In silico, in vitro, and in vivo investigation of antioxidant potential and toxicity of ethyl ferulate.

By submitting this manuscript, each author certifies that they have made a direct and substantial contribution to the work reported in the manuscript. In this manuscript the conception, design, investigation, acquisition of data and analysis, interpretation of data and writing of the article were conducted by author Camila Bomfim de Sá under the guidance of professors Margareth de Fátima Formiga Melo Diniz, Hilzeth de Luna Freire Pessôa and Caliandra Maria Bezerra Luna Lima, who also approved the final version of the manuscript. Professor Damião Pergentino de Sousa and his student Mayara Castro de Morais performed the production, synthesis and chemical characterization of ethyl ferulate (EF). Professor Abrahão Alves de Oliveira Filho assessed the in silico tests. PhD student Andressa Brito Lira participated in the critical review of the text for important intellectual content and assisted in the in vitro antioxidant activity and cytotoxicity tests. Kardilandia Mendes de Oliveira participated in acute oral toxicity tests evaluating the biochemical parameters. Students, Tafaela Dias and Cinthia Rodrigues Melo also assisted in the acute oral toxicity testing and preparing of slides for histopathological analysis. Pathologist Alexandre Rolim da Paz analyzed the histopathology results. EF, a phenolic compound of the large class of phenylpropanoids, is derived from ferulic acid and is produced both naturally and synthetically. Its principal pharmacological activities are: anti-inflammatory and antioxidant activity. This study aimed to investigate the in silico, in vitro and in vivo toxicity and antioxidant activity of EF. The in silico prediction showed more than 20 biological activities as well as good absorption at the biological membranes and no theoretical toxicity. However, EF presented high environmental toxicity. EF presented low hemolytic potential and exerted protective activity for the erythrocyte membrane for only blood type O. EF presented antioxidant activity against H2O2 at all concentrations and all blood types, but no effect against phenylhydrazine, being unable to prevent its oxidative effects. In the acute nonclinical toxicological trial, the treated animals presented behavioral changes (e.g., sedation). Feed intake was higher for the 2000 mg/kg group, but with no significant difference in weight change. The biochemical parameters presented no differences between treated and control animals, and the organs remained intact with no change. Thus, EF presents a low toxic profile and this study provides important information about the toxicity of this compound, suggesting future safe use.

Effect of p-cymene and rosmarinic acid on gastric ulcer healing - Involvement of multiple endogenous curative mechanisms.

BACKGROUND: p-Cymene and rosmarinic acid are secondary metabolites found in several medicinal plants and spices. Previous studies have demonstrated their anti-inflammatory, antioxidant, and cytoprotective effects. PURPOSE: To evaluate their gastroduodenal antiulcer activity, gastric healing and toxicity in experimental models. METHODS: Preventive antiulcer effects were assessed using oral pre-treatment on HCl/ethanol-induced gastric lesions and cysteamine-induced duodenal lesions models. Gastric healing, the underlining mechanisms and toxicity after repeated doses were carried out using the acetic acid-induced gastric ulcer rat model and oral treatment for 14 days. RESULTS: In the HCl/ethanol-induced gastric ulcer and cysteamine-induced duodenal injury, p-cymene and rosmarinic acid (50-200 mg/kg) decreased significantly the ulcer area, and so prevented lesions formation. In the acetic acid-induced ulcer model, both compounds (200 mg/kg) markedly reduced the ulcerative injury. These effects were related to an increase in the levels of reduced glutathione (GSH) and interleukin (IL)-10, and due to a decrease in malondialdehyde (MDA), IL-1ß, tumor necrosis factor (TNF)-α, total and mitochondrial reactive oxygen species (ROS) levels. Downregulation of factor nuclear kappa B (NFκB) and enhanced expression of suppressor of cytokine signaling (SOCS)3 were also demonstrated. Furthermore, positive vascular endothelial growth factor (VEGF), metalloproteinase (MMP)-2, and cyclooxygenase (COX-2)-stained cells were increased in treated groups. Treatment also upregulated the platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor (TGF)-ß and epidermal growth factor receptor (EGFR) in gastric tissues. In isolated gastric epithelial cells this healing effect seems to be linked to a modulation of apoptosis, proliferation, survival and protein phosphorylation, such as the extracellular signal-regulated kinases (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK). Oral toxicity investigation for 14 days revealed no alterations in heart, liver, spleen, and kidneys weight nor the biochemical and hematological assessed parameters. p-Cymene and rosmarinic acid also protected animals from body weight loss maintaining feed and water intake. CONCLUSIONS: Data altogether suggest low toxicity, antiulcer and gastric healing activities of p-cymene and rosmarinic acid. Antioxidant and immunomodulatory properties seem to be involved in the curative effect as well as the induction of different factors linked to tissue repair.

Toxicological evaluation in silico and in vivo of secondary metabolites of Cissampelos sympodialis in Mus musculus mice following inhalation.

The ethanolic extract of the leaves of Cissampelos sympodialis showed great pharmacological potential, with inflammatory and immunomodulatory activities, however, it showed some toxicological effects. Therefore, this study aims to verify the toxicological potential of alkaloids of the genus Cissampelos through in silico methodologies, to develop a method in LC-MS/MS verifying the presence of alkaloids in the infusion and to evaluate the toxicity of the infusion of the leaves of C. sympodialis when inhaled by Swiss mice. Results in silico showed that alkaloid 93 presented high toxicological potential along with the products of its metabolism. LC-MS/MS results showed that the infusion of the leaves of this plant contained the alkaloids warifteine and methylwarifteine. Finally, the in vivo toxicological analysis of the C. sympodialis infusion showed results, both in biochemistry, organ weights and histological analysis, that the infusion of C. sympodialis leaves presents a low toxicity.

Non-clinical acute and chronic toxicity evaluations of Cissus sicyoides L. (Vitaceae) hydroalcoholic leaf extract.

Cissus sicyoides (Cs) has been traditionally used to treat diabetes and belongs to the family Vitaceae, and is known as "vegetable insulin". This study aimed to evaluate the acute and chronic non-clinical toxicity of hydroalcoholic extract from the leaves of Cissus sicyoides (EHA-Cs). The acute test was performed in Wistar rats, administering a single dose of 40.5 mg/kg. Behavioral parameters for pharmacological screening were observed to detect signs of Central Nervous System activity; consumption of daily food and water, and weight evaluation. After day 14, the animals were euthanized and blood samples were collected for laboratory analyses of hematological and biochemical parameters. The chronic tests were administered in doses of 4.5, 13.5 and 40.5 mg/kg. The same parameters were observed together with body temperature, glucose, exploration activity (test on the open field), and motor activity (diagnostic tests on the Rota-rod). For the group given the highest dosage during the study, histopathological examinations of vital organs were performed. For acute toxicity, there were no CNS level effects, changes in water and food consumption, or hematologic parameters. However, there was a significant decrease in weight gain for the treated females. Biochemical analyses of the treated animals presented increased levels of AST (aspartate aminotransferase) in females, uric acid levels in females and males, and amylase in males. In the chronic toxicity tests, water consumption was higher for females (at the dosages of 13.5 and 40.5 mg/kg) and for males (at 40.5 mg/kg). At the dosages of 4.5 and 13.5 mg/kg, feed consumption increased for females, while for males it decreased along with weight gain. Blood analysis presented an increase in albumin and changes in erythrocytes and hemoglobin for males (at the dose of 13.5 mg/kg). Glycemia in females (13.5 mg/kg dose) was significantly less, presenting only slight drops at the other doses. The changes were reversible in the satellite group. EHA-Cs revealed a relatively low toxicity profile (at the popular use dose), and only small changes in hematological and biochemical parameters at the dose of 13.5 mg/kg (3x the popular use dosage). In addition, EHA-Cs did not promote histological changes in vital organs such as the heart, lungs, liver and kidneys.

Isopropyl Caffeate: A Caffeic Acid Derivative-Antioxidant Potential and Toxicity.

Phenolic compounds, among them isopropyl caffeate, possess antioxidant potential, but not without toxicity and/or adverse effects. The present study aimed to evaluate the antioxidant activity and toxicity of isopropyl caffeate through in silico, in vitro and in vivo testing. The results showed that isopropyl caffeate presents no significant theoretical risk of toxicity, with likely moderate bioactivity: GPCR binding, ion channel modulation, nuclear receptor binding, and enzyme inhibition. Isopropyl caffeate induced hemolysis only at the concentrations of 500 and 1000 µg/ml. We observed types A and O erythrocyte protection from osmotic stress, no oxidation of erythrocytes, and even sequestrator and antioxidant behavior. However, moderate toxicity, according to the classification of GHS, was demonstrated through depressant effects on the central nervous system, though there was no influence on water and food consumption or on weight gain, and it did present possible hepatoprotection. We conclude that the effects induced by isopropyl caffeate are due to its antioxidant activity, capable of preventing production of free radicals and oxidative stress, a promising molecule with pharmacological potential.