Journal of Ethnopharmacology 155 (2014) 30–38

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Review

Evaluation of toxicity of Calophyllum brasiliense stem bark extract by in vivo and in vitro assays Mariana Canevari Oliveira a, Larissa Maria Scalon Lemos a, Ruberlei Godinho de Oliveira a, Evandro Luiz Dall'Oglio b, Paulo Teixeira de Sousa Júnior b, Domingos Tabajara de Oliveira Martins a,n a Department of Basic Sciences in Health, Faculty of Medicine, Federal University of Mato Grosso (UFMT), Av. Fernando Correa da Costa, no. 2367, Boa Esperança, Cuiabá, Mato Grosso 78060-900, Brazil b Natural Products Chemistry Research Laboratory, Department of Chemistry, Institute of Exact and Earth Sciences, Federal University of Mato Grosso (UFMT), Av. Fernando Correa da Costa, no. 2367, Boa Esperança, Cuiabá, Mato Grosso, 78060-900, Brazil

art ic l e i nf o

a b s t r a c t

Article history: Received 13 March 2014 Received in revised form 31 May 2014 Accepted 4 June 2014 Available online 13 June 2014

Ethnopharmacological relevance: Calophyllum brasiliense Camb., Clusiaceae, is commonly known as “guanandi” and its stem bark is used in Brazilian traditional medicine to treat rheumatism, vein problems, hemorrhoids and gastric ulcers. The aim of this study was to evaluate the toxicity of hexane extract of Calophyllum brasiliense stem bark (HECb) using in vitro and in vivo experimental models. Materials and methods: In vitro toxicity was evaluated by Alamar Blue cytotoxicity assay and micronucleus test, using Chinese hamster ovary (CHO-k1) epithelial cells. in vivo toxicity was evaluated by oral acute and subchronic toxicity assays. In the oral acute toxicity screening, a single dose of HECb was administered to mice at doses ranging from 250 to 1000 mg/kg. In the subchronic study, HECb was administered orally for 30 days to Wistar rats at doses of 100 mg/kg and 500 mg/kg. Phytochemical analyses were performed by HPLC/UV–vis, secondary metabolites were quantified by spectrophotometric methods. Results: HECb presented IC50 ¼ 119.9474.31 mg/mL after a 24 h cytotoxicity test using CHO-k1 cells, showing low cytotoxicity. However, when the cells were exposed to HECb for 72 h, the IC50 value was 8.3972.00 mg/mL, showing in this case, a pronounced cytotoxic effect. In the oral acute toxicity studies, doses up to 500 mg/kg of HECb did not cause any changes in both male and female mice. At 1000 mg/kg, male mice showed signs typical of depression and stimulation that were reversed at 72 h. Besides, female mice were more sensitive to the toxic effect of HECb at 1000 mg/kg, which initially presented typical agitation signals, followed by depression signals, leading to death of all the animals at 24 h. In subchronic assay with rats, HECb administered orally at doses of 100 and 500 mg/kg did not cause significant changes in all clinical parameters evaluated. Histopathological analyses showed no deleterious effect in the vital organs of rats. Preliminary phytochemical analysis revealed the presence of phenolic compounds, steroids, and volatile coumarins. Analysis by HPLC showed two major peaks characteristic of chromanones. Conclusions: In vitro toxicological tests showed that HECb exhibited cytotoxicity especially after 72 h of exposition, and mutagenicity on the highest tested dose. The in vivo studies demonstrated that HECb produced some toxicity signs at the highest dose tested, particularly, in the acute toxicity test but showed no significant signs of toxicity in the subchronic assay. Based on these and previous pharmacological studies, it is possible to say that HECb did not exhibit significant toxicity at its effective dose. This suggests that HECb is relatively safe in humans at its effective dose. & 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Calophyllum brasiliense Chromanones in vivo toxicity in vitro toxicity

Contents 1. 2.

n

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Corresponding author. Tel.: þ 55 65 3615 88 62. E-mail address: [email protected] (D.T. de Oliveira Martins).

http://dx.doi.org/10.1016/j.jep.2014.06.019 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.

M.C. Oliveira et al. / Journal of Ethnopharmacology 155 (2014) 30–38

31

2.1. 2.2. 2.3. 2.4. 2.5. 2.6. 2.7. 2.8. 2.9. 2.10.

Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Cell culture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Plant material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Obtention of extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Preliminary phytochemical screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Fingerprint HPLC analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Cytotoxicity assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Micronucleus assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Hippocratic screening test. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Subchronic toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 2.10.1. Histopathological analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 2.11. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 3. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 3.1. Preliminary phytochemical screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 3.2. Fingerprint HPLC analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 3.3. Cytotoxicity by Alamar Blue assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 3.4. Micronucleus test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 3.5. Hippocratic screening test. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 3.6. Subchronic toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 3.6.1. Histopathological analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

1. Introduction The development of a drug involves preclinical and clinical trials in order to ensure its effectiveness and safety. Toxicity studies are conducted in the first preclinical stage, since a compound with high levels of toxicity cannot be used as a drug. Calophyllum brasiliense Camb. (Clusiaceae) is the most abundant species of the Calophyllum genus in Latin America. It occurs from Mexico to Brazil, at elevations from 5 m to 1200 m, in damp and swampy soil (Mesía-Vela et al., 2001). This specie is found both in the dense primary forest and in sequences at secondary stages, such as barns and coppices. The species is scattered widely but discontinuously, generally occurring in large clusters that sometimes form homogeneous populations. It is a tree that usually reaches heights of up to 20 m and diameters of 20–50 cm, while specimens in the Amazon region may display measurements up to twice as large (Lorenzi, 1992). Popularly known as “guanandi”, “olandi”, and “jacareúba” among many other names, Calophyllum brasiliense presents varied uses, including construction, carpentry, shipbuilding, manufacturing of wine barrels, bee keeping, tree planting squares, biodiesel production, and veterinary and folk medicine (Lorenzi, 1992). Some studies have shown its usefulness in the recovery of degraded areas (Montagnini and Cusack, 2004). In Brazil, Calophyllum brasiliense is being cultivated in a sustainable fashion to address reforestation. In fact, some companies have embarked on commercial cultivation but usually its bark is discarded. Calophyllum brasiliensis has proved to be a rich source of bioactive substances. These include chromanones (Stout et al., 1968; Cottiglia et al., 2004; Caneppele et al., 2008), coumarins (Ito et al., 2003; Reyes-Chilpa et al., 2004), xanthones (Sartori et al., 1999; Ito et al., 2002), triterpenoids (Reyes-Chilpa et al., 2004) and bioflavonoids (Silva et al., 2001). In folk medicine, ethnopharmacological records show that the stem bark infusion is used as an anti-inflammatory (Jesus et al., 2009), as well as for treatment of rheumatism, vein related problems, hemorrhoids, and gastric ulcers (Gasparotto et al., 2005; GuarimNeto, 2006). The yellow resin from the stem bark is used to treat pain, inflammation, diabetes, hypertension, herpes, and rheumatism

(Corrêa, 1978; Guarim-Neto, 1987). Reported pharmacological activities of the extracts, fractions, and isolated compounds from Calophyllum brasiliense have demonstrated it as having anti-ulcer (Sartori et al., 1999; Souza et al., 2009; Lemos et al., 2012), anti-Helicobacter pylori (Souza et al., 2009), antifungal (Silva-Júnior et al., 2009), antiHIV (Huerta-Reyes et al., 2004), anti-nociceptive (Silva et al., 2001), anti-Epistein-Barr virus (Ito et al., 2002), anti-neoplastic (Ito et al., 2003), antibacterial (Reyes-Chilpa et al., 2004; Silva-Júnior et al., 2009), antiparasitic (Reyes-Chilpa et al., 2008; Honda et al., 2010), antispasmodic (Emendörfer et al., 2005), angiotensin converting enzyme (ACE) inhibitory (Braga et al., 2007), and immunomodulatory (Philippi et al., 2010) properties. The evidences describing the biological effects of Calophyllum brasiliense are increasing, testifying to its great potential as an herbal medicine. Therefore, it is extremely important to evaluate its safety by studying its toxic, genotoxic, and mutagenic potentials. From the stem bark of Calophyllum brasiliense, a hexane extract (HECb), fractions, and isolated compounds were obtained. Anti-ulcer activity has been reported for HECb (Sartori et al., 1999), its dichloromethane fraction (Souza et al., 2009) and its chromanones-rich fraction (Lemos et al., 2012). In addition, antimicrobial effects have been reported against gram-positive bacteria and fungi for HECb and its ethyl acetate fraction (Silva-Júnior et al., 2009). Furthermore, the anti-Helicobacter pylori of HECb and its chromanones-rich fraction have been demonstrated by Souza et al. (2009) and Lemos et al. (2012) respectively. Cottiglia et al. (2004) studied the antibacterial and cytotoxic effects of brasiliensophyllic and isobrasiliensophyllic acids isolated from HECb on normal and tumor cells. Their studies showed the effects of these isolates on gram-positive bacteria as well, but had no demonstrated cytotoxicity effects in the test systems. However, to the best of our knowledge, there are no studies concerning HECb toxicity, despite the existence of several studies involving biological activities for its fractions and isolated compounds. This work therefore, was aimed to carry out pre-clinical toxicological investigation of HECb, using in vitro and in vivo experimental models in order to evaluate its potential toxicity, cytotoxicity, and mutagenic effects considering its pharmacological effectiveness and potential use in phytotherapy.

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2. Materials and methods 2.1. Animals Male albino Wistar rats (150–200 g) and Swiss webster mice of both sexes (25–30 g) from the Central Animal House of Federal University of Mato Grosso (UFMT) were used. The animals were maintained in propylene cages at 25 71 1C in a 12 h light–dark cycle and were provided with free access to standard pellet chow and water. Procedures concerning animal treatments and experiments in this study were reviewed and approved by the Animal Use Ethics Committee of the UFMT (No. 23108.020402/09-6). 2.2. Cell culture Chinese hamster ovary (CHO-k1) epithelial cells from Rio de Janeiro Cell Bank Collection were cultured in DMEM (Dulbecco's modified Eagle's Medium) (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% fetal bovine serum (FBS) (Cultilab, Campinas, SP, Brazil), at 37 1C in a humidified atmosphere with 5% CO2 and 90% air. Cellular viability was evaluated by Trypan blue (Sigma-Aldrich, St. Louis, MO, USA) assay. 2.3. Plant material Calophyllum brasiliense was collected in Cuiabá, Mato Grosso, Brazil, GPS coordinates: 151380 40.8″S and 561030 05.6″W, in July 2007. The plant was identified and authenticated by Ms. Harri Lorenzi of the Plantarum Institute in São Paulo, Brazil, and a voucher specimen (No. 37.993) was deposited at the Herbarium of UFMT. Calophyllum brasiliense is neither in the endangered species list, nor it is in the federal conservation units; therefore its collection does not require permission, according to the Brazilian legislation (Art. 10 IN 157/07 of the Brazilian Institute of Environment and Natural Resources – IBAMA/MMA). 2.4. Obtention of extract Calophyllum brasiliense stem bark (564 g) was cleaned, dried at 40 1C to constant weight, and later triturated in an electric mill (model TE-625 Tecnal, Piracicaba, SP, Brazil) with a sieve of a mesh size of 40. The dried powder obtained was macerated with hexane (1:5 w/v) at room temperature for 7 days. After this period, the macerate was filtered through a filter paper, and concentrated in a rotary evaporator (model MA-037 – FANEM, São Paulo, SP, Brazil) at 40 1C under reduced pressure (600 mmHg). The residual solvent was eliminated in an incubator at 40 1C to obtain HECb (10.4% yield) which was protected from light and stored at 4 1C. For the experimental procedures, HECb was emulsified in 2% Tween 80 in distillated water which was used as vehicle. 2.5. Preliminary phytochemical screening In order to identify the major phytochemical constituents of HECb, it was subjected to a qualitative wet chemistry analysis by a standard screening test, based on colored reactions, precipitation, and foaming tests. The conventional protocol for detecting the presence of alkaloids, flavonoids, saponins, tannins, xanthones, steroids, anthocyanin, triterpenoids, catechins, chalcones, phenols, resins, and volatile coumarins was used (Matos, 1988). 2.6. Fingerprint HPLC analysis High-performance liquid chromatography (HPLC) analysis was performed with a Pro Star 210/215 Solvent Delivery Module Pump,

connected to a Pro Star 325 UV–vis detector, a Pl-ELS 2100, and a HPLC column Microsorb 300-5, C-4, 250 mm  4.6 mm (Varian, Palo Alto, California, USA). Mobile phase for the HPLC analysis was isocratic 90% of A (acetonitrile) and 10% of B (0.25% trifluoroacetic acid aqueous solution), flow rate 1.0 mL/min for 80 min, 20 mL of injection volume. Detection was at 285 nm, flow gas 1.4 SLM. All solvents used were of HPLC grade. The HPLC chromatograms of HECb were obtained using Workstation software toolbars. 2.7. Cytotoxicity assay CHO-k1 cells were plated in 96-well plates (2  104 cells/well) in 200 mL of DMEM medium (Sigma-Aldrich, St. Louis, Montana, USA) supplemented with 10% fetal bovine serum  FBS (Cultilab, Campinas, SP, Brazil), and incubated overnight at 37 1C in a humidified atmosphere with 5% CO2. Then, the cells were treated with HECb (3.125-200 mg/mL, serial dilution) at the same conditions. Doxorubicin (Sigma-Aldrich, St. Louis, Montana, USA) 0.0058–58 mg/mL, serial dilution) was used as a positive control. For the negative control, some wells had the same amount of medium. After 24 h or 72 h of incubation at the same conditions, the treatments were removed and 200 mL of 10% Alamar Blue (Invitrogen®, Life Technologies, USA) was added to each well and incubated again for 5 h. The conversion of resazurin to resorufin by the cells was measured by fluorescence at 540 nm (oxidized state) and at 620 nm (reduced state) in microplate reader Multiskan EX Microplate (Thermo Scientific, Tewksbury, Massachusetts, USA). The cell viability was expressed as inhibitory concentration at 50% inhibition (IC50 7SEM). 2.8. Micronucleus assay The cytokinesis-block micronucleus (MN) test was performed according to Fenech (2000) modified. CHO-k1 cells at the fourth passage were plated at a density of 1  106 cells/bottle and incubated overnight at 37 1C, 5% CO2 atmosphere to reach the semiconfluent stage. Then, they were treated with HECb (11.8, 35.3, and 106 mg/mL). One bottle received only Ham F12þ Dulbecco's modified eagle medium (DMEM) while another bottle received doxorubicin (0.03 mg/mL) as negative and positive mutagenic controls, respectively. After 24 h of treatment, the cells received 4.5 mg/mL of cytochalasin B, a cytokinesis blocker drug. After 24 h of incubation at the same conditions, the micronucleus test was performed. All drugs and media were from Sigma-Aldrich, St. Louis, Montana, USA and Alamar blue were from Life Technologies - USA. After trypsinization, the cells were centrifuged, the supernatant was discarded, and 5 mL of 1% cold sodium citrate was added to the cell pellet, which was then gently resuspended. After 15 s, 5 mL of freshly prepared fixative (methanol/acetic acid 3:1) and 4 drops of 37% formaldehyde were added. The cell suspension was centrifuged for 5 min at 1000 rpm (Centrifuge model Excelsa II, 206 BL, FANEMs, São Paulo, Brazil). The supernatant was discarded, and the fixation process was repeated two more times, without formaldehyde. After the third fixation, around 1 mL of the supernatant was retained to permit resuspension of the cells. The cell suspension was dripped onto clean glass slides that had previously been frozen to dry. Then, the suspension was fixed with methanol and stained with Giemsa (1:20 in phosphate buffer) for 4 min. One thousand binucleated cells per slide were analyzed with intact nuclei of equal size, similar pattern of staining cytoplasm, intact membrane, and distinguishable from adjacent cells, excluding apoptotic and necrotic cells. We considered the micronuclei that had the same morphology and color of the main nuclei, with 1/16–1/3 the main nuclei diameter, unrefringent and not overlapping or connected to the main nuclei. The presence of buds and

M.C. Oliveira et al. / Journal of Ethnopharmacology 155 (2014) 30–38

33

Fig. 1. Analysis by high performance liquid chromatography (HPLC) of the hexane extract of Calophyllum brasiliense stem bark.

dicentric bridges (PNP) in the binucleated cells was analyzed. To evaluate the frequency, MN/1000 binucleated cells (including PNP and buds) were used. Nuclear division index (NDI) was calculated by NDI ¼ ½ð1  mononuclear cellsÞ þð2  binuclear cellsÞ þ ð3  multinuclear cellsÞ=N; where N is total cells number. 2.9. Hippocratic screening test The effect of the HECb on the general behavior of conscious animals was evaluated in male and female mice, by using three animals per group. One control animal in each group received only the vehicle. Signs and symptoms were observed after oral administration (p.o.) of HECb (250, 500, and 1000 mg/kg). Following treatment, the animals were observed at 0, 15, 30, 60 min and after 4, 8, 24, and 48 h and then once daily for 14 days, for clinical signs and mortality (Malone and Robichaud, 1962). 2.10. Subchronic toxicity The subchronic toxicity study was performed daily through single oral administration to male Wistar rats (150–200 g), six per group, of vehicle (10 mL/kg) or HECb (100 and 500 mg/kg), over 30 days (Chan et al., 1982). In this period, animals were monitored daily for any changes in food and water consumption, any additional behavioral or clinical signs of toxicity, and for mortality. At the end of 30 days, blood samples were collected from the inferior venae cavae for biochemical and hematological evaluations. For the histopathological analyses, the brain, heart, lungs, liver, stomach, spleen, and kidneys were collected, preserved in 10% buffered formalin solution, processed, and stained with hematoxylin and eosin (Boabaid et al., 2008). 2.10.1. Histopathological analyses Organ samples from each animal were further processed for histopathology, performed at the Laboratory of Veterinary Pathology of UFMT. Samples were fixed in 10% formalin, infiltrated in paraffin, sectioned to a thickness of 5 mm and stained with hematoxylin and eosin for microscopic analysis. The tissues were analyzed in optical

Fig. 2. Chinese hamster ovary cells (CHO-k1) exposed to varying concentrations of hexane extract of Calophyllum brasiliense stem bark (HECb) and doxorubicin (Dox) for (a) 24 h and (b) 72 h.

Table 1 Averages for aberrations in Chinese hamster ovary cells (CHO-k1) treated with hexane extract of Calophyllum brasiliense stem bark (HECb), doxorubicin (DOX), and normal control without treatment.

Normal control HECb 11.8 mg/mL HECb 35.3 mg/mL HECb 106.0 mg/mL DOX 0.03 mg/mL

MNa

PNPa

BUDa

NDI

14.8 71.4 16.7 70.9 27.3 71.5 36.6 70.8nn 90.6 77.6nnn

7.7 7 0.7 5.3 7 0.5 7.7 7 1.3 13.6 7 0.8nnn 37.6 7 1.2nnn

5.2 7 0.9 5.7 7 0.4 5.3 7 0.8 9.7 7 0.2nnn 30.8 7 1.1nnn

1.71 1.77 1.73 1.71 1.90

a The aberrations counting was performed in 1000 binucleated cells per slide. MN: micronuclei; PNP: dicentric bridges; BUD: buds; NDI: nuclear division index. Results expressed as mean 7SEM. Kruskal  Wallis test followed by Dunn. nn po 0.01. nnn po 0.001 versus normal control.

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microscope by a person blind to the treatments. The organs were analyzed for their general structure, degenerative changes, necrosis evidence and signs of inflammation. 2.11. Statistical analysis Data are presented as mean 7standard error of mean (X 7 SEM). The parametric data were analyzed by one-way analysis of variance (ANOVA), and the statistical significance among the groups was determined by Student–Newman–Keuls and Dunn's test. The level of significance for all the comparisons was p o0.05. The IC50 was determined by linear regression relating the percentage of inhibition versus the logarithm of the concentrations tested, assuming a confidence level of 99% (p o0.01) for the curve obtained. For the in vitro assays that do not involve statistical analysis, the mean 7 SEM of three independent experiments runned in duplicate was used.

3. Results 3.1. Preliminary phytochemical screening The preliminary phytochemical analysis of the HECb revealed the presence of phenolic compounds, steroids, and volatile coumarins. 3.2. Fingerprint HPLC analysis Fig. 1 shows the HPLC/ultraviolet–visible (UV–vis) chromatogram of HECb, which showed two principal peaks: the most intense peak at retention time (RT) 32.955 min and a second intense peak at RT 29.663 min. The other peaks are low signal.

3.4. Micronucleus test Table 1 shows the absolute values of MN, PNP, and BUD aberrations, and nuclear division index (NDI), obtained for the normal control (vehicle), HECb (11.8, 35.3, and 106.0 mg/mL), and doxorubicin (0.03 mg/mL). The results showed significant increase only at 106 mg/mL of HECb by 147.3%, 76.6% and 86.5% for MN (p o0.05), PNP (p o 0.001) and BUD (po 0.001), respectively, when compared with vehicle. Doxorubicin showed increase of 512.2% for MN (p o 0.001), 388.3% for PNP (p o0.001) and 492.3% for BUD when compared to vehicle.

3.5. Hippocratic screening test The HECb doses tested (250 mg/kg and 500 mg/kg) did not lead to any sign of toxicity or change in general behavior in female rats, whereas the dose of 500 mg/kg decreased the motor activity of 2 of the 3 male rats, although it was only in the first hour after drug administration. In females, the highest dose (1000 mg/kg) caused dyspnea, decreased motor activity, exophthalmos, eye swelling, tearing, evacuation and decrease urine excretion, passivity, sedation, tremors, decreased flight reaction, loss of screen grip, and throes 15 min after drug administration and resulted in 100% mortality within 24 h. In males, the same dose (1000 mg/kg) caused decreased motor activities by 1–24 h, diuresis increase by 24–72 h, evacuation increase by 4–72 h, and tremor of the tail by 24–72 h and no death was observed. The results are shown in Table 2. There was no alteration of relative weight of organs.

3.6. Subchronic toxicity 3.3. Cytotoxicity by Alamar Blue assay The exposition of CHO-k1 cells to HECb for 24 h results on IC50 ¼117.07 4.31 mg/mL (Fig. 2a), suggested that the extract can be considered non-cytotoxic, but the cell survival curve for 72 h of exposition (Fig. 2b) showed pronounced cytotoxicity of HECb, with IC50 ¼8.4 72.00 mg/mL (Froelich et al., 2007). The positive control doxorubicin shows IC50 4200 mg/mL and IC50 ¼0.1 70.01 mg/mL for 24 h and 72 h, respectively.

The results from the treatment of rats with the vehicle or HECb (100 and 500 mg/kg) over 30 days showed no significant changes in all biochemical and hematological parameters evaluated (Table 3), except for the thrombocytosis in the HECb 500 mg/kg group (p o0.01). Animals treated with HECb (100 and 500 mg/kg, p.o.) showed no change in body weight, food and water intake, and urine and feces excretion, when compared to the vehicle group (Table 4).

Table 2 Effect of oral administration of hexane extract of Calophyllum brasiliense (HECb) on the general behavior in mice. HECb (mg/kg p.o.)a

Behavioral effectsb Males

250 500 1000

a b

Single dose. Observed for 14 days.

 No changes  Motility decrease in 2/3, 1 h up to 2 h  Motility decrease in 3/3, 1 h up to 24 h  Increase evacuation in 3/3, 4 h up to 72 h  Increased urination in 3/3, 24 h up to 72 h  Tail tremor in 1/3, 24 h up to 72 h.

Females

 No changes  No changes  Agitation in 1/3, 15 min up to 8 h  Exophthalmos in 2/3, 30 min up to 1 h  Motility decrease in 3/3 1 h up to 24 h  Increased urine coloration in 3/3, 1 h up to 24 h  Passivity and decreased flight reaction in 1/3, 4 h up to 24 h  Dyspnea in 1/3, 4 h up to 24 h  Loss of paw grasping in 1/3 at 8 h  Coarse tremors in 1/3, 8 h up to 24 h  Decreased urination in 3/3, 8 h up to 24 h  Decrease evacuation in 2/3 at 24 h  Eye edema and tearing in 3/3 at 24 h  Fine tremor in 1/3 at 24 h  Spasms during sleep in 1/3 at 24 h  Sedation in 1/3 at 24 h  Death in 3/3 at 24 h

M.C. Oliveira et al. / Journal of Ethnopharmacology 155 (2014) 30–38

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Table 3 Effect of oral subchronic administration (30 days) of hexane extract of Calophyllum brasiliense (HECb) in male rats on various biochemical and hematological parameters. Parametera

Vehicle

HECb (mg/kg p.o.) 100

500

Biochemical parameters Glucose (mg/dL) Urea (mg/dL) Creatinine (mg/dL) Aspartate amino transferase (IU/L) Alanine amino transferase (IU/L) Alkaline phosphatase (IU/L) Total bilirubin (mg/dL) Direct bilirubin (mg/dL) Indirect bilirubin (mg/dL) Total Cholesterol (mg/dL) Triglycerides (mg/dL) Total proteins (mg/dL) Total proteins (mg/dL)

180.8 7 44.1 51.5 7 10.7 0.51 7 0.04 80.5 7 9.7 52.2 7 9.6 490.5 7 125.6 0.068 7 0.03 0.031 7 0.014 0.036 7 0.026 97.3 7 19.7 71.0 7 18.1 5.26 7 0.81 5.26 7 0.81

204.0 749.9 47.3 77.1 0.50 70.06 86.2 72.7 58.5 78.4 476.3 7196.8 0.06 70.029 0.013 70.015 0.046 70.035 97.0 722.0 56.0 713.1 6.03 70.33 6.03 70.33

Hematological parameters Red blood cells (106/mm3) Hemoglobin (g/dL) Hematocrit (%) MCV (fL) MCH (pg) MCHC (%) Platelets (103/mm3) Total leukocyte (103/mm3)

7.65 7 0.57 15.25 7 1.02 43.41 7 3.09 56.83 7 1.72 19.93 7 /0.62 35.11 7 0.24 729.5 7 42.85 5.68 7 1.00

7.50 70.13 15.28 70.59 43.38 71.68 58 72.36 20.3570.71 35.21 70.19 828.6 753.77 6.3571.22

Lymphocyte Relative (%) Absolute (103/mm3)

73.83 7 2.48 4.20 7 0.82

71.5 74.76 4.52 70.86

74.83 7 6.01 5.577 1.79

Monocyte Relative (%) Absolute (103/mm3)

4.5 7 2.4 0.25 7 0.14

4.3371.03 0.27 70.06

4.667 2.25 0.357 0,21

Neutrophil Relative (%) Absolute (103/mm3)

18.83 7 2.71 1.06 7 0.22

23 74 1.47 70.42

17.5 7 5.95 1.197 0.18

Eosinophil Relative (%) Absolute (103/mm3)

1.0 7 0 0.05 7 0.01

1.16 70.40 0.0770.02

1.0 7 0 0.077 0.02

Basophil Relative (%) Absolute (103/mm3)

0.16 7 0.4 0.01 7 0.02

0 70 0 70

0.337 0.51 0.02 7 0.04

a

204.17 46.9 46.0 7 3.6 0.55 7 0.05 78.8 7 8.0 477 11.8 458.0 7 176.2 0.077 0.035 0.02 7 0.017 0.05 7 0.04 102.0 7 4.1 73.8 7 47.3 6.4 7 0.50 6.4 7 0.50 7.92 7 0.41 15.43 7 0.44 44.117 1.25 55.667 2.42 19.517 0.75 34.96 7 0.37 909.667 32.081;nn 7.4 7 2.22

Results are means 7 SEM. One-way ANOVA, followed by Student  Newman  Keuls test. p o0.01 versus vehicle.

nn

3.6.1. Histopathological analyses Histopathological evaluation of all the organs (brain, heart, lungs, liver, stomach, spleen, and kidneys) displayed no relevant macroscopic or histological changes in animals that received HECb (at both doses), except a multifocal pulmonary edema associated with peribronchiolar lymphocytic infiltrate in the vehicle and treated groups.

4. Discussion In order to determine the safety of medicinal plants for human use, toxicological evaluation must be carried out using various experimental tests. The present work was therefore focused on evaluating potential toxicity of HECb. In this case, Alamar Blue assay in CHO-k1 cells was employed to assess potential citotoxicity of HECb. Alamar Blue is a redox indicator that acts through the cell respiratory chain. It is absorbed by passive diffusion only by viable cells and is reduced by mitochondria or microsomes in the cytosol, thus permitting the calculation of IC50 (Severin et al., 2010).

The IC50 value for 24 h of cell exposition to HECb was greater than 50 mg/mL, thus it is considered to have low cytotoxicity. However, when evaluated at 72 h of cell exposure, HECb could be considered highly cytotoxic, since its IC50 value was lower than 50 mg/mL (Froelich et al., 2007). Although in vitro tests are useful as an initial toxicity prescreen, the result from this test alone is not a proof of the compound toxic effect. Moreover, cytotoxicity studies require additional tests, such as mutagenicity and/or genotoxicity tests, in order to investigate the real cytotoxic potential of a drug (Houghton et al., 2007). Micronucleus test is a bioassay used in toxicological screening to access genotoxic effects. The test is recommended by OECD 487 (OECD, 2009), and it is based on the counting of micronuclei in dividing cells, resulting in chromosome breakage (clastogenicity) or whole chromosomes unable to migrate to the poles by spindle during anaphase (aneugenesis). Bridges linking nuclei on the binucleated cells are also evaluated, as are buds in the main nucleus. It is believed that these structures can also be formed during the S-phase of cell replication through gene amplification (Schubert and Oud, 1997). The dicentric bridges and buds provide

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M.C. Oliveira et al. / Journal of Ethnopharmacology 155 (2014) 30–38

Table 4 Effect of oral subchronic administration (30 days) of hexane extract of Calophyllum brasiliense (HECb) on various parameters. Parametera

Treatment period (days) D0

D5

D10

D15

D20

D25

D30

Control (vehicle) Body weight (g) Body weight gain (g) Water consumption (mL) Food intake (g) Feces (g) Urine (mL)

179.9 7 19.8 — — — — —

190.9 7 20.1 11.0 7 6.1 67.2 7 17.1 69.17 6.6 35.57 5.0 23.8 7 5.9

185.2 718.3  5.7 712.4 110.5 733.6 76.7 77.1 44.7 75.2 39.5 712.9

223.5 7 16.1 38.2 7 7.2 139.2 7 28.5 109.8 7 10.2 56.6 7 9.1 45.7 7 14.8

234.4 7 18.2 10.9 7 3.8 111.2 7 38.0 93.8 7 12.1 36.17 15.7 27.2 7 13.3

253.6 7 18.6 19.17 4.6 124.8 7 21.0 103.4 7 4.2 37.6 7 20.4 22.7 7 10.4

259.9 7 21.6 6.4 7 8.1 151.6 7 71.9 101.9 7 6.7 70.97 5.9 21.2 7 15.4

HECb 100 mg/kg Body weight (g) Body weight gain (g) Water consumption (mL) Food intake (g) Feces (g) Urine (mL)

171.8 7 45.8 — — — — —

178.17 43.0 6.3 7 13.2 76.8 7 4.8 70.47 13.4 34.3 7 6.9 31.3 7 2.9

175.4 736.5  2.7 716.2 126.0 719.6 72.9 78.1 44.7 74.0 43.7 711.7

209.5 7 41.1 34.17 7.6 143.6 7 16.9 105.0 7 13.7 55.9 7 9.4 45.0 7 4.8

224.17 41.0 14.5 7 9.2 130.8 7 20.9 96.4 7 10.0 45.3 7 3.4 31.7 7 6.6

239.4 7 33.8 15.3 7 10.5 135.6 7 18.3 100.5 7 9.3 42.8 7 15.6 26.8 7 6.8

254.17 29.2 14.7 7 12.6 142.5 7 12.9 101.6 7 6.3 76.88 7 7.4 29.3 7 11.5

HECb 500 mg/kg Body weight (g) Body weight gain (g) Water consumption (mL) Food intake (g) Feces (g) Urine (mL)

170.67 36.4 — — — — —

173.17 30.2 2.5 7 11.2 76.8 7 10.0 65.5 7 8.1 34.9 7 3.6 31.8 7 5.0

181.7 726.5 8.6 76.7 120.0 78.8 76.5 73.3 44.0 75.2 41.3 76.8

217.4 7 28.6 35.77 9.9 148.67 17.8 112.0 7 13.9 54.17 13.2 49.77 14.8

231.17 18.7 13.7 7 11.2 1417 12.6 100.7 7 6.4 45.8 7 5.7 40.0 7 8.8

249.8 7 21.0 18.6 7 9.2 140.8 7 10.2 104.47 5.5 36.3 7 9.9 35.0 7 8.6

261.6 7 12.3 11.8 7 13.1 140.3 7 15.0 95.4 7 9.6 85.17 4.8 30.5 7 5.3

D0: day of treatment; D5: 5th day; D10: 10th day; D15: 15th day; D20: 20th day; D25:25th day; D30: 30th day. a

Results are means 7SEM. One-way ANOVA.

an additional evaluation of the chromosomal rearrangement (Fenech, 2000). Micronucleus tests in this work were performed with a cytokinesis blocker (CBMN). To ensure that the evaluated cells have a completed division process before the blocking, it was necessary to retain the test treatments long enough to fulfill one cell cycle. When cells are incubated with the cytokinesis blocker, the separation of daughter cells is avoided, resulting in binucleated cells to be evaluated in the assay (OECD, 2009). Mutagenesis test was performed with concentrations of 11.6, 35.3, and 106.0 mg/mL of HECb, that represent the IC50 and two lower concentrations, respecting the maximum recommended concentration for testing in vitro mutagenic potential of 500.0 mg/mL, when not limited by solubility or cytotoxicity (OECD, 2009). The results showed that, when compared to normal controls, only IC50 dose of HECb presented significant difference in occurrences of micronuclei, bridges, and buds. Treatment with HECb did not interfere with cell proliferation, since the ratio of nuclear division (NDI) of the treated cells remained unchanged compared to the control. According to Bochkov et al. (1976) and Committee-17 (1975), a compound can only be conclusively classified as clastogenic when more than one test protocol is performed. Fenech (2000) argues that the correct interpretation of the results of mutagenicity tests involves the analysis of various parameters and not just an increase in one. And also considering that in vitro tests do not fully represent the conditions of in vivo interactions with other metabolic factors, we therefore conclude that the present result of the mutagenic test is not enough to make a definite statement about the mutagenic potential of HECb, and that other models of evaluating mutagenicity will be carried out in future. Since metabolism of a substance by an organism requires interplay of uptake, distribution, metabolism and excretion of the substance in question, besides hormonal and other signaling pathways, we therefore proceeded to evaluate HECb using in vivo toxicity assay. In this work, despite the potential cytotoxic effect showed by HECb in the in vitro test, particularly with 72 h exposure, the

in vivo results for both acute and subchronic assays did not demonstrate any relevant signs of toxicity up to 500 mg/kg dose. The toxic effects were observed from 1000 mg/kg, with less intensity for males than for females, for which deaths were observed. It is found in the scientific literature that males and females have different responses from great variety of drugs, mainly due to hormones produce, especially the estrogen, by its effect on B and T lymphocytes cells equilibrium and cytochrome P450 enzymes metabolism (Ahmed et al., 1999, Sin et al., 2007). Females are usually more susceptible to autoimmune diseases and drugs adverse events for women are usually serious (Miller, 2001). These could help explain the results found in this work. Moreover, previous studies by our group showed HECb at the dose of 100 mg/kg was highly effective as antimicrobial (SilvaJúnior et al., 2009), anti-ulcer, and anti-Helicobacter pylori (Lemos et al., 2012). Therefore, at its effective dose, HECb can be considered to be devoid of toxicity. Its toxicity appeared at doses 10 times higher than the effective dose. In sub-chronic toxicity tests in rats, the group treated with the highest HECb dose presented thrombocytosis as the only change. Despite the significant, but a non-dose dependent increase in platelets, the value is within the physiological range (837– 1455  103/mm3) for Wistar rats (Melo et al., 2012). Moreover, the reactive or secondary thrombocytosis is due to an external cause and can be seen in a variety of clinical conditions, including inflammation, chronic infections, trauma, acute bleeding, cancer, or splenectomy, and rarely cause more serious complications (Skoda, 2009). All these clinical situations would also cause other hematological and/or biochemical changes, which were not observed in these groups. Furthermore, this change was not followed by any clinical sign or histopatological changes at the tested doses. Thus, considering the normality of the remaining parameters, and considering that, although it is expected that thrombocytosis increases the risk of thrombosis, there is little evidence of a direct link between these facts (Vannucchi and Barbui, 2007), and therefore HECb showed no typical signs and clinical symptoms of toxicity when administered over 30 days in rats.

M.C. Oliveira et al. / Journal of Ethnopharmacology 155 (2014) 30–38

In histological evaluations, the only change observed was pulmonary edema with multifocal peribronchiolar lymphocytic infiltrate found in the lungs of these animals. This is possibly due to inadequate air inspired by these animals; these changes were found in all animals, treated with HECb and vehicle, and therefore did not result from its administration (Beserra et al., 2010; Tasaki et al., 2008). The toxic signs showed in in vitro tests by the HECb may be due to the phenolic compounds detected with the phytochemical analysis. It is known that phenolic compounds can be hematotoxic and hepatotoxic, and may provoke mutagenesis and carcinogenesis (Michalowicz and Duda, 2007). The chromatogram showed two major peaks; these are presumably the two isomeric chromanones: brasiliensic and isobrasiliensic acids and they probably are responsible, at least in part, for the HECb toxic effects observed in the in vitro and in vivo tests, especially at high concentrations or doses (Lemos et al., 2012). Stout et al. (1968) found that Calophyllum brasiliense stem bark produces a greenish acid gum that consisted primarily (approximately 95%) of two isomeric chromanones: brasiliensic and isobrasiliensic acids (ratio 7:3), corroborating the hypothesis that the two peaks observed in the chromatogram of HECb are the brasiliensic and isobrasiliensic acids.

5. Conclusions In vitro toxicological tests showed that HECb exhibit cytotoxicity only at the highest concentrations tested. The in vivo studies demonstrated that HECb produced some toxicity signs at the highest dose tested, particularly, in the acute toxicity test but showed no signs of significant toxicity in the subchronic assay. Based on this and previous pharmacological studies referred in the literature, it is possible to say that HECb did not show significant toxicity in its effective dose. This suggests that HECb is safe in humans at its effective dose.

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