Avaliação das propriedades cicatrizantes do extrato de Garcinia brasiliensis e modulação da proteína Anexina A1 em modelo de lesão cutânea
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Data
2022-03-04
Autores
Souza, Helena Ribeiro
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Universidade Estadual Paulista (Unesp)
Resumo
O uso de plantas medicinais remonta à antiguidade. Com o advento da ciência moderna, vários compostos químicos dessas plantas foram identificados e suas aplicações e eficácias comprovadas. No entanto, existem muitas plantas medicinais usadas por populares em diversas partes do mundo ainda pouco investigadas. Dentre essas espécies, encontra-se a Garcinia brasiliensis, planta de porte arbóreo nativa da América do Sul, conhecida por suas propriedades anti-inflamatórias e antitumorais e estudada em algumas condições clínicas. Nesse trabalho, o objetivo foi identificar alguns compostos do metabolismo secundário da G. brasiliensis, testar a atividade antioxidante e citotoxicidade de soluções extrativas da planta, bem como, avaliar essas soluções em lesão cutânea ainda pouco explorada, em modelos in vivo e in vitro. Para isso, folhas de espécime da G. brasiliensis foram coletadas, secadas e trituradas para serem infundidas em etanol 70%. Após retirada do etanol dessa solução extrativa foi obtido o Extrato Puro (EP), do qual, por meio de fracionamento líquido-líquido, foram produzidas as Frações Acetato de Etila (FAE) e Hexânica (FH). EP, FAE e FH foram pesquisados para verificação da presença de terpenos, compostos fenólicos e alcaloides por meio de testes colorimétricos e cromatográficos (HPTLC), enquanto, a atividade antioxidante foi avaliada por meio da descoloração do radical livre DPPH. A citotoxicidade das amostras em diferentes concentrações foi analisada em teste de hemólise em solução glicosilada (5%) de sangue humano (4%) e em teste in vivo/in ovo da membrana corioalantoide de ovos de Gallus gallus fertilizados. Os resultados dessas análises indicaram a presença de compostos de interesse farmacológico no EP e FAE e baixa citotoxidade nessas amostras, que foram submetidas a teste de suas propriedades microbicidas contra Staphylococcus aureus. Embora somente EP tenha apresentado atividade microbicida em concentração maior que 50%, a presença de compostos metabolicamente ativos, alta capacidade antioxidante e baixa citotoxicidade da FAE foram importantes para seleção dessas duas soluções extrativas para o modelo de lesão cutânea. Desse modo, EP e FAE foram incorporadas em formulações, que após testes de estabilidade, foram testadas em feridas de 5 mm no dorso de ratos Wistar, infectadas ou não com S. aureus (25 µL 105). Após o tratamento tópico de 6 dias, EP e FAE demonstraram controlar o processo de formação de prurido, modularam a expressão da proteína Anexina A1, relacionada ao processo inflamatório, e reduziram a quantidade de células imunomarcadas para Gasdermina-D, relacionada ao processo de morte celular piroptótica, e aumentaram a expressão de MCP-1. Em conjunto, os dados mostram importante perfil anti-inflamatório bem como potencial terapêutico do EP e da FAE no processo de regeneração cutânea, inclusive em lesões infectadas.
The use of medicinal plants dates back to antiquity. With the advent of modern science, several chemical compounds from these plants have been identified and their applications and effectiveness proven. However, there are many medicinal plants used by people in different parts of the world that are still poorly investigated. Among these species is Garcinia brasiliensis, a tree-sized plant native to South America, known for its anti-inflammatory and antitumor properties and studied under some clinical conditions. In this work, the objective was to identify some compounds from the secondary metabolism of G. brasiliensis, test the antioxidant activity and cytotoxicity of extractive solutions of the plant, as well as evaluate these solutions under clinical conditions still little explored, in models in vivo and in vitro. For this purpose, specimen leaves of G. brasiliensis were collected, dried and ground to be infused in 70% ethanol. After removing the ethanol from this extractive solution, the Pure Extract (PE) was obtained, from which, by means of liquid-liquid fractionation, the Ethyl Acetate (EAF) and Hexanic (HF) fractions were produced. PE, EAF and HF were investigated to verify the presence of terpenes, phenolic compounds and alkaloids by means of colorimetric and chromatographic tests (HPTLC), while the antioxidant activity was evaluated by means of DPPH free radical decolorization. The cytotoxicity of samples at different concentrations was analyzed in a hemolysis test in glycosylated solution (5%) of human blood (4%) and in an in vivo/in ovo test of the chorioallantoic membrane of fertilized eggs of Gallus gallus. The results of these analyzes indicated the presence of compounds of pharmacological interest in PE and EAF and also low cytotoxicity in these samples, which were tested for their microbicidal properties against Staphylococcus aureus. Although only PE showed microbicidal activity at a concentration greater than 50%, the presence of metabolically active compounds, high antioxidant capacity and low cytotoxicity of EAF were important for selecting these two extractive solutions for the skin lesion model. Thus, PE and EAF were incorporated into formulations, which, after stability tests, were tested on 5 mm wounds on the back of Wistar rats, infected or not with S. aureus. After the 6-day topical treatment, PE and EAF demonstrated to control the pruritus formation process, modulated the expression of the Annexin A1 protein, related to the inflammatory process, and reduced the amount of immunomarked cells for Gasdermin-D, related to the death process pyroptotic cell, and increse the expression of MCP-1. Together, the data show an important anti-inflammatory profile and therapeutic potential of PE and EAF in the process of skin regeneration, including in infected lesions.
The use of medicinal plants dates back to antiquity. With the advent of modern science, several chemical compounds from these plants have been identified and their applications and effectiveness proven. However, there are many medicinal plants used by people in different parts of the world that are still poorly investigated. Among these species is Garcinia brasiliensis, a tree-sized plant native to South America, known for its anti-inflammatory and antitumor properties and studied under some clinical conditions. In this work, the objective was to identify some compounds from the secondary metabolism of G. brasiliensis, test the antioxidant activity and cytotoxicity of extractive solutions of the plant, as well as evaluate these solutions under clinical conditions still little explored, in models in vivo and in vitro. For this purpose, specimen leaves of G. brasiliensis were collected, dried and ground to be infused in 70% ethanol. After removing the ethanol from this extractive solution, the Pure Extract (PE) was obtained, from which, by means of liquid-liquid fractionation, the Ethyl Acetate (EAF) and Hexanic (HF) fractions were produced. PE, EAF and HF were investigated to verify the presence of terpenes, phenolic compounds and alkaloids by means of colorimetric and chromatographic tests (HPTLC), while the antioxidant activity was evaluated by means of DPPH free radical decolorization. The cytotoxicity of samples at different concentrations was analyzed in a hemolysis test in glycosylated solution (5%) of human blood (4%) and in an in vivo/in ovo test of the chorioallantoic membrane of fertilized eggs of Gallus gallus. The results of these analyzes indicated the presence of compounds of pharmacological interest in PE and EAF and also low cytotoxicity in these samples, which were tested for their microbicidal properties against Staphylococcus aureus. Although only PE showed microbicidal activity at a concentration greater than 50%, the presence of metabolically active compounds, high antioxidant capacity and low cytotoxicity of EAF were important for selecting these two extractive solutions for the skin lesion model. Thus, PE and EAF were incorporated into formulations, which, after stability tests, were tested on 5 mm wounds on the back of Wistar rats, infected or not with S. aureus. After the 6-day topical treatment, PE and EAF demonstrated to control the pruritus formation process, modulated the expression of the Annexin A1 protein, related to the inflammatory process, and reduced the amount of immunomarked cells for Gasdermin-D, related to the death process pyroptotic cell, and increse the expression of MCP-1. Together, the data show an important anti-inflammatory profile and therapeutic potential of PE and EAF in the process of skin regeneration, including in infected lesions.
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Palavras-chave
Bacupari, Compostos fenólicos, Inflamação, Cicatrização cutânea, Piroptose, Gasdermina-D, Phenolic compounds, Inflammation, Wound healing, Pyroptosis, Gasdermin-D