5.4 AVALIAÇÃO DA TOXICIDADE in vitro E in vivo
5.4.2 Avaliação da Toxicidade Aguda
A toxicidade aguda foi avaliada na dose de 2000 mg/kg por via oral em camundongos fêmeas como preconizado pela OECD (2001). Os animais tratados por via oral com os EH das folhas e das cascas do caule de C. leptophloeos na dose de 2000 mg/kg não apresentaram alterações comportamentais, neurológicas, morbidade e mortalidade durante o período de observação (14 dias). O peso corporal e dos órgãos (coração, baço, fígado, rins e cérebro) não foram estatisticamente diferentes entre o grupo teste e o grupo controle (grupo salina) (Tabela 18).
As análises bioquímicas não mostraram alterações nos níveis séricos das enzimas hepáticas (ALT e AST), que são consideradas biomarcadores e indicam danos hepáticos quando os níveis séricos de AST e ALT estão alterados (RAMAIAH, 2011). Também, não foi observado alterações dos níveis de creatinina sérica que é um biomarcador das funções renais e quando aumentados indicam sinais de toxicidade (KAMRAN et al, 2018). Assim, foi verificado que os EH das folhas e das cascas do caule não causaram nenhuma alteração indicativa de danos hepáticos ou renais. Outros parâmetros bioquímicos como proteína total, colesterol, glicose, ureia foram analisados e não apresentaram diferença estatística significante entre os grupos testes e os grupos controles (grupo salina) (Tabela 18).
Portanto, uma vez que não foi observado nenhum sinal clínico de toxicidade aguda, de acordo com a OECD 423, sugere-se que os extratos apresentam baixa toxicidade e possui um LD50 (dose letal) superior a 2000 mg/kg (OCDE, 2001).
Tabela 18 – Efeito dos EH das folhas e das cascas do caule de C. leptophloeos no peso corporal e peso corporal relativo em camundongos tratados por via oral.
Peso corporal (g) Grupo salina Extrato das folhas Extrato das cascas Peso inicial 33.670 ± 2.5220 34.200 ± 1.2080nd 32.020 ± 1.0130nd
Peso final 35.550 ± 2.2760 35.980 ± 1.9650nd 33.440 ± 1.3460nd
Peso corporal relativo (g/10g de peso corporal)
Coração 0.0196 ± 0.003050 0.0192 ± 0.002864nd 0.0190± 0.003240nd
Baço 0.0186 ± 0.002510 0.0172 ± 0.004438nd 0.0192 ± 0.003834nd
Rins 0.0376 ± 0.001517 0.0356 ± 3.962000nd 0.0368 ± 0.002490nd
Fígado 0.1394 ± 0.014150 0.1368 ± 0.006723nd 0.1374 ± 0.011630nd
Cérebro 0.0400 ± 0.002739 0.0392 ± 0.002387nd 0.0388 ± 0.004764nd
Legenda: Valores expressos como média ± desvio padrão (n = 5), grupos tratados com extratos comparados ao grupo tratado com solução salina. nd, indica não diferença estatística em comparação com o grupo de solução salina. Fonte: Resultados Experimentais.
Tabela 19 – Efeito dos EH das folhas e das cascas do caule de C. leptophloeos nos parâmetros bioquímicos em camundongos tratados por via oral.
Dosagens Grupo salina Extrato das folhas Extrato das cascas Albumina (g/dL) 2.040 ± 0.1008 1.994 ± 0.0991nd 2.058 ± 0.0598nd Proteína total (g/dL) 4.422 ± 0.2987 4.444 ± 0.2812nd 4.387 ± 0.2963nd Colesterol (mg/dL) 62.000 ± 9.0990 60.800 ± 9.9100nd 66.000 ± 8.809nd Glucose (mg/dL) 89.830 ± 3.5450 87.200 ± 8.1360nd 87.500 ± 9.854nd Ureia (mg/dL) 39.000 ± 7.2660 37.800 ± 3.9620nd 40.170 ± 5.345nd Creatinina (mg/dL) 0.236 ± 0.0196 0.268 ± 0.0408nd 0.256 ± 0.0467nd Alanina aminotransferase (U/L) 48.170 ± 4.1190 40.200 ± 5.2150* 43.170 ± 3.1890 nd Aspartato aminotransferase (U/L) 36.300 ± 0.3300 31.600 ± 2.0800 nd 34.200 ± 8.1340nd
Valores expressos como média ± desvio padrão (n = 5), grupos tratados com extratos comparados ao grupo tratado com solução salina. * p <0,05 comparou amostra com grupo salino estéril. nd, indica não diferença estatística em comparação com o grupo de solução salina estéril.
6 CONCLUSÕES
As análises fitoquímicas clássicas e modernas mostraram a diferença do perfil fitoquímico das folhas e cascas do caule, em que as folhas são ricas em flavonoides e as cascas do caule em taninos condensados.
Foi possível identificar 16 substâncias presentes no extrato das folhas e 8 substâncias no extrato das cascas por CL-MS e por FIA-ESI-IT-MS/MS;
A Cromatografia de Partição Centrífuga (CPC) se mostrou eficiente para o isolamento e purificação de flavonoides e procianidinas a partir do EH das folhas e fração BuOH das cascas do caule;
A técnica de CLAE-DAD-ELSD se mostrou satisfatória para análise qualitativa e quantitativa de flavonoides e procianidinas presentes nos extratos;
O extrato das folhas a 100 µg/mL e o extrato das cascas do caule nas concentrações de 1, 10, 100 e 200 µg/mL demonstraram efeito anti-inflamatório in vitro no ensaio de óxido nítrico induzido por LPS.
No modelo de edema de pata induzido por carragenina, o extrato das folhas nas doses de 200 e 400 mg/kg e das cascas do caule nas doses de 100, 200 e 400 mg/kg inibiu significativamente a atividade edematogênica seguido da redução da mieloperoxidase (MPO);
No modelo de bolsa de ar induzido por zimosam, os extratos das folhas e das cascas do caule nas doses de 100, 200 e 400 mg/kg, reduziram a migração celular, a concentração de proteínas totais, a mieloperoxidase (MPO) e os níveis de malondialdeído (MDA) e da citocina pró-inflamatória TNF-α e aumentou os níveis da citocina anti-inflamatória IL-10;
O extrato das folhas bem como sua fração BuOH possuem efeito bactericida contra as cepas clínicas e de referência de Bacillus cereus;
O extrato das cascas e as frações AcOET e BuOH nas concentrações de 20 a 100 µg/mL possuem efeito fungistático e bactericida frente as cepas clínicas e de referência de fungos do gênero candida e bactérias Gram-positivas;
O composto isolado procianidina dimérica do tipo B obtido do extrato das cascas do caule apresentou atividade antifúngica contra cepas clínicas e referência de Candida spp. e Trichosporon asahii;
O estudo de toxicidade mostrou que os extratos das folhas e das cascas do caule na dose 2000 mg/kg por via oral não apresentou toxicidade aguda;
A triagem realizada pelo estudo in silico para isovitexina e procianidina dimérica do tipo B indicou possíveis ligantes relacionados à atividade anti-inflamatória e antimicrobiana, respectivamente, corroborando com os resultados obtidos nos estudos in vivo e in vitro.
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