Evaluation of toxicity of Calophyllum brasiliense stem bark extract by in vivo and in vitro assays

Abstract

Ethnopharmacological relevanceCalophyllum 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 methodsIn 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 1000mg/kg. In the subchronic study, HECb was administered orally for 30 days to Wistar rats at doses of 100mg/kg and 500mg/kg. Phytochemical analyses were performed by HPLC/UV–vis, secondary metabolites were quantified by spectrophotometric methods. ResultsHECb presented IC50=119.94±4.31µg/mL after a 24h cytotoxicity test using CHO-k1 cells, showing low cytotoxicity. However, when the cells were exposed to HECb for 72h, the IC50 value was 8.39±2.00µg/mL, showing in this case, a pronounced cytotoxic effect. In the oral acute toxicity studies, doses up to 500mg/kg of HECb did not cause any changes in both male and female mice. At 1000mg/kg, male mice showed signs typical of depression and stimulation that were reversed at 72h. Besides, female mice were more sensitive to the toxic effect of HECb at 1000mg/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 500mg/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. ConclusionsIn vitro toxicological tests showed that HECb exhibited cytotoxicity especially after 72h 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.