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Journal of Ethnopharmacology 175 (2015) 9–13

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Journal of Ethnopharmacology

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

Toxicity study about a medicinal : A contribution to the Brazilian Unified Health System (SUS)

A.Z. Ameni a, O.A. Latorre a, L.M.B. Torres b, S.L. Górniak a,n

a Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87. CEP: 05508-270 - Cidade Universitária, São Paulo, SP, Brazil b Botanic Institute of São Paulo,Av. Miguel Estéfano, 3687, Água Funda, CEP: 04301-012, São Paulo, São Paulo, Brazil

article info abstract

Article history: Ethnopharmacological relevance: Casearia sylvestris S.w () is catalogued by the Brazilian Unified Received 18 February 2015 Health System as a plant of interest for the Brazilian population with the purpose of treating in- Received in revised form flammatory disorders, such as pain and gastrointestinal disorders based on the folk use and some lit- 31 July 2015 erature about efficacy; however, no toxicological studies concerned the safety of extract fluid of this plant Accepted 21 August 2015 have been reported. Available online 3 September 2015 Aim of the study: The present study was carried out to evaluate the acute and subchronic toxicity of the Keywords: hydroethanolic extract fluid (FE) obtained from leaves of C. sylvestris in Wistar rats. Casearia sylvestris Materials and methods: In the acute toxicity test three female Wistar rats were treated with a single dose Toxicity evaluation of FE (2000 mg/kg) administered by oral gavage and observed for 14 days in order to identify signs of Acute toxicity toxicity or death. In subchronic toxicity study animals received, by daily gavage three doses 60, 120 and Repeated dose toxicity Medicinal 240 mg/kg of the FE of the plant for 28 and 90 days. The animals were observed daily for clinical signs and mortality. Body weight and food consumption were measured weekly and at the end of treatment were analysed hematological, biochemical and histopathological parameters. Also was analysed the cellularity of bone marrow and spleen. Moreover, phytochemical analysis by HPLC–PDA–ESI þ/MS and CG/MS/EI was carried out to qualify the constituents of the extract. Results: The results of acute study indicated that the LD50 is higher than 2000 mg/kg and at 28 and 90 day oral toxicity showed that there were no toxic effects detected in any of the parameters evaluated: body weight and relative organ weight, general behavioral changes, haematological and biochemical parameters and histopathological examination. The analysis by HPLC–PDA–ESI þ/MS and CG/MS/EI identified the flavonoids rutin, quercetin and luteolin and also chlorogenic on the extract. Conclusion: Based on this study the hydroethanolic fluid extract of C. sylvestris could be safe even when used over a long period for therapeutic uses proposed by the Brazilian Unified Health System. & 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction have led to the inclusion of the use of medicinal plants into the public health system. Currently, 47 medicinal plants are approved In Brazil the use of medicinal plants has an important role in to be prescribed in the Brazilian Unified Health System (SUS) primary health care, mainly in the communities without easily based on ethnopharmacology data (ANVISA, 2011). However, access to medicines. Since 2006, the Brazilian government defined while there is a widespread use of herbal medicine, little has been national policies on the use of herbal medicines in The National done to establish its safety (Carvalho et al., 2009). In this manner, Policy of Integrative and Complementary Practices (PNPIC) and although in Brazil, the National Health Surveillance Agency (AN- The National Policy of Medicinal Plants and Herbal Medicines VISA) is developing standards for the regulation for the pharma- ceutical companies submit data on efficacy and safety of herbal (PNPMF). These documents encourage research into and the de- medicines, presently, information and reliable data are scarce and velopment of herbal medicines while prioritizing the protection of often contradictory. Thus, it should be considerate that there is a biodiversity and promoting greater access to safe and effective lack of minimum information necessary for the correct use of treatments (Carvalho et al., 2011). Furthermore, these documents medicinal plants and herbal medicine, which makes an easy target for self-medication without liability and also it is necessary to n Corresponding author. Fax: þ55 11 3091 7829. develop protocols of studies and better evaluate the safety of E-mail address: [email protected] (S.L. Górniak). natural products and herbal medicines, thereby providing a

http://dx.doi.org/10.1016/j.jep.2015.08.027 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved. 10 A.Z. Ameni et al. / Journal of Ethnopharmacology 175 (2015) 9–13 rational use of them (Alexandre et al., 2005). TMCS), and the analysis was performed using an Agilent GC 6890 gas In this context, our research group performed a toxicity study chromatograph coupled to an Agilent MSD 5973N mass spectrometer about Casearia sylvestris, which is catalogued by the SUS as a plant column (HP5 30 m, 0.25 mm, 0.25 Vm), which was operated in the of interest for the Brazilian population with the purpose of treating electronic impact (EI) mode (70 eV). The temperatures of the injector inflammatory disorders, such as pain and gastrointestinal dis- and detector ports were kept at 230 °Cand250°C, respectively, and orders, such as gastritis and ulcers. In addition, it is used as a ci- theionsourcewaskeptat200°C.Heliumwasusedasthecarriergasat catrizing agent in skin diseases, with its only restriction being a flow rate of 1.0 ml/min. The analysis was performed using the fol- against use during pregnancy, according to the “Formulário de lowing program: 5 min isothermal heating (70 °C), a gradient of 5 °C/ Fitoterápicos da Farmacopéia Brasileira” (ANVISA, 2011). Moreover, min to 310 °C, and a final minute of heating at 310 °C. The temperature there are studies showing different uses of it as an antimicrobial was then equilibrated before automatic injection of the next sample. (Da Silva et al., 2008), analgesic and anti-inflammatory (Ruppelt The mass spectra were recorded at 2 scans/s with a scanning range of et al., 1991), antivenom (Cavalcante et al., 2007; Cintra-Fran- 50–650 m/z. Data from chromatograms and mass spectra were ana- s cischinelli et al., 2008), cytotoxic effects against tumor cells (Ito- lyzed using the program ChemStation (Agilent )andcomparedwith kawa et al., 1990; Morita et al., 1991; Oberlies et al., 2002) and the literature (NIST and WILEY 275 L libraries) and data obtained from gastric antiulcer activities (Basile et al., 1990; Sertié et al., 2000). authentic standards. Despite the widespread use of C. sylvestris, controlled clinical studies have not been conducted and there are paucity of data 2.3. Animals related to the evaluation of the toxicity of this plant. Therefore, the aim this study was to evaluate the possible toxic effects of the fluid Eight three Male and female Wistar rats, 60 days years old, bred extract of C. sylvestris (FE) on preclinical studies, in Wistar male in the Department of Pathology at the School of Veterinary Med- rats, as acute oral toxicity and repeated dose 28 and 90 day oral icine and Animal Sciences, University of São Paulo, were used. The toxicity based on the “Guide to studies of pre-clinical toxicity of rats were randomly assigned to the control and treatment groups, herbal medicines” edited by ANVISA (2004). individually housed, maintained under controlled temperature conditions (22–25 °C), relative humidity (50–65%) and lighting (12 h/12 h light/dark cycle). Before the test was conducted, the 2. Material and methods animals were kept in their cages five days to allow for acclimati- zation to the laboratory conditions. Drinking water and standard s 2.1. Plant material diet (Nuvilab-CR1 , Nuvital Nutrientes LTDA) were provided ad libitum. All procedures were performed following the Guide for the Fresh leaves of C. sylvestris Sw (Salicaceae) were collected at the Care and Use of Laboratory Animals, NIH publication n°85–23 and University of São Paulo (Parque Esporte para Todos), São Paulo, SP, were reviewed and approved by the Bioethics Committee of the Brazil, in February 2009. A voucher herbarium specimen was de- FMVZ-USP (process n°1738/09). posited at the Botanic Institute of São Paulo/SP, under reference number 430190. 2.4. Acute toxicity study The leaves of C. sylvestris were dried at room temperature. Next, this material was powdered, and the hydroethanolic fluid extract The single-dose acute oral toxicity test was performed ac- extract (FE) was obtained by percolation, following Brazilian Offi- cording to OECD Guideline 423 (OECD, 2001). cial Pharmacopeia 1st edition instructions. The all material for the Three female Wistar rats were treated with a single dose of FE study was prepared in one batch lyophilized and kept frozen at (2000 mg/kg) administered by oral gavage. Next, the rats were 20 °C until use and was resuspended in distilled water at dif- closely observed during the first day, hourly, and once daily ferent doses immediately before administration. thereafter for a total of 14 days in order to identify signs of toxicity or death. The body weight and food consumption of each rat were 2.2. Phytochemicals analysis measured every 3 days throughout the observation period. At the end of this period, all survivors were killed to examine macro- 2.2.1. HPLC–PDA–ESIþ/MS analysis scopic alterations in their vital organs. The spectrometric analysis was performed using a Shimadzu© HPLC system consisting of a LC-10 pump and a SPD-M10A PDA 2.5. Subchronic 28 and 90 day toxicity studies detector coupled to an ESI/MS. All experiments were carried out in positive ion mode with a mass spectrometer Esquire 3000 Ion trap In the present study, the doses used were determinate from the (Bruker Daltonics©, Billerica, MA, USA). PDA signal was acquired lowest dose (60 mg/kg) with which the extract presented phar- scanning wavelengths between 190 e 800 nm while total ion macological activities (antiulcer activity) in rats (Basile et al., chromatogram (TIC) mode was employed for MS analysis. Analysis 1990), and from this dose, applied a factor of 2 and 4 times the of extracts by analytical HPLC was performed using a Varian Pur- therapeutic dose to established the medium and high doses. suit C18 column (150 4.6 mm i.d.; 5 Vm) maintained at 25 °C. For each experiment, forty male Wistar rats were randomly Solvent system: A: 0.1% acetic acid, B: acetonitrile (HPLC gradua- allocated into four groups (n¼10) and daily treated, for 28 or 90 tion). A gradient was performed as following: 0' [5]; 0'–15' [28]; days, by oral gavage, the following doses of FE: 0 mg/kg (Co), 15'–55' [68]; 55'–60' [100]; in a flow rate of 1 mL/min. Injection 60 mg/kg (FE 60), 120 mg/kg (FE 120) and 240 mg/kg (FE 240). volume of 20 VL of a sample concentration of 1 mg/mL in H2O. The animals were observed daily for clinical signs and mor- ESI–MS conditions: positive ion mode scan range: 50–1000 m/z; tality. Body weight and food consumption were measured weekly throughout the study period. At the end of treatment, all rats were Nebulizer gas (N2) 27 psi; dry gas (N2) 7 ml/min; dry temperature 32 °C and HV capillary 4000 V. anesthetized intraperitoneally (ip) with ketamine (50 mg/kg) and xylazine (5 mg/kg) to collect blood samples by cardiac puncture. 2.2.2. GC/MS/EI analysis Vacutainers containing EDTA and vacutainers that were dry were The C. sylvestris extract and authentic standards of rutin, quercetin used to process blood for complete blood count (CBC) and serum and chlorogenic acid (Sigma-Aldrichs) were derivatized with N,O-bis biochemistry, respectively. Next, liver, spleen and thymus were (trimethylsilyl)trifluoroacetamide and 1% trimethylchlorosilane (BSTFA/ harvested and weighed so that relative weight could be calculated A.Z. Ameni et al. / Journal of Ethnopharmacology 175 (2015) 9–13 11 and histopathology could be evaluated. In addition, one femur which data were similar of authentic standards. from each rat was collected, to verify bone marrow cellularity as These flavonoids have been reported to act in the gastro- well as from as well spleen. intestinal tract showing cytoprotective anti-ulcerogenic efficacy and antispasmodic, antidiarrheal, antioxidant and antiinflamatory 2.5.1. Complete blood count properties (Borrelli and Izzo, 2000; Di Carlo et al., 1999; Havsteen, The complete blood count was performed using an automated 2002; La Casa et al., 2000) suggesting they could be correlate with hematology analyzer Vet ABCTM Animal Blood Counter (Horiba, medicinal activity proposed to this plant. Furthermore, studies ABX Diagnostics). Hematological evaluations included red blood about C. sylvestris also showed the presence of phenolic com- cell count (RBC), hemoglobin concentration (HGB), hematocrit pounds, mainly flavonoids with rutin in constitution, which can be (HCT), mean corpuscular volume (MCV), mean corpuscular he- pointed as chemical markers (Silva et al., 2006). moglobin (MCH), mean corpuscular hemoglobin concentration Future studies will be conducted in order to verify if flavonoids (MCHC), red cell volume distribution (RDW), blood platelet count found in plant are the main responsible for the gastroprotective e/ (PLT) and white blood cell count (WBC). or anti-inflammatory effects and to standardization this extract.

2.5.2. Serum biochemistry 3.2. Acute toxicity study Blood samples collected without anticoagulant were allowed to stand for 45 min at room temperature before being centrifuged at The acute toxicity results, with the limit test single dose of 4000 rpm for 10 min. The serum from each sample was recovered 2000 mg/kg of the C. sylvestris extract, in the present study did not and placed at 20 °C until required. show any signs of toxicity. All rats treated with the FE survived on Creatinine, urea, uric acid, chloride, total proteins, albumin, the 14-day observation period. No adverse clinical signs were glucose, triglycerides, total cholesterol, aspartate aminotransferase observed. Body weight and food consumption measurements did (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) not reveal any difference, and no alterations were observed at and gamma glutamyl transferase (GGT) were evaluated using an necropsy. automatic chemistry analyzer CELM SBA-200 (Cia. Equipadora de Taking into account that the Globally Harmonized System of Laboratórios Modernos, S.A., Brasil). Classification and Labelling of Chemicals (GHS) assumes that substances with values higher than 2000 mg/kg are classified as 2.5.3. Histopathology having relatively low acute toxicity, we can consider C. sylvestris FE Histopathological analyses were performed on several organs: safe. Similarly, the low toxicity of C. sylvestris had already been liver, spleen, thymus and kidney. They were fixed in 10% neutral described by Basile et al. (1990), which showed that the LD 50 was formalin and routinely processed for paraffin embedding. Sections higher than 1840 mg/kg. (5 mm thick) were prepared and stained with hematoxylin and eosin (HE). 3.3. Subchronic 28 and 90 day toxicity studies

2.6. Statistical analysis C. sylvestris has been prescribed by SUS to treat gastrointestinal disorders; therefore, we established the initial dose for the sub- The data were analyzed using GraphPad Prism 4.00s software chronic studies based on the Basile et al. (1990) study that showed (GraphPad Software, Inc., San Diego, CA). Homogeneity of variance that the antiulcerogenic effective dose is 57.5 mg/kg. was tested with Barlett's test. If variance was homogeneous, the The studies with doses of 60, 120 and 240 mg/kg/day of fluid data were subjected to one-way analysis of variance (ANOVA) extract C. sylvestris for 28 and 90 days show that the treated rats followed by Dunnett's test for multiple comparisons, otherwise, presented no signs of behavioral changes or clinical toxic signs, as data were analyzed by the Kruskal–Wallis nonparametric ANOVA indicated by the lack of alterations in body weight (P40.05) and followed by Dunn's test. All data were expressed as the mean7SD, food consumption (P40.05) (Fig. 1), hematological (P40.05) and differences were considered to be statistically significant at (Table 1) relative organ weight (P40.05) (Table 2), and histo- po0.05. pathological parameters compared to control animals. On the other hand, some alterations were observed in the biochemical values in rats treated with 120 and 240 mg/kg C. 3. Results and discussion sylvestris extract, treated for 28 days as an increase in total pro- teins (Po0.01 post test Dunnet) and albumin (Po0.05 post test 3.1. Phytochemical analysis Dunn's) and decrease in triglyceride (Po0.01 post test Dunnet) creatinine (Po0.05 post test Dunn's) and chloride levels (Po0.01 Data analysis by HPLC-PDA-ESI/MSþ system of chromatogram post test Dunnet) (Table 3). Rats treated with 240 mg/kg C. syl- profile of C. sylvestris extract at 254 nm, which showed 18 chro- vestris extract for 90 days showed significant reductions in crea- matographic peaks with respective retention time (Rt). The total tinine (Po0.05 post test Dunn's) and total cholesterol (Po0.01 ion currents (TIC) of FA components and the positive ion ESI post test Dunnet) while animals treated with 120 mg/kg showed spectrum of rutin showed an abundant [Mþ H]þ ion peak at m/z significant reductions in total cholesterol and increases in chloride 611 (M¼610, C27H30O6), ion peak at m/z 303.0 corresponding to levels (Table 4). quercetin [M þ H]þ, the aglycone moiety of rutin and the peak at The result of decrease in triglyceride levels corroborates with a m/z 465 resulted from loss of rhamnose from rutin similar of study conducted by Werle et al. (2009) which showed that the standard at Rt¼26.1 min. The positive ion ESI spectrum of hydroalcoholic extract of C. sylvestris at 500 mg/kg lowers blood chlorogenic acid at Rt¼16.3 min was identified by mass spectrum triglyceride levels, and can indicate this plant as a candidate to a data with fragment ions m/z 355 [M þH]þ (M¼354, C16H18O9) natural trygliceride reducer. At the same way, the results suggest and m/z 163 (100%) [MC9H7O3]. The luteolin glucoside was that this extract could be a reductor of cholesterol and creatinine. identified at Rt¼33.7 min; m/z¼645, 639, 286 [luteolin aglycone However, futures experiments should be conducted to better M þH þ, M¼286, C15H10O6]. Data analysis by GC/MS/EI after de- evaluate this possibility. rivatization identified the quercetin at Rt¼49.646 min, m/z¼575 In the same manner, although biochemical analysis of serum (%) e 487 and rutin Rt¼49.486, m/z¼543, 207, 133(90%), 73(100 %) collected showed fluctuations of multiple parameters in those 12 A.Z. Ameni et al. / Journal of Ethnopharmacology 175 (2015) 9–13

500 Co Co FE 60 425 FE 120 FE 60 FE 120 400 FE 240 FE 240 375 400

350 Body weightBody (g)

Body weight (g) weight Body 325 300 1 5 9 13 17 21 25 29 1 2 3 4 5 6 7 8 9 10 11 12 Days weeks

40 30

30 20 20

10 10 Daily feed consumption (g) Daily feed consumption (g) 0 0 Co FE 60 FE 120 FE 240 Co FE 60 FE 120 EF 240

Fig. 1. Body weight and food consumption for the rats treated with C. sylvestris hydroethanolic extract: 0 (Co), 60 (FE 60), 120 (FE 120) and 240 (FE 240) mg/kg for 28 days (A and B) and for 90 days (C and D). No statistical differences were detected between the treated 60, 120 and 240 EF and control groups (n¼10) at every treatment.

Table 1 Table 2 Complete blood count of rats treated daily, for 28 and 90 days, with C. sylvestris Relative lymphoid organs weight of rats treated daily for 28 and 90 days with C. hydroethanolic extract: 0 (Co), 60 (FE 60), 120 (FE 120) and 240 (FE 240) mg/kg. sylvestris hydroethanolic extract: 0 (Co), 60 (FE60),120 (FE120) and 240 (FE240) mg/ kg (mean7SD, n¼10). 28 DAYS Co FE60 FE120 FE240 28 DAYS Lymphoid organs (g) Co FE60 FE120 FE 240 HCT (%) 41.0 73.1 40.872.7 40.571.4 41.7 73.0 HGB (g/dl) 14.470.7 14.570.6 14.370.6 14.970.5 Spleen 0.2170.022 0.2170.028 0.2170.03 0.2270.025 RBC (106/mm3) 8.370.6 8.270.5 8.370.4 8.570.5 Thymus 0.0870.021 0.0870.020 0.0970.01 0.0970.01 WBC (103/mm3) 5.971.0 5.671.0 5.670.9 5.171.0 90 DAYS PLT (103/mm3) 648.1756.9 649.0764.6 637.9765.0 612.1777.7 Lymphoid organs Co FE60 FE120 FE 240 MCV (fm3) 48.873.5 50.071.7 48.871.5 48.871.3 (g) MCH (pg) 17.4 70.8 17.870.8 17.270.5 17.470.7 MCHC (g/dl) 34.871.7 35.671.6 35.471.4 35.871.9 Spleen 0.1870.008 0.1970.020 0.1870.028 0.1970.020 90 DAYS Thymus 0.0670.008 0.0570.014 0.0570.009 0.0670.009 Co FE60 FE120 FE240

HCT (%) 36.772.9 36.871.6 37.5 73.7 36.476.3 7 7 7 7 HGB (g/dl) 13.9 0.9 14.1 0.5 14.1 1.2 14.0 2.0 In the same manner, no alterations were observed in relative RBC (106/mm3) 6.470.5 6.470.3 6.470.7 6.371.1 4 WBC (103/mm3) 3.871.3 4.071.2 3.970.9 4.070.8 organ weight, histology of the thymus and spleen (P 0.05), or in PLT (103/mm3) 617.4 787.5 638.8769.2 600.67223.5 565.47167.0 the cellularity of the spleen and bone marrow (P40.05) (Table 5), 7 7 7 7 MCV (fm3) 57.6 0.7 57.9 1.1 56.4 1.0 57.8 1.2 suggesting that this extract did not produce immunotoxic effects. MCH (pg) 21.9 70.5 22.170.6 21.371.2 22.471.5 MCHC (g/dl) 38.170.9 38.470.7 37.872.0 38.772.3

Data are presented as mean7SD (n¼10). HCT¼Hematocrit, HGB¼Hemoglobin, RBC¼Red blood cels, WBC¼White blood cels, PLT¼Platelet, MCV¼Mean corpus- 4. Conclusion cular volume, MCH¼Mean corpuscular hemoglobin, MCHC¼Mean corpuscular hemoglobin concentration. In summary, these data suggest that the hydroethanolic extract animals treated with C. sylvestris, these parameters remained of C. sylvestris could be safe even when used over a long period. within the normal ranges for Wistar rats (Bihun and Bauck, 2004; Moreover, this study is an important contribution to the rational Sharp and Regina, 1998). Furthermore, no clinical data were ob- use of medicinal plants on SUS. Future studies about pharmaco- served to substantiate these results. In addition, one must consider kinetics and pharmacodynamic and drug-interactions are sug- that animals treated with different doses of this plant showed no gested for better knowledge of therapeutic safe use. signs of systemic toxicity, neither was affected the general welfare, growth or normal development. A.Z. Ameni et al. / Journal of Ethnopharmacology 175 (2015) 9–13 13

Table 3 References Serum biochemical levels of rats treated daily, for 28 days, with C. sylvestris hy- droethanolic extract: 0 (Co), 60 (FE60), 120 (FE120) and 240 (FE240) mg/kg. Alexandre, R.F., Garcia, F.N., Simões, C.M.O., 2005. Evidence-based herbal medicine. Part 2. Phytopharmaceuticals elaborated with artichoke, horse chestnut, gin- Biochemistry Co FE60 FE120 FE 240 seng and passion flower. Acta Farm. Bonaer. 24, 310–314. parameteres ANVISA, 2004. Agência Nacional de Vigilância Sanitária. Ministério da Saúde. Agência Nacional de Vigilância Sanitária (Anvisa). Resolução - RE nº90/2004. AST (U/L) 205.0734.1 180.5722.9 183.3720.2 205.4740.7 Normas para estudos toxicológicos de produtos fitoterápicos Diário Oficial da ALT (U/L) 48.9275.4 49.9277.4 52.0179.7 64.22735.8 República Federativa do Brasil, Poder Executivo. Brasília, DF. 16 de março de. 7 7 7 7 GGT (U/L) 0.06 0.20 0.25 0.33 0.19 0.30 0.38 0.45 ANVISA, 2011. Agência Nacional de Vigilância Sanitária Formulário de Fitoterápicos 7 7 7 7 ALP (U/L) 180.1 36.7 186.4 25.0 216.9 66.1 190.3 46.5 da Farmacopéia. Brasil. 7 7 7 nn 7 nn Total protein (g/ 5.98 0.5 6.40 0.4 7.21 0.6 6.94 0.4 Basile, A.C., Sertie, J.A.A., Panizza, S., Oshiro, T.T., Azzolini, C.A., 1990. Pharmacolo- dl) gical assay of Casearia sylvestris. I: Preventive anti-ulcer activity and toxicity of 7 7 7 n 7 Albumin (g/dl) 4.71 0.38 4.78 0.15 5.21 0.29 4.78 0.21 the leaf crude extract. J. Ethnopharmacol. 30, 185–197. Glucose (mg/dl) 124.8712.2 131.1712.8 129.879.8 133.2711.8 Bihun, C., Bauck, L., 2004. Basic anatomy, physiology, husbandry, and clinical Urea (mg/dl) 50.7073.1 50.3075.0 51.1075.1 50.2075.8 techniques. In: Carpenter, K.E.Q.W. (Ed.), Ferrets, Rabbits, and Rodents, Second n n Creatinin (mg/dl) 0.4970.04 0.4970.04 0.3970.04 0.4070.03 ed. W.B. Saunders, Saint Louis, pp. 286–298 (Chapter 28). Uric Acid (mg/dl) 1.5470.37 1.8271.25 1.2970.43 1.5670.38 Borrelli, F., Izzo, A.A., 2000. The plant kingdom as a source of anti-ulcer remedies. Cholesterol (mg/ 37.5472.7 39.2574.4 36.417 5.5 36.7474.8 Phytother. Res. 14, 581–591. dl) Carvalho, A.C.B., Dos Santos, L.A., Silveira, D., 2009. Regulation of plants and herbal Triglycerides (mg/ 192.8762.0 185.7738.2 127.3745.6nn 124.5723.0nn medicines in Brazil. Blacpma. Boletín Latinoamericano y del Caribe de Plantas dl) Medicinales y Aromáticas 8, 7–11. Chloride (meq/L) 128.278.8 124.277.3 111.0 79.0nn 98.60711.2 nn Carvalho, A.C.B., Perfeito, J.P.S., e Silva, L.V.C., Ramalho, L.S., de Oliveira Marques, R. F., Silveira, D., 2011. Regulation of herbal medicines in Brazil: advances and Data are presented as mean7SD (n¼10). ALP, alkaline phosphatase; AST, aspartate perspectives. Bras. J. Pharm. Sci. 47, 467–474. aminotransferase; ALT, alanine aminotransferase. Cavalcante, W.L.G., Campos, T.O., Dal Pai-Silva, M., Pereira, P.S., Oliveira, C.Z., Soares, n A.M., Gallacci, M., 2007. Neutralization of snake venom phospholipase A2 po0.05 post test Dunn. nn toxins by aqueous extract of Casearia sylvestris () in mouse neu- po0.01 post test Dunnett. romuscular preparation. J. Ethnopharmacol. 112, 490–497. Cintra-Francischinelli, M., Silva, M.G., Andréo-Filho, N., Cintra, A.C.O., Leite, G.B., Table 4 Cruz-Höfling, M.A., Rodrigues-Simioni, L., Oshima-Franco, Y., 2008. Effects of Serum biochemical levels of rats treated daily for 90 days, with C. sylvestris hy- commonly used solubilizing agents on a model nerve-muscle synapse. Lat. Am. droethanolic extract (FE): 0 (Co), 60 (FE60), 120 (FE120) and 240 (FE240) mg/kg. J. Pharm. 27, 721–726. Da Silva, S.L., Chaar, J.D.S., Damico, D.C.S., Figueiredo, P.D.M.S., Yano, T., 2008. An- Biochemistry Co FE60 FE120 FE 240 timicrobial activity of ethanol extract from leaves of Casearia sylvestris. Pharm. parameteres Biol. 46, 347–351. Di Carlo, G., Mascolo, N., Izzo, A.A., Capasso, F., 1999. Flavonoids: old and new as- AST (U/L) 115.2751.2 121.7728.2 108.0721.7 116.5723.9 pects of a class of natural therapeutic drugs. Life Sci. 65, 337–353. fi fl ALT (U/L) 55.5721.3 45.376.3 42.974.1 49.378.9 Havsteen, B.H., 2002. The biochemistry and medical signi cance of the avonoids. – GGT (U/L) 0.770.82 0.470.52 0.570.53 0.270.42 Pharmacol. Ther. 96, 67 202. ALP (U/L) 87.7714.4 83.278.4 99.5726.1 89.8711.9 Itokawa, H., Totsuka, N., Morita, H., Takeya, K., Iitaka, Y., Schenkel, E.P., Motidome, Total Protein (g/ 7.9770.52 8.0670.40 7.9170.22 8.0470.18 M., 1990. New antitumor principles, casearins A-F, for Casearia sylvestris Sw. – dl) (Flacourtiaceae). Chem. Pharm. Bull. 38, 3384 3388. La Casa, C., Villegas, I., Alarcón De La Lastra, C., Motilva, V., Martín Calero, M.J., 2000. Albumin (g/dl) 5.3470.54 5.8070.83 5.5070.70 5.2670.38 Evidence for protective and antioxidant properties of rutin, a natural flavone, Glucose (mg/dl) 242.5720.5 224.4721.5 229.8711.2 241.6714.2 against ethanol induced gastric lesions. J. Ethnopharmacol. 71, 45–53. Urea (mg/dl) 77.778.1 76.077.9 79.177.8 77.0 76.0 Morita, H., Nakayama, M., Kojima, H., Takeya, K., Itokawa, H., Schenkel, E.P., Moti- Creatinin (mg/dl) 1.2970.21 1.2170.09 1.1970.14 1.1470.04n dome, M., 1991. Structures and cytotoxic activity relationship of casearins, new Uric acid (mg/dl) 1.8770.63 1.8870.54 2.1070.98 2.0570.53 clerodane diterpenes from Casearia sylvestris Sw. Chem. Pharm. Bull. 39, Cholesterol (mg/ 44.6374.1 44.1474.8 33.7573.9nn 35.6773.0nn 693–697. dl) Oberlies, N.H., Burgess, J.P., Navarro, H.A., Pinos, R.E., Fairchild, C.R., Peterson, R.W., 7 7 7 7 Triglycerides (mg/ 241.6 52.3 255.9 110.4 265.7 80.2 245.1 49.9 Soejarto, D.D., Farnsworth, N.R., Kinghorn, A.D., Wani, M.C., Wall, M.E., 2002. dl) Novel bioactive clerodane diterpenoids from the leaves and twigs of Casearia 7 7 7 nn 7 Chloride (meq/L) 142.0 16.0 143.6 13.2 166.6 21.1 154.2 14.2 sylvestris. J. Nat. Prod. 65, 95–99. Ruppelt, B.M., Pereira, E.F., Gonçalves, L.C., Pereira, N.A., 1991. Pharmacological 7 ¼ Data are presented as mean SD (n 10). ALP, alkaline phosphatase; AST, aspartate screening of plants recommended by folk medicine as anti-snake venom – I. aminotransferase; ALT, alanine aminotransferase. Analgesic and anti-inflammatory activities. Memorias do Instituto Oswaldo n Po0.05 post test Dunn. Cruz 86 (Suppl 2), 203–205. nn Po0.01 post test Dunnett. OECD, 2001. Organization for economic cooperation and development. Guidelines for the Testing of Chemicals: Acute Oral Toxicity—Acute Toxic Class Method. OECD Guideline. Paris. 423. Table 5 Sertié, J.A.A., Carvalho, J.C.T., Panizza, S., 2000. Antiulcer activity of the crude extract Cellularity of spleen and bone marrow of rats treated daily, for 28 and 90 days with from the leaves of Casearia sylvestris. Pharm. Biol. 38, 112–119. C. sylvestris hydroethanolic extract: 0 (Co), 60 (FE60), 120 (FE120) and 240 (FE240) Sharp, P.E., Regina, M.C.L., 1998. Laboratory Rat Patrick E. Sharp, Marie C. La Regina. 7 ¼ mg/kg (mean SD, n 10). CRC Press, 240 pp, ISBN 9780849325656. Silva, M.A.Sd, Ming, L.C., Pereira, A.M.S., Bertoni, B.W., Batistini, A.P., Pereira, P.S., 28 DAYS 2006. Phytochemical and genetic variability of Casearia sylvestris Sw. from Sao Cellularity Co FE60 FE120 FE 240 Paulo State Atlantic forest and Cerrado populations. Revista Brasileira de Plantas Medicinais 8, 159–166. 7 Spleen (cellsx10 ) 38.0719.1 34.9717.0 32.3716.3 44.5731.9 Werle, A.L.B., Zanetti, G.D., Ceron, C.S., Melânia, P., 2009. Evaluation of the activity of 8 Bone marrow (cellsx10 ) 16.1 712.4 15.6712.9 11.977.8 12.277.4 Casearia sylvestris Swartz (Flacourtiaceae) on rats serum triglyceride. Avaliação 90 DAYS da atividade de Casearia sylvestris Swartz (Flacourtiaceae) sobre os níveis Cellularity Co FE60 FE120 FE 240 séricos de triglicerídeos em ratos 19, 400–402.

Spleen (cellsx107) 3.672.3 3.672.1 2.671.4 3.072.6 Bone marrow (cellsx108) 2.171.9 2.071.14 2.370.9 1.871.0

Acknowledgment

We are thankful to CAPES (1406395) for the financial support.