Brasiliensic acid: in vitro cytotoxic and genotoxic, in vivo acute toxicity and in silico pharmacological prediction of a new promising molecule.

Brasiliensic acid (Bras) is a chromanone isolated from Calophyllum brasiliense Cambèss. bark extracts with confirmed potential activity on gastric ulcer and Helicobacter pylori infection. This study aimed to investigate the in vitro and in vivo toxicity of Bras and molecular docking studies on its interactions with the H. pylori virulence factors and selected gastric cancer-related proteins. Cytotoxicity was evaluated by alamarBlue© assay, genotoxicity by micronucleus and comet assays, and on cell cycle by flow cytometry, using Chinese hamster epithelial ovary cells. Bras was not cytotoxic to CHO-K1 cells, and caused no chromosomal aberrations, nor altered DNA integrity. Furthermore, Bras inhibited damages to DNA by H2O2 at 1.16 µM. No cell cycle arrest was observed, but apoptosis accounted for 31.2% of the cell death observed in the CHO-K1 at 24 h incubation of the IC50. Oral acute toxicity by Hippocratic screening test in mice showed no relevant behavioral change/mortality seen up to 1,000 mg/kg. The molecular docking approach indicated potential interactions between Bras and the various targets for peptic ulcer and gastric cancer, notably CagA virulence factor of H. pylori and VEGFR-2. In conclusion, Bras is apparently safe and an optimization for Bras can be considered for gastric ulcer and cancer.Communicated by Ramaswamy H. Sarma.

Diversity, geographical distribution, and prevalence of Entamoeba spp. in Brazil: a systematic review and meta-analysis.

The genus Entamoeba includes a variety of widely distributed species adapted to live in the digestive tracts of humans and a large variety of animals of different classes. The objective of this study was to investigate the prevalence, distribution, and molecular epidemiology of Entamoeba spp. in different classes of hosts in Brazil. Studies that analyzed hosts from several classes, including humans and domestic, wild, or captive animals, were considered. The pooled prevalence of Entamoeba spp. was calculated using the random-effects model. A total of 166 studies on humans and 16 on animals were included. The prevalence of Entamoeba spp. in the Brazilian population was 22% (95% CI: 21-24). The state with the highest prevalence was Paraiba with 72%, followed by Federal District with 53%, and Rondonia with 50%. In immunocompromized patients, the prevalence was 18%, and cancer (36%) was the most prevalent cause of immunosuppression. The prevalence of Entamoeba spp. in animal hosts was 12% (95% CI: 7-17). Captive wild animals and domestic farm animals showed the highest prevalence, with 16% and 15%, respectively. The species found more often were E. coli (86.5%), E. dispar (7.9%), and E. histolytica (3.1%). In conclusion, a high prevalence (22%) of Entamoeba spp. was found in the Brazilian population, with a prevalence of up to 50% mainly in the northern, northeastern, and central-western regions. The pathogenic species E. histolytica is distributed in most Brazilian regions, with significant prevalence percentages. Among animals, unidentified Entamoeba species were most prevalent in mammals.

TITLE: Diversité, répartition géographique et prévalence d'Entamoeba spp. au Brésil : revue systématique et méta-analyse. ABSTRACT: Le genre Entamoeba comprend une variété d'espèces largement distribuées, adaptées à vivre dans le tube digestif des humains et une grande variété d'animaux de différentes classes. L'objectif de cette étude était d'étudier la prévalence, la distribution et l'épidémiologie moléculaire d'Entamoeba spp. dans différentes classes d'hôtes au Brésil. Les études qui ont analysé les hôtes de plusieurs classes, y compris les humains et les animaux domestiques, sauvages ou captifs, ont été prises en compte. La prévalence combinée d'Entamoeba spp. a été calculée à l'aide du modèle à effets aléatoires. Au total, 166 études sur l'homme et 16 sur les animaux ont été incluses. La prévalence d'Entamoeba spp. dans la population brésilienne était de 22 % (IC à 95 % : 21­24). L'état avec la prévalence la plus élevée était Paraiba avec 72 %, suivi du District fédéral avec 53 % et Rondonia avec 50 %. Chez les patients immunodéprimés, la prévalence était de 18 % et le cancer (36 %) était la cause la plus fréquente d'immunosuppression. La prévalence d'Entamoeba spp. chez les hôtes animaux était de 12 % (IC à 95 % : 7­17). Les animaux sauvages en captivité et les animaux domestiques d'élevage ont affiché la prévalence la plus élevée, avec respectivement 16 % et 15 %. Les espèces trouvées le plus souvent étaient E. coli (86,5 %), E. dispar (7,9 %) et E. histolytica (3,1 %). En conclusion, une prévalence élevée (22 %) d'Entamoeba spp. a été trouvée dans la population brésilienne, allant jusqu'à 50 % principalement dans les régions du nord, du nord-est et du centre-ouest. L'espèce pathogène E. histolytica est répartie dans la plupart des régions du Brésil, avec des pourcentages de prévalence importants. Parmi les animaux, les espèces d'Entamoeba non identifiées étaient les plus répandues chez les mammifères.

Evaluation of genotoxicity and subchronic toxicity of the standardized leaves infusion extract of Copaifera malmei Harms in experimental models.

ETHNOPHARMACOLOGICAL RELEVANCE: Copaifera malmei Harms (Fabaceae), known mainly as óleo-mirim, is a native and endemic plant found in the states of Mato Grosso and Goiás of Brazil. The plant's leaves infusion is popularly used by riverine communities of the northern Araguaia microregion, Mato Grosso, Brazil, for the treatment of gastric ulcers and inflammatory diseases of the respiratory tract. The gastric antiulcer activity of the standardized leaves infusion extract of Copaifera malmei (SIECm) in rodents has been reported. The objective of this study was to advance the investigation of the safety profile of SIECm by evaluating the genotoxicity and subchronic toxicity using in vitro and in vivo experimental models. MATERIALS AND METHODS: SIECm was prepared by infusion, by incubating the powdered dried leaves material in boiled water for 15min. In vitro genotoxicity of SIECm (10, 30 or 100µg/mL) was assessed by micronucleus and comet tests using Chinese hamster ovary (CHO-k1) epithelial cells. The evaluation of subchronic toxicity profile was performed by daily oral administration of SIECm (100, 400 or 1000mg/kg) to Wistar rats for 30 days. Clinical observations of toxicological related parameters were done every 6 days. After the treatment period, blood was collected for hematological and biochemical analysis, and some organs were removed for macroscopic and histopathological analysis. RESULTS: In the micronucleus assay, SIECm demonstrated anti-mutagenic activity. In the comet assay, SIECm presented anti-genotoxic effect preventing DNA damage at all the three concentrations tested with pre-treatment, while the same effect was only observed in the co-treatment at the lowest concentration. Post-treatment with SIECm increased the genetic damage induced by hydrogen peroxide (H2O2) at the highest concentration. In the subchronic toxicity test, few changes were observed, such as increase in feed consumption in the group of animals treated with 100mg/kg of the SIECm, which reversed after 6 days. There were no macroscopic, histological and relative weights changes in the organs of animals treated with SIECm. No toxicologically relevant changes were observed in the hematological analysis. Subchronic administration of SIECm reduced levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in animals treated with 100mg/kg and serum triglyceride levels at 400 and 1000mg/kg. However, the hematological and biochemical changes observed are within the physiological ranges for this animal species. CONCLUSION: The results demonstrate that SIECm is not genotoxic, and does not present toxicity when used orally for up to 30 days. In addition, it showed protection to the genetic damage induced by H2O2. The SIECm therefore has a high safety margin for therapeutic use.

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

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=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.

Evaluation of antiulcer activity of chromanone fraction from Calophyllum brasiliesnse Camb.

ETHNOPHARMACOLOGICAL RELEVANCE: Calophyllum brasiliense Camb. (Clusiaceae), popularly known as 'guanandi', is found in the tropical areas and swampy lands. The latex exuding from its bark is used in the treatment of gastric ulcer in folk medicine. Several active compounds have been isolated from its stem bark among them, are the chromanone acids. Therefore, this study aimed to evaluate antiulcer activity and probable mechanism(s) of action of a fraction containing a mixture of chromanone acids (BI), derived by column chromatography fractionation of the hexane extract of the stem bark of Calophyllum brasiliense (HECb), using experimental in vitro and in vivo models. MATERIALS AND METHODS: Ulcer was induced by oral administration of ethanol (75%, v/v) and indomethacin (50mg/kg). Malondialdehyde, reduced glutathione and catalase activity was measured in stomach tissue after ethanol induced ulcer. In order to evaluate the effect of BI on nitric oxide, ulcer was induced by ethanol in l-NAME pretreated animals. Anti-Helicobacter pylori activity was verified in disk diffusion and broth microdilution in vitro assays, using cagA+ and vacA+ Helicobacter pylori strains. RESULTS: BI prevented the gastric ulceration caused by ethanol and indomethacin treatments. Its gastroprotective mechanism in ethanol-induced ulcer was partly due to reduction of MDA and CAT levels in the gastric tissue. BI did not affect the GSH levels and its gastroprotective effect was not reversed by pretreatment with l-NAME. BI showed anti-Helicobacter pylori in the both assays. CONCLUSION: The results indicate that BI is partly responsible for the HECb antiulcer and anti-Helicobacter pylori effects.