title: Evaluation of toxicity of Calophyllum brasiliense stem bark extract by in vivo and in vitro assays.
abstract: BACKGROUND
  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.
  
  
  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=119.94±4.31 µg/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 8.39±2.00 µg/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 24h. 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.
authors.affiliation: 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.
authors.forename: Mariana Canevari
authors.initials: MC
authors.lastname: Oliveira
authors.affiliation: 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.
authors.forename: Larissa Maria Scalon
authors.initials: LM
authors.lastname: Lemos
authors.affiliation: 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.
authors.forename: Ruberlei Godinho
authors.initials: RG
authors.lastname: de Oliveira
authors.affiliation: 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.
authors.forename: Evandro Luiz
authors.initials: EL
authors.lastname: Dall'Oglio
authors.affiliation: 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.
authors.forename: Paulo Teixeira
authors.initials: PT
authors.lastname: de Sousa Júnior
authors.affiliation: 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. Electronic address: taba@terra.com.br.
authors.forename: Domingos Tabajara
authors.initials: DT
authors.lastname: de Oliveira Martins
mesh_terms: D000818:Animals; D001938:Brazil; D016466:CHO Cells; D029759:Calophyllum / Q000737:chemistry*; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D005260:Female; D006586:Hexanes / Q000737:chemistry; D006801:Humans; D020128:Inhibitory Concentration 50; D008297:Male; D008519:Medicine, Traditional / Q000009:adverse effects / Q000379:methods; D051379:Mice; D024301:Plant Bark; D010936:Plant Extracts / Q000008:administration & dosage / Q000633:toxicity*; D051381:Rats; D017208:Rats, Wistar; D012997:Solvents / Q000737:chemistry; D013045:Species Specificity; D018675:Toxicity Tests / Q000379:methods*
chemical_list: D006586:Hexanes; D010936:Plant Extracts; D012997:Solvents; C026385:n-hexane
keywords: Calophyllum brasiliense; Chromanones; in vitro toxicity; in vivo toxicity