Abstract: FITOPROT, which contains curcuminoids and Bidens pilosa L. extract, is an innovative mucoadhesive formulation indicated for the topical treatment of chemoradiotherapy-induced oral mucositis (OM) in patients with advanced and visible oral squamous cell carcinoma. The formulation is used as a mouthwash directly on tumor tissue of patients with advanced neoplasms, without triggering cancer cell proliferation or tumor invasiveness. Thus, the aim of this study was to evaluate the biological effects of FITOPROT on an oral squamous cell carcinoma cell line (SCC-4). The viability of SCC-4 cells was assessed after exposure to FITOPROT using MTT reduction assay. The effects of the mucoadhesive formulation on cell cycle progression and cell death parameters were evaluated using flow cytometry. In addition, the inflammatory profile of the tumor cells was evaluated using the cytometric bead array (CBA) assay. FITOPROT promoted a concentration-dependent decrease in cell viability and cell cycle arrest at the G2/M phase (p < 0.05). Mitochondrial membrane potential was also altered after exposure to the formulation (p < 0.05), in parallel with a reduction in VEGF and IL-8 production (p = 0.01 and p = 0.05, respectively). In summary, the results indicate that FITOPROT reduces SCC-4 cell viability, promotes cell cycle arrest, modulates mitochondrial membrane potential, and exhibits antiangiogenic and anti-inflammatory properties, thus indicating its potential for topical use in patients with OM and visible tumors in the mouth.

This study evaluated the safety of Lafoensia pacari A. St.-Hil. (Lythraceae) stem bark hydroalcoholic extract (LPE) through acute and subchronic toxicological assessments in mice. In the acute toxicity evaluation, a single 2000 mg/kg oral dose of LPE was administered to mice and clinical observations were conducted for 14 days. For subchronic toxicity, LPE doses (6.25-1000 mg/kg) were administered orally for 28 days and biochemical, hematological, histopathological analyses and renal and liver expression of Ki-67 were carried out. The acute oral toxicity evaluation of LPE showed no toxicity in mice and it was was classified as category 5 (LD50>2000-5000 mg/kg). In a repeated dose 28-day toxicity study, LPE (100-1000 mg/kg) led to an increase in reticulocytes, which suggests a possible proliferative effect on blood cells. In addition, LPE (400-1000 mg/kg) of produced alterations in biochemical parameters, although no microscopic changes were found in the organs analyzed. A normal expression of the Ki-67 cell proliferation indicator was observed in the kidney and liver tissues, which suggests that LPE does not bring about changes in the proliferative activity of these organs. In conclusion, LPE should be used with caution, particularly in larger doses over longer periods and also in combination with other medication.

ABSTRACT This study evaluated the antifungal activity and cytotoxicity profile of the ellagitannin punicalagin, a compound extracted from the L. pacari A. St.-Hil (Lythraceae) leaf, against Cryptococcus neoformans species complex. Minimum inhibitory concentrations (MIC) were checked using the broth microdilution method. Minimum fungicidal concentrations (MFC) and time of death were used to confirm the antifungal activity of the compound. The in vitro cytotoxicity of punicalagin was tested in BALB/c3T3 fibroblasts and A549 human lung cancer cell line, while the hemolytic potential was tested on sheep erythrocytes. The morphological changes induced in yeast strains by the presence of punicalagin were also analyzed. Tested on eight isolates of the C. neoformans complex punicalagin showed MIC of 0.5 to 4.0 μg/mL and MFC> 256 μg/mL. Punicalagin also demonstrated a good growth inhibitory activity in time-kill curves, but it was not able to achieve a statistically significant reduction of fungal growth suggesting a fungistatic effect of the compound. In vitro cytotoxicity studies using the two cell lines showed that punicalagin has low activity on these cells and no activity on sheep erythrocytes. Morphological changes were seen in the yeasts strains studied when treated with punicalagin. Therefore, punicalagin is a potential antifungal for important pathogenic yeasts and presents a low cytotoxicity profile associated with no hemolytic effects.

Abstract In this study, the potential antileukemic activity of grandisin, a lignan extracted from Piper solmsianum, was evaluated against the leukemic line K562. The cytotoxicity of grandisin (0.018 to 2.365 µM) was evaluated in K562 and normal peripheral blood lymphocytes by Trypan Blue Exclusion and MTT methods after 48h exposure to the drug. In both methods, cellular viability was concentration-dependent and the IC50 values were lower than 0.85µM. Analysis of K562 cells after treatment with grandisin showed that the cell cycle was arrested in the G1 phase with a 12.31% increase, while both S and G2 phases decreased. Morphological studies conducted after the exposure of K562 to grandisin revealed changes consistent with the apoptosis process, which was confirmed by anexin V stain and caspase activation. Thus, lignan grandisin showed antileukemic activities against the K562 cell line and the cell death process occurred via apoptosis.

Este estudo investigou os efeitos quimioprotetores do extrato alcoólico dos frutos da Punica granatum L. (Punicaceae) (EPG) em camundongos expostos ao cromo hexavalente [Cr(VI)]. Os animais foram pré-tratados com o EPG (25, 50 ou 75 mg/kg/dia) durante 10 dias e subsequentemente expostos a uma dose subletal de Cr(VI) (30 mg/kg). A frequência de eritrócitos policromáticos micronucleados na medula óssea foi investigada e os níveis de Cr(VI) foram quantificados nos rins, fígado e plasma. Para a análise de sobrevida, os camundongos foram previamente tratados com EPG durante 10 dias e expostos a única dose letal de Cr(VI) (50 mg/kg). A exposição à dose subletal de Cr(VI) induziu aumento significativo na frequência de células micronucleadas. Entretanto, o tratamento profilático com EPG levou à redução de 44,5% (25 mg/kg), 86,3% (50 mg/kg) e 64,2% (75 mg/kg) na incidência de micronúcleo. Além disso, a dose de 50 mg/kg de EPG produziu maior efeito quimioprotetor, uma vez que a taxa de sobrevivência foi de 90%, quando comparada àquela do grupo não tratado. Nesses animais, quantidades reduzidas de cromo foram detectadas nos materiais biológicos, em comparação com os outros grupos. Em conjunto, os resultados demonstram que o EPG exerce efeito protetor contra a genotoxicidade induzida pelo Cr(VI).

This study investigated the chemoprotective effects of Punica granatum L. (Punicaceae) fruits alcoholic extract (PGE) on mice exposed to hexavalent chromium [Cr(VI)]. Animals were pretreated with PGE (25, 50 or 75 mg/kg/day) for 10 days and subsequently exposed to a sub-lethal dose of Cr(VI) (30 mg/kg). The frequency of micronucleated polychromatic erythrocytes in the bone marrow was investigated and the Cr(VI) levels were measured in the kidneys, liver and plasm. For the survival analysis, mice were previously treated with PGE for 10 days and exposed to a single lethal dose of Cr(VI) (50 mg/kg). Exposure to a sub-lethal dose of Cr(VI) induced a significant increase in the frequency of micronucleated cells. However, the prophylactic treatment with PGE led to a reduction of 44.5% (25 mg/kg), 86.3% (50 mg/kg) and 64.2% (75 mg/kg) in the incidence of micronuclei. In addition, the 50 mg/kg dose of PGE produced a higher chemoprotective effect, since the survival rate was 90%, when compared to that of the non-treated group. In these animals, reduced amounts of chromium were detected in the biological materials, in comparison with the other groups. Taken together, the results demonstrated that PGE exerts a protective effect against Cr(VI)-induced genotoxicity.

Se não for tratado de forma adequada e eficientemente, os resíduos produzidos pela indústria química podem contaminar o meio-ambiente. Utilizar fungos capazes de degradar compostos orgânicos (por exemplo, fenol), pode ser um método para tratar efluentes proeminentes de insústrias farmacêuticas, em particular, os fungos da podridão branca. O objetivo deste trabalho foi tratar um efluente farmacêutico pelo fungo Pycnoporus sanguineus. Três amostras de efluentes foram coletadas em uma indústria farmacêutica. A produção de enzimas como a lacase e manganês peroxidase foi determinado. A sua produção durante o tratamento com o fungo P. sanguineus atingiu concentrações máximas de 4,48 U.mL-1 (Efluente 1), 8,16 U.mL-1 (Efluente 2), 2,8 U.mL-1 (Efluente 3) e 0,03 Abs.min-1 (efluente 2), respectivamente, durante 96 horas de tratamento biológico. Efeitos genotóxicos dos efluentes brutos e tratados também foram investigados usando o ensaio em que foi avaliada a formação de micronúcleos (MN) na medula óssea de camundongos. Os resultados mostraram que o tratamento biológico reduziu a frequência de MN, de um modo dependente da dose, em comparação com amostras não tratadas. A diminuição de cerca de 20% e 45% da concentração de compostos fenólicos foi observada em todo o tratamento, o que confirma que a produção de lacase pode ser relacionada com a degradação de compostos tóxicos presentes no efluente. Por conseguinte, a biodegradação do fungo P. sanguineus parece um método promissor para a mineralização de compostos recalcitrantes presentes em efluentes farmacêuticos.

If not properly and efficiently treated, wastes produced by the chemical industry can contaminate the environment. Using fungi able to degrade organic compounds (e.g. phenol) seems to be a prominent method to treat pharmaceutical wastewaters, in particular, the white-rot fungus. The aim of this work was to treat pharmaceutical effluent by the Pycnoporus sanguineus fungus. Three effluent samples were collected in a pharmaceutical industry. The production of enzymes such as laccase and manganese peroxidase was determined. Their production increased throughout the treatment with the P. sanguineus fungus, reaching maximum concentration of 4.48 U.mL-1 (Effluent 1), 8.16 U.mL-1 (Effluent 2), 2.8 U.mL-1 (Effluent 3) and 0.03 Abs.min-1 (Effluent 2), respectively, during 96 hours of biological treatment. Genotoxic effects of the raw and treated effluents were also investigated using the in vivo mouse bone marrow micronucleus (MN) assay. Results showed the biological treatment reduced the frequency of MN, in a dose-dependent manner, when compared to untreated sample. The decreasing of around 20% and 45% of phenolics concentration was observed throughout the treatment, confirming that laccase production can be related to the degradation of toxic compounds present in the effluent. Therefore, the biodegradation by the P. sanguineus fungus seems a promising method for the mineralization of recalcitrant compounds present in pharmaceutical effluents.

A leucemia mielóide crônica (LMC) é uma doença mieloproliferativa clonal, que apresenta resistência à apoptose. A introdução do mesilato de imatinibe revolucionou o tratamento da LMC, porém a resistência ao imatinibe pode ser desenvolvida em qualquer tempo e, inevitavelmente, leva à progressão da doença. Synadenium umbellatum Pax. pertence à família Euphorbiaceae e é usado popularmente no Brasil para o tratamento do câncer. A citotoxicidade das Euphorbiaceae está associada com a capacidade dessas plantas e seus compostos bioativos em induzir apoptose em células tumorais. Portanto, este trabalho teve como objetivo investigar a citotoxicidade e os mecanismos de morte induzidos por S. umbellatum em células leucêmicas. A citotoxicidade de S. umbellatum foi avaliada pelo ensaio de exclusão do azul de tripano e a análise do ciclo celular foi feita por citometria de fluxo. Os mecanismos envolvidos na morte celular das células K-562 foram investigados por microscopia óptica e por citometria de fluxo. Os resultados demonstraram que S. umbellatum é citotóxico para células leucêmicas de uma maneira dependente da concentração. A análise morfológica revelou que o tratamento com S. umbellatum induziu as célula K-562 à morte por via apoptótica. Além disso, os dados indicam aumento de ERO S, alterações no potencial de membrana mitocondrial, externalização da fosfatidilserina e ativação de caspase 9. Em conjunto, os resultados demonstram que S. umbellatum promoveu retenção do ciclo celular das células K-562 e induziu estas células à morte por apoptose.

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disease that shows apoptosis resistance. The introduction of imatinib mesylate has revolutionized the treatment of CML, but imatinib resistance may develop at any time and inevitably leads to disease progression. Synadenium umbellatum Pax. belongs to the Euphorbiaceae family and is popularly used in Brazil for the treatment of cancer. The cytotoxicity of Euphorbiaceae is associated with the ability of these plants and their bioactive compounds to induce apoptotic tumor cell death. Therefore, we aimed to investigate the cytotoxicity and the mechanisms of death induced by S. umbellatum extract in leukemic cells. S. umbellatum cytotoxicity was evaluated by trypan blue exclusion assay and flow cytometric analysis of the cell cycle; the mechanisms involved in K-562 cell death were investigated by light microscopy and flow cytometry. The results demonstrate that S. umbellatum is cytotoxic to leukemic cells in a concentration-dependent manner. Morphological analysis revealed that S. umbellatum treatment induced K-562 cell death by an apoptotic pathway. Furthermore, data indicate ROS overproduction, alterations in mitochondrial membrane potential, phosphatidylserine externalization and activation of caspase 9. Taken together, the results demonstrate that S. umbellatum extract arrested the cell cycle and triggered apoptosis at several levels in K-562 cells.

In this study, Annona coriacea Mart., Annonaceae, was examined for possible toxic effects on brain, liver and kidney of mice exposed to crude extract of the seeds (CESAN) of this plant. CESAN was administered by gavage for four days at doses of 12.5, 25, 50, and 100 mg/kg/day. Significant changes on liver were observed, which showed reduction in the number of hepatocytes per area and increase the apoptotic index in the exposed groups, and changes in the cytoplasm and nucleus of these cells and reduced consumption of water and feed in these animals. For the other studied areas, brain and kidneys showed no changes in the parameters used in this study. The results suggest hepatotoxic effects of CESAN, but without damage to brain and kidneys in this experiment, showing a toxic potential to this species, as to the Annonaceae family.

No presente estudo investigamos o potencial do extrato etanólico das folhas da Punica granatum (PGFO) e do extrato etanólico dos frutos da Punica granatum (PGFR) de induzir mutagenicidade ou de proteger contra efeitos genotóxicos induzidos pela ciclofosfamida (CF). Camundongos machos Swiss foram tratados por 10 dias, via oral, com PGFO ou PGFR (12,5, 25, 50 e 75 mg/kg/dia), previamente a exposição à CF (i.p. 200 mg/kg) 24 horas após término do tratamento. Observamos que os animais tratados por 10 dias com ambos os extratos (12,5, 25, 50 e 75 mg/kg/dia) demonstraram a frequência de micronúcleo policromático eritrocitário (MNPCE) similar ao grupo controle. Quando aos efeitos protetores dos extratos foram investigados, a análise dos dados revelou que, independentemente da dose ou do extrato usado, a administração oral por 10 dias, previamente à exposição, reduziu, de forma dose-dependente, a frequência de MNPCE induzidos pela CF, em todos os grupos estudados. As maiores reduções foram observadas com PGFR nas doses de 50 e 75 mg/kg. Em conjunto, sob as condições testadas, camundongos tratados com P. granatum demonstraram ausência de efeitos mutagênicos e, de forma dose-dependente, efeitos protetores contra os danos oxidativos do DNA induzidos pela CF.

In the present study, the ability of Punica granatum ethanolic leaf extract (PGL) and Punica granatum ethanolic fruit extract (PGF) to induce mutagenicity or to modulate the genotoxic effects induced by the alkylating agent cyclophosphamide (CP) was evaluated. Swiss male mice were treated by gavage for 10 days with PGL or PGF (12.5, 25, 50, and 75 mg/kg/day) prior to exposure to CP (i.p. 200 mg/kg), 24 h after the end of the treatment. Initial observations revealed that normal mice treated with both extracts (12.5, 25, 50, and 75 mg/kg/day) showed a similar micronucleated polychromatic erythrocyte (MNPCE) frequency to that of the control group. Investigation of the protective effect of PGL and PGF based on data analysis revealed that, irrespective of dose or extract, oral administration of PGL or PGF for 10 days prior to exposure had reduced, in a dose-dependent manner, the frequency of MNPCE induced by CP in all groups studied. Higher reductions were observed at PGF doses of 50 and 75 mg/kg. Taken together, these results demonstrate that mice treated with P. granatum showed an absence of mutagenic effects and dose-dependent protective effects against CP-induced oxidative DNA damage.

O Synadenium umbellatum Pax. (Euphorbiacea) é uma planta nativa da África tropical conhecida como "cola-nota", "avelós", "cancerola", "milagrosa", dentre outros. A planta é utilizada pela população brasileira como detentora de propriedades antiinflamatória, analgésica, dentre outras. Foram avaliados os efeitos depressores sobre o sistema nervoso central (SNC) do extrato etanólico das folhas de Synadenium umbellatum (EES) e de suas frações - hexânica (FH), clorofórmica (FC) e metanol/água (FM). Vários testes foram utilizados em camundongos machos albinos (Mus musculus), dentre eles, o sono induzido por barbitúrico, campo aberto e o teste do rota-rod. O EES foi testado nas doses de 25, 50 e 100 mg/kg, enquanto que a FH foi testada na dose de 10 mg/kg, a FC na dose de 20 mg/kg e a FM na dose de 25 mg/kg. O EES e as frações FH e FC, mas não a FM, apresentaram um possível efeito depressor sobre o SNC, visto que foram capazes de aumentar o tempo parado e diminuir o número de bolos fecais no campo aberto, além de potencializarem o sono induzido por barbitúrico. No teste do rota-rod, observou-se que o EES e as frações não foram capazes de causar incoordenação motora ou relaxamento muscular. Assim, conclui-se que o extrato etanólico e as frações FH e FC do Syandenium umbellatum Pax. possuem possível efeito depressor sobre o SNC.

Synadenium umbellatum Pax. (Euphorbiacea) is a native plant from tropical Africa known as "cola-nota", "avelós", "cancerola", "milagrosa", among others. The plant is used by Brazilian folks for having anti-inflammatory and analgesic properties, among others. It was evaluated the depressor effects over the central nervous system (CNS) of the ethanolic extract of the leaves of Synadenium umbellatum (EES) and its fractions - hexane (HF), chloroformic (CF) and methanol/water fractions(MF). Several tests were used in Swiss mice (Mus musculus), among them, barbiturate-induced sleep, open field and rota-rod test. The EES was tested at 25, 50 and 100 mg/kg doses, while the HF was tested at 10 mg/kg dose, the CF at 20 mg/kg dose and the MF at 25 mg/kg dose. The EES and the HF and the CF fractions, but not the MF, have presented a possible depressor effect over the CNS once they were able to increase the stop time and decrease the number of fecal boli on the open field, besides they were able to potencialize the barbiturate-induced sleep. The rota-rod test showed that the EES and the fractions were not able to cause motor incoordination or muscle relax. Therefore, we conclude that Synadenium umbellatum Pax. have a possible depressor effect over the CNS.

No presente estudo investigamos o potencial citotóxico e mutagênico, in vitro e in vivo, respectivamente, do extrato etanólico de Synadenium umbellatum (EESU) sobre células da medula óssea de camundongos. A citotoxicidade in vitro foi avaliada por meio da exposição de células da medula óssea de animais normais a diferentes concentrações (40-0,312 mg/mL) do EESU, por 12, 24 ou 48 h, utilizando os testes de redução do MTT e o de exclusão do azul de tripano. O ensaio de micronúcleo foi realizado para investigar potenciais efeitos mutagênicos do EESU (10, 25 ou 50 mg/kg/dia) sobre a medula óssea de camundongos. Os animais foram expostos a uma única dose, por via oral, e 24 h após à exposição, sacrificados para realização do estudo (n=5/grupo). Os resultados obtidos demonstraram que o EESU possui potencial efeito citotóxico e mutagênico, de forma dose-dependente, sobre as células da medula óssea de camundongos, in vitro e in vivo, respectivamente. Maiores estudos são necessários para expandir o conhecimento acerca do potencial toxicológico/farmacológico do EESU.

In the present study we investigated, in vitro and in vivo, the cytotoxic and mutagenic potential of the Synadenium umbellatum ethanolic extract (SUEE) on the bone marrow cells of mice. In vitro cytotoxicity was assayed incubating bone marrow cells with different concentrations (40-0.312 mg/mL) of SUEE for 12, 24 or 48 h using the MTT tetrazolium reduction test and the trypan blue exclusion test. The micronuclei assay was performed to determine mutagenic effects of mice orally exposed to different doses of SUEE (10, 25 or 50 mg/kg/day) in 24 h. Our results demonstrated that SUEE has, in a dose-dependent manner, potential cytotoxic and mutagenic effects on the bone marrow cells of mice. Further studies are needed to expand the knowledge of the toxicological/pharmacological potential of the SUEE.