. Ardenghi et al. (2006) showed that the antinociceptive action of the triterpene niga-ichigoside F1 isolated from Rubus imperialis appears to be related to the dopaminergic, cholinergic, glutamatergic, tackykininergic and oxinitrergic systems, supporting the ethnomedical use of this plant. In addition to these studies, it was demonstrated more recently that Rubus imperialis had an antiviral activity against herpes and rabies virus, with 50% effective cytotoxicity (EC50) ranging from 60 to 170 mg/ mL of the extract concentrations (Muller et al., 2007). To our knowledge, there is no reported genetic damage associated with the use of Rubus imperialis. Consequently, the present study was undertaken to evaluate the potential genotoxic effects of its aerial parts' extract in terms of induction of DNA damage in peripheral blood leukocytes and liver, and induction of micronuclei in bone marrow cells of Swiss albino mice in vivo. Furthermore, the possible antigenotoxicity of this extract on cyclophosphamide (CPA)-induced DNA damage was determined.

2. Material and methods 2.1. Chemicals The agent cyclophosphamide (CPA) (Sigma, CAS 50-18-0) was used as the DNA damaging agent in the comet assay and micronucleus test using Swiss mice. It was dissolved in phosphate buffer, pH 6. Other main 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), sodium salt N-lauroyl sarcosinate (L-5125 – Sigma) and ethylenediaminetetraacetic acid (EDTA – Merck). 2.2. Plant material The plant material was collected in August 2008 at Treze de Maio (latitude 281 330 28″S and longitude 0491 080 48″W) state of Santa Catarina, Brazil, and was identified by Dr. Ademir Reis. A voucher specimen was deposited at the Barbosa Rodrigues Herbarium under code VC-Filho012. 2.3. Preparations of extracts, fractions and isolation of compounds Dried and powdered aerial parts of Rubus imperialis (1.10 kg), were exhaustively extracted at room temperature (r.t.). The macerated ones were filtered and concentrated under reduced pressure, using a rotatory evaporator, yielding crude methanolic extract (MeOH). The extract (127.5 g; 12.7%) was then suspended in MeOH–water mixture (9:1) and successively partitioned with n-hexane, chloroform and ethyl acetate furnishing 5.32, 8.39 and 9.25 g, respectively. Each fraction separately was chromatographed on a silica-gel column (0.04–0.063 mm, 2.5  50 cm2, Merck for flash chromatography) or using an open column silicagel column (0.063–0.20 mm, 2.5  50 cm2, Merck) and eluted with a gradient of CHCl3–MeOH or Hexane–acetone (100--0) to give several fractions. Similar fractions were combined and the purity was examined by thin layer chromatography (TLC) using Merck silica gel pre-coated aluminum plates, with a layer thickness of 200 μm and several solvent systems of different polarities. The compounds were identified by NMR spectral data in comparison with authentic samples and the literature data. 2.4. Phytochemical profile by LC–MS conditions The nano-LC system used consisted of a Shimadzu Prominence Nano HPLC System (Shimadzu, Japan) with an autoinjector for

695

solvent delivery and sample introduction. An Amazon-ETD (Electron Transfer Dissociation), LC–MS/MS mass spectrometer with an electrospray ionization source (ESI) was used as a detector. Separation was performed on a 2.1  100 mm2 C18 column packed with 1.8 μm particles (Zorbax 300SBC18, Agilent).The mobile phase consisted of acetonitrile:formic acid 0.1%, 60/40 v/v, and was delivered at a flow rate of 0.6 mL/min at room temperature. Analyst software (version 1.4, Applied Biosystems/MDS SCIEX, Toronto, Canada) was used for the control of equipment, acquisition and data analysis. Scans were performed in a negative mode and declustering potential was optimized. 2.5. Animals and dosing The experiments were carried out using 12-week-old male Swiss albino mice (Mus musculus), weighing 25–30 g. The animals were acquired from the Universidade Estadual Paulista (UNESP), Botucatu, São Paulo State, Brazil, and housed in polyethylene boxes in a climate-controlled environment (257 74 1C, 557 75% humidity) with a 12-h light/dark cycle (7:00 a.m. to 7:00 p.m.). Food (Nuvilab CR1, Nuvital) and water were available ad libitum. The mice were divided into 8 experimental groups of 6 animals each. Rubus imperialis extract was dissolved in 1% Tween 80 aqueous solution and administered in a single dose of 0.3 mL by gavage at concentrations of 50, 250 and 500 mg/kg body weight. Considering that there are no reports of doses used in folk medicine by humans, the doses were chosen on the basis of hypoglycemic activity at 300 mg/kg in rats (Kanegusuku et al., 2002) and of antinociceptive activity at 200 mg/kg in mice (Niero et al., 1999). The negative control group received 1% Tween 80 aqueous solution by gavage, and the positive control group received an intraperitoneal injection of cyclophosphamide (CPA) at 50 mg/kg body weight. The animals used in this study were sacrificed by cervical dislocation without anesthesia to avoid possible alterations in the DNA damage analysis. The Animal Bioethics Committee of the Faculdade de Medicina de Marília (CEP/ FAMEMA, Marília, São Paulo state, Brazil) approved the present study on 10 November 2009 (protocol number 666/09), in accordance with federal government legislations on animal care. 2.6. Comet assay The comet assay (SCGE) was carried out by the method described by Speit and Hartmann (1999) and reviewed by Burlinson et al. (2007). Peripheral blood samples from the tail vein were obtained from six Swiss mice of each group, at 4 and 24 h after treatment and before euthanasia. After sacrifice of the animals, liver cells samples were washed in saline solution, in an ice bath. A small portion (diameter of about 4 mm) was transferred to a Petri dish containing 1 mL of Hank's solution (pH 7.5) and then homogenized gently with a small pair of tweezers and a syringe to remove clumps of cells. An aliquot of 20 mL was removed from the supernatant of each cell type to determine cell viability. Cell counting was performed using a hemocytometer. Cell viability was determined by trypan blue dye exclusion. The number of trypan blue-negative cells was considered to be the number of viable cells and was greater than 90%. Another equal aliquot of cells from each animal was mixed with 120 mL of 0.5% low melting point agarose at 37 1C, and rapidly spread onto two microscope slides per animal, pre-coated with 1.5% normal melting point agarose. The slides were coverslipped and allowed to gel at 4 1C for 20 min. The coverslips were gently removed and the slides were then immersed in cold, freshly prepared lysing 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 g solid NaOH, 890 mL of distilled water and 1% sodium lauryl sarcosine), plus 1 mL of Triton X-100 (Merck) and 10 mL of dimethyl sulfoxide (Merck). The

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slides, which were protected from light, were allowed 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, prepared from a stock solution of 10 N NaOH and 200 mM, pH 10.0, EDTA) at 4 1C for 20 min prior to electrophoresis, to allow DNA unwinding. 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 were then submerged in a neutralization buffer (0.4 M Tris–HCl, pH 7.5) for 15 min, dried at room temperature and fixed in 100% ethanol for 10 min. The slides were dried and stored overnight or longer, before staining. For the staining process, the slides were briefly rinsed in distilled water, covered with 30 mL of 1  ethidium bromide staining solution prepared from a 10  stock (200 mg/ml) and coverslipped. The material was evaluated immediately at 400  magnification, using a fluorescence microscope (Olympus BX 50) with a 515–560 nm excitation filter and a 590 nm barrier filter. Only individual nucleoids were scored. The extent and distribution of DNA damage indicated by the SCGE assay was evaluated by examining at least 100 randomly selected and non-overlapping cells (50 cells per coded slide) per animal in a blind analysis (six mice per group). These cells were scored visually, 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 of the DNA in the tail or with a very wide tail, were excluded from the evaluation because they probably 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 the number of cells in each class by the damage class. 2.7. Micronucleus test The assay was carried out following standard protocols as recommended by Schmid (1976) and Krishna and Hayashi (2000). The same six male mice per group as those used in the comet assay were also used for this protocol. The bone marrow from one femur was flushed out using 2 mL of saline (0.9% NaCl) and centrifuged at 1000 rpm for 7 min. The supernatant was discarded and smears were made on slides. The slides were coded for a “blind” analysis, fixed with methanol and stained with Giemsa. For the analysis of the micronucleated cells, 2000 polychromatic erythrocytes (PCE) per animal were scored to determine the clastogenic/aneugenic property of the extract. To detect possible cytotoxic effects, the PCE/NCE (normochromatic erythrocytes) ratio in 200 erythrocytes/animal was calculated (Gollapudi and McFadden, 1995). The cells were blindly scored using a light microscope at 1000  magnification. The mean number of micronucleated polychromatic erythrocytes (MNPCE) in individual mice was used as the experimental unit, with variability (standard deviation) based on differences among animals within the same group. 2.8. Percentage reduction The percentage reduction of genotoxic agent-induced damage by Rubus imperialis extract was calculated according to Waters et al. (1990), using the following formula: %Reduction ¼

AB  100 AC

where A corresponds to the score or MNPCE mean observed in the treatment with CPA (positive control), B corresponds to score or MNPCE mean observed in the antigenotoxic treatment (extract

plus CPA) and C corresponds to the score or MNPCE mean in the control. 2.9. Statistical analysis After verifying for normal distribution (normality test KS performed using GraphPad Instats software), the data obtained from the comet assay were submitted to analysis of variance (ANOVA) and the Tukey–Kramer multiple comparison test, and the data obtained from the micronucleus assay were submitted to the analysis of variance test (ANOVA) with linear regression, both using the GraphPad Prism 5 software (version 3.01). The results were considered statistically significant at P o0.05.

3. Results and discussion Genetic toxicology tests are assays designed to detect compounds that induce genetic damage directly or indirectly. DNA damage can result in gene mutations, loss of heterozygosity, and chromosome aberrations. These events may play an important role in many malignancies and may also induce inheritable effects leading to birth defects. Thus, identifying genotoxic effects of any agent is important for the risk/benefit assessment of its potential use in humans. Since no documented results are available about the potential genetic toxicity of Rubus imperialis and its possible chemoprevention against cyclophosphamide DNA-damage agent, studies have been initiated by us to verify this question. In this study, the animals were subjected to acute treatment via gavage of the extract. This treatment regimen and the administration method were designed because they were closer to the form that the extract is ingested by the population. The alkaline single cell gel electrophoresis (SCG) assay, also known as comet assay, is a rapid and sensitive technique for quantifying DNA damage in mammalian cells, detecting single strand breaks and alkali labile sites in individual cells (Collins et al., 2008). In the comet assay performed in the present study, Rubus imperialis extract did not induce DNA damage at the tested doses for the three cell samples, with no significant difference in the mean scores obtained, when compared with those obtained for the control. An exception was observed to the peripheral blood 24 h sample, at 500 mg/kg dose, that presented a significant DNA damage increase (Table 1). When cells were exposed to three doses of the test compound, the majority of cells examined on slides did not show any DNA damage (class 0), with very few nucleoids presenting class 1 DNA damage. These findings suggest no genotoxic effects of the Rubus imperialis extract, at least at low doses, on the cells analyzed. As expected, the animals treated with CPA exhibited a higher DNA damage index (Po0.001) compared to the control group. Simultaneous treatment showed a significant reduction in the extent of DNA strand breaks for all cell types exposed to the three doses (50, 250 and 500 mg/kg) of the extract plus CPA, compared with the CPA-treated group alone (Table 2). In all the cell types analyzed, the percentage of reductions were higher than 64%, showing a potent chemopreventive effect of the Rubus imperialis extract. However, the increase in dose of the extract results in a reduction in the chemoprevention against CPA genotoxicity, demonstrating an inverse dose–response prevention of the extract. Some studies analyzing other plant extracts have also demonstrated an inverse dose–dependent relationship (Knasmuller et al., 2002; Almeida et al., 2012). As such, the lack of a dose–response relationship might be attributed to the activation of different cell mechanisms on the Rubus imperialis dose tested.

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Table 1 DNA migration in the comet assay for the assessment of genotoxicity of Rubus imperialis extract in different cells of male Swiss mice in vivo (mean 7SD). Treatments and cells analyzed

Totald

Comet class

Scores

0

1

2

3

Peripheral blood (4 h sample) Control RIE 50 mg/kg RIE 250 mg/kg RIE 500 mg/kg CPA 50 mg/kg

5.337 2.16 3.667 1.63 12.337 3.83 5.83 71.83 39.16a 78.93

96.83 7 6.30 96.337 1.63 87.67c 7 3.83 94.17 71.83 60.83a 7 8.93

5.007 1.78 3.667 1.63 9.50 73.61 5.007 2.36 31.50a 7 10.15

0.167 0.40 0.00 70.00 1.337 1.50 0.3370.51 5.33b 7 5.42

0.167 0.40 0.00 70.00 0.50 7 0.83 0.50 7 0.83 3.337 4.63

6.00 73.40 3.677 1.63 13.6776.08 7.177 2.13 52.17a 7 18.73

Peripheral blood (24 h sample) Control RIE 50 mg/kg RIE 250 mg/kg RIE 500 mg/kg CPA 50 mg/kg

13.337 1.63 7.83 7 3.92 10.167 2.92 40.5a 7 9.13 66.66a 7 7.11

86.677 1.63 92.17 73.92 89.83 7 2.92 59.50a 7 9.13 32.83a 7 7.88

13.177 1.83 5.667 1.96 8.007 3.63 37.67a 7 8.91 65.17a 7 6.30

0.00 70.00 0.50 7 1.22 1.83 7 1.94 2.667 2.65 1.50 7 1.37

0.00 70.00 1.66 72.33 0.667 1.21 1.66 70.40 0.00 70.00

13.177 1.83 11.677 8.71 13.6776.21 43.50a 7 9.77 68.16a 7 8.08

Liver Control RIE 50 mg/kg RIE 250 mg/kg RIE 500 mg/kg CPA 50 mg/kg

8.007 1.89 14.667 3.26 22.40 78.53 16.337 6.89 57.83a 7 10.30

92.007 1.89 85.337 3.26 77.60c 7 8.53 84.007 6.75 42.17a 7 10.30

7.3371.63 12.6774.92 17.20 7 9.20 14.83 7 4.99 36.50a 7 20.38

0.667 0.81 0.667 1.63 3.60 7 2.07 1.007 1.26 15.50b 7 16.50

0.00 70.00 1.337 2.33 1.60 7 2.60 0.337 0.81 5.83c 77.67

8.677 2.42 18.007 7.32 29.20 7 9.83 17.83 7 7.23 90.00a 7 29.79

a

Significantly different from the negative control (P o 0.001). Significantly different from the negative control (Po 0.01). Significantly different from the negative control (Po 0.05). d Total number of damaged cells (class 1þ 2þ3); RIE: Rubus imperialis extract; CPA: cyclophosphamide. b c

Table 2 DNA migration in the comet assay for the assessment of antigenotoxicity of Rubus imperialis extract in different cells of male Swiss mice in vivo (mean 7 SD). Treatments and cells analyzed

Totald

Comet class 0

1

2

3

Scores

Reduction (%)

Peripheral blood (4 h sample) Control RIE 50 mg/kg þ CPA 50 mg/kg RIE 250 mg/kg þ CPA 50 mg/kg RIE 500 mg/kg þ CPA 50 mg/kg CPA 50 mg/kg

5.33a 7 2.16 7.00a 7 2.28 16.83a 7 3.65 16.33a 7 5.35 39.167 8.93

96.83a 7 6.30 93.00a 7 2.28 83.17a 7 3.65 83.67a 7 5.35 60.83 78.93

5.00a 7 1.78 7.00a 7 2.28 15.83a 7 3.97 12.17a 7 2.22 31.50 7 10.15

0.16b 7 0.40 0.00b 7 0.00 1.00c 71.26 2.50 72.42 5.337 5.42

0.16 70.40 0.007 0.00 0.007 0.00 1.667 3.20 3.337 4.63

6.00a 7 3.40 7.00a 7 2.28 17.83a 73.76 22.16a 713.27 52.17 7 18.73

– 97.83 74.37 64.99 –

Peripheral blood (24 h sample) Control RIE 50 mg/kg þ CPA 50 mg/kg RIE 250 mg/kg þ CPA 50 mg/kg RIE 500 mg/kg þ CPA 50 mg/kg CPA 50 mg/kg

13.33a 7 1.63 17.33a 75.92 21.16a 7 8.37 19.33a 7 4.80 66.66 7 7.11

86.67a 7 1.63 82.67a 7 5.92 78.83a 7 8.37 80.67a 7 4.80 32.83 77.88

13.17a 7 1.83 15.67a 74.27 18.50a 75.61 12.17a 7 3.43 65.177 6.30

0.007 0.00 1.3371.50 2.50 73.33 5.83 74.16 1.50 7 1.37

0.007 0.00 0.337 0.51 0.16 70.40 1.337 1.21 0.007 0.00

13.17a 71.83 19.33a 7 8.11 24.00a 7 11.81 27.83a 7 9.49 68.167 8.08

– 88.79 80.30 73.34 –

Liver Control RIE 50 mg/kg þ CPA 50 mg/kg RIE 250 mg/kg þ CPA 50 mg/kg RIE 500 mg/kg þ CPA 50 mg/kg CPA 50 mg/kg

8.00a 7 1.89 15.83a 7 4.75 22.50a 79.37 20.83a 75.67 57.83 7 10.30

92.00a 7 1.89 84.17a 7 4.75 77.50a 7 9.37 79.17a 7 5.67 42.177 10.30

7.33a7 1.63 13.50b 7 2.88 21.3379.62 15.17b 7 6.11 36.50 7 20.38

0.66b 7 0.81 2.16c 7 2.40 1.16b 7 0.98 4.00c 7 3.40 15.50 7 16.50

0.007 0.00 0.16 70.40 0.00c 70.00 1.667 1.63 5.83 7 7.67

8.67a 7 2.42 18.33a 77.23 23.66a 79.22 28.17a 7 6.79 90.007 29.792

– 88.12 81.56 76.02 –

a

Significantly different from the cyclophosphamide positive control (Po 0.001). Significantly different from the cyclophosphamide positive control (P o0.01). c Significantly different from the cyclophosphamide positive control (Po 0.05). d Total number of damaged cells (class 1þ 2þ3); RIE: Rubus imperialis extract; CPA: cyclophosphamide. b

In the present study, the in vivo micronucleus test was performed on mammalian bone marrow cells treated with Rubus imperialis extract. The micronucleus (MN) test is one of the most widely applied assays to test new compounds, in vivo and in vitro, for regulatory purposes (OECD, 1997; OECD, 2009). Micronucleus (MN) formation results either from chromosome breakage (clastogenicity) or aneuploidy. Table 3 shows the micronucleus test results obtained in male Swiss mice treated with Rubus imperialis extract: the number of micronucleated polychromatic erythrocytes (MNPCE) of each animal and means, for untreated controls and

treated animals. The results obtained showed a statistically significant increase in the average number of MNPCE in the two high doses tested of the plant extract (P o0.001), indicating a clastogenic/aneugenic effect of this extract at high doses. As expected, animals treated with cyclophosphamide (CPA) alone showed a high frequency of MNPCE in bone marrow cells when compared to the control (Po 0.001). In addition, oral administration of different doses of Rubus imperialis extract followed with the injection of CPA led to a significant reduction in the frequency of MNPCE when compared with the group treated only with CPA. This reduction

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Table 3 Number of micronucleated polychromatic erythrocytes (MNPCE) observed in the bone marrow cells of male (M) Swiss mice treated with a Rubus imperialis extract, and respective controls. 2000 cells were analyzed. SD ¼ standard deviation of the mean. Treatments

Vehicle control (Tween) Rubus imperialis (50 mg/kg) Rubus imperialis (250 mg/kg) Rubus imperialis (500 mg/kg) Cyclophosphamide (CPA) (50 mg/kg) Rubus imperialis extract (50 mg/kg þ CPA) Rubus imperialis extract (250 mg/kg þ CPA) Rubus imperialis extract (500 mg/kg þCPA) a b

Number of MNPCE per animal

MNPCE mean 7 SD

PCE/NCE mean 7 SD

Reduction (%)

M1

M2

M3

M4

M5

M6

02

04

02

05

02

02

2.83 7 1.32

1.01 70.06

–

05

03

06

04

06

07

5.00 7 0.89

1.02 70.11

–

10

15

06

09

08

09

9.50 7 3.01a

1.0770.08

–

10

12

09

06

07

10

9.00 7 2.19a

0.99 70.03

–

a

1.00 70.05

–

24

16

20

18

14

11

17.16 7 4.57

03

05

04

03

02

02

3.16 7 1.16b

1.00 70.04

97.69

01

01

00

03

03

02

1.66 7 1.21b

1.01 70.06

108.16

00

b

1.02 70.05

108.16

02

03

02

02

01

1.66 7 1.03

Significantly different from the negative control (P o0.001). Significantly different from the cyclophosphamide (CPA) (Po 0.001).

Fig. 1. Molecular structures of compounds isolated from Rubus imperialis: (1) stigmasterol, (2) niga-ichigoside F1), (3) tormentic acid, (4) 3,30 methoxy, 4-O-xylose ellagic acid, (5) 2β, 3β, 19α-trihydroxyursolic acid).

ranged from 97.69% to 108.16% and indicated a potent chemoprevention of the extract against the clastogenic/aneugenic effects of CPA (Table 3). Doses of 250 and 500 mg/kg of Rubus extract resulted in a same reduction in mutagenicity induced by CPA. The ratio of polychromatic erythrocytes to normochromatic erythrocytes (Table 3) analyzed in Swiss mice bone marrow cells treated with CPA or extract was not significantly different from the negative control group (P 40.05), indicating absence of cytotoxicity of different treatments under the conditions employed. Studies involving the genotoxic or mutagenic potential of plants from Rubus genus are very scarce in the literature. The mutagenic potential of Rubus idaeus acetone extract was investigated in vitro by the Ames test. The results obtained did not show any mutagenic effects as well as no cytotoxicity against caco-2 cells (Kreander et al., 2006). The above results are in agreement with the results obtained in our study, when absence of genotoxic, mutagenic and cytotoxic effects of Rubus imperialis were also verified. The major compounds of the phytochemical profile of Rubus imperialis aerial parts extract performed in the present study are

presented in the Fig. 1. The compounds were identified by NMR spectral data in comparison with authentic samples and the literature data, and were identified as stigmasterol (1), nigaichigoside F1 (2), tormentic acid (3) 3,30 methoxy, 4-O-xylose ellagic acid (4) and 2β, 3β, 19α-trihydroxyursolic acid (5). The phytochemical profile using the LC–MS system has shown saponins and steroidal compounds (Fig. 2). Moreover, derivatives from ellagic acid can be found in the profile. The main compounds detected were niga-ichigoside (m/z 667) and tormentic acid (m/z 487), this one analyzed by MS/MS spectrometry (Fig. 3). These results are according to the literature for this plant genus. The MS/MS fragmentation has shown a pattern, which is in accordance with the literature (Ramirez, 2012). Ion 486 can be obtained by the loss of one hydrogen (C28, –COOH group) or even from the hydrogen from the group CH2OH (C23). The ion with m/z 459 is due to the lost of a –CO group (m/z 28), and finally, the ion with m/z 441, due to the loss of the group COO– and two hydrogens. The main fragments were 485 m/z by [M–2H]-; 459 m/z by [M–H–CO]- and 441 m/z by [M–2H–CO2]  .

A.B.C.R. Alves et al. / Journal of Ethnopharmacology 153 (2014) 694–700 Intens. [%]

699

-All MSn, CID, 0.1-1.1min #(7-108)

487.2

100 80 469.2

60

503.2

40 617.5 425.2

20 300.8 0

300

340.8

371.1

390.8

350

441.2

407.2

400

455.2

450

500

550

663.5

633.2

561.9

515.3

600

650

m/z

Fig. 2. Phytochemical profile of the methanolic extract from leaves of Rubus imperialis using LC–MS.

Intens. [%]

-MS2(487.0), 0.1-1.0min #(2-44) 485.0

100 80 60 40

441.0

20

459.0

421.0 340.7

314.6

368.9

0 300

325

350

409.0

375.0 385.0

375

400

471.0 425

450

475

500

525

m/z

Fig. 3. MS/MS analyses of the ion with m/z 487, identified as tormentic acid.

To our knowledge, there are no studies evaluating the genetic toxicity of the above compounds or other extract plants from Rubus genus. The use of mutagenic drugs for cancer chemotherapy, with cyclophosphamide (CPA), is a routine practice (Doppalapudi et al., 2007). Therefore, identifying chemopreventive agents is important for the risk/benefit assessment of their potential use in humans. In the present study, the three tested doses of Rubus imperialis extract followed with the injection of CPA led to a significant reduction of the genotoxicity of the CPA alone. The two major compounds identified in the Rubus imperialis extract studied in the present study, niga-ichigoside and tormentic acid, were isolated from Rubus coreanus and also attenuated the cytotoxicity of other effective chemotherapeutic agent cisplatin, in renal epithelial LLCPK cells (Kim et al., 2011). Tormentic acid also promoted antiproliferative activities on renal, prostate and melanoma cancer cell lines (Loizzo et al., 2013). We believe that this data and our preliminary results may serve as a stimulus to the pharmaceutical industry for development of Rubus imperialis-derivated products for the prevention of diseases such as cancer or to protect healthy cells during chemotherapy with CPA. However, complementary studies are necessary— once mutagenic effects of the extract alone at high doses were also observed. The Rubus imperialis extract had its genotoxic and clastogenic/ aneugenic potentials evaluated for the first time in the present work. The results indicated that the mixture of compounds found in this extract did not induce a significant increase in the mean number of cells with DNA strand breaks or micronuclei, when given at single doses of 50 mg/kg body weight. However, in spite of one positive data for clastogenic/aneugenic activity at high doses of 250 and 500 mg/kg of the extract, the results obtained indicated that caution with its use is necessary and that further studies need be done to evaluate the safety of the high doses or subchronic/chronic consumption of this extract.

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