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Atividade antibacteriana da espécie africana Cleistochlamys Kirkii Oliv : isolamento e caracterização de constituintes bioativos
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O trabalho científico apresentado nesta dissertação teve como principal objetivo contribuir para a validação do uso da espécie Cleistochlamys kirkii (Annonaceae) no tratamento de doenças infeciosas, na medicina tradicional de Moçambique.
Para tal, recolheu-se a parte da planta usada pelas populações – casca da raiz – e procedeu-se ao estudo fitoquímico bioguiado do extrato metanólico. Da fração do extrato metanólico solúvel em diclorometano (a que exibiu maior atividade antibacteriana) foram isolados e caracterizados dez compostos, dos quais uma nova flavanona C-benzilada, denominada cleistina (2.4), à qual foi atribuída a estrutura 5,7-di-hidroxi-6-(4’’’-hidroxibenzil)-8-(2’’-hidroxibenzil)-(2S)-fenil-croman-4-ona. Ainda do grupo das flavanonas, foram também isoladas três estruturas C-benziladas [a chamanetina (2.1), a isochamanetina (2.2) e a dichamanetina (2.3)]. De realçar o isolamento pela primeira vez no reino Plantae, de um alcaloide indólico com o núcleo da dioxopiperazina, a echinulina (2.5). Isolou-se ainda uma acetogenina, a cis-solamina (2.6), duas lactonas α,β-insaturadas [o cleistenolido (2.7) e o acetilmelodorinol (2.8)], um triterpeno tetracíclico, o policarpol (2.9) e um composto fenólico denominado benzofenona (2.10). As estruturas dos compostos isolados foram caracterizadas através dos seus dados espetroscópicos (IV, MS e RMN uni- e bidimensional).
A avaliação da atividade antibacteriana dos extratos/frações foi realizada pelo método da microdiluição em meio líquido, em estirpes de Staphylococcus aureus sensíveis (ATCC 6538) e resistentes (ATCC 700699, ATCC 43866 e “wild-type” MRSA, CIP 106760 MRSA/VISA) e numa estirpe de Enterococcus faecalis resistente à vancomicina (ATCC 51299, VRE). Para avaliar a atividade antibacteriana dos compostos isolados, procedeu-se à determinação da concentração mínima inibitória (CMI) e da concentração mínima bactericida (CMB) sobre um painel alargado de bactérias sensíveis e resistentes aos antibióticos, que incluiu, além das estirpes referidas, o Bacillus subtilis ATCC 6633 (Gram-positivo) e estirpes Gram-negativas, Salmonella typhymurium ATCC 13311 e Pseudomonas aeruginosa ATCC 9027.
O composto que exibiu maior atividade foi a dichamanetina (2.3) que se apresentou como um potente agente antibacteriano sobre um painel alargado de estirpes de Staphylococcus aureus sensíveis (CMI 2 μg mL-1, CMB 4 μg mL-1) e resistentes à meticilina, incluindo uma estirpe “wild-type” (CMI 1- 4 μg mL-1, CMB 4-8 μg mL-1) e com sensibilidade intermédia à vancomicina (CMI 2 μg mL-1, CMB 4 μg mL-1). Este composto foi ainda fortemente ativo quer para a estirpe de Enterococcus faecalis resistente à vancomicina (CMI 8 μg mL-1) quer para o Bacillus subtilis (CMI 4 μg mL-1). De realçar que os valores de CMI apresentados por este composto em estirpes de Staphylococcus aureus resistentes à meticilina foram da mesma ordem de grandeza dos valores do antibiótico de referência vancomicina (CMI 2 μg mL-1). Outro composto com atividade antibacteriana relevante foi o cleistenolido (2.7), que apresentou valores de CMI entre 8-15 μg mL-1 contra estirpes de Staphylococcus aureus MRSA (ATCC 43300 e “wild type”) e de 31 μg mL-1 quer para as restantes estirpes ensaiadas desta espécie quer para o E. faecalis e o B. subtilis.
De acordo com os resultados obtidos, os compostos mais ativos poderão constituir protótipos promissores para o desenvolvimento de novos antibióticos contra estirpes resistentes.
The main aim of this work was to contribute to the scientific validation of the traditional use of the specie Cleistochlamys kirkii (Annonaceae) on the treatment of infectious ailments in Mozambique. To achieve this goal, the plant part used by native populations was collected (root bark) and a bioassay-guided phytochemical study was performed on the methanolic extract. From the dichloromethane soluble fraction of the methanol extract (that displayed major antibacterial activity) ten pure compounds were isolated and characterized, namely a new C-benzylated flavanone, named cleistine [5,7-di-hydroxy-6-(4’’’-hydroxybenzyl)-8-(2’’-hydroxybenzyl)-(2S)-phenyl-chroman-4-one] (2.4). Three other known C-benzylated flavanones were also isolated and identified as chamanetin (2.1), isochamanetin (2.2) and dichamanetin (2.3). A dioxopiperazine alkaloid [echinulin (2.5)] was isolated for the first time in the Plantae kingdom. An acetogenin, cis-solamin (2.6), two α,β-unsaturated lactones [cleistenolide (2.7) and acetylmelodorinol (2.8)], a tetracyclic triterpene, polycarpol (2.9) and a phenolic compound identified as benzophenone (2.10) were also isolated. Compounds structures were established by spetroscopic analysis (IR, MS and one- and two dimensional NMR). The evaluation of the antibacterial activity of the extracts/fractions was carried out by using the microdilution method on sensitive (ATCC 6538) and resistant (ATCC 700699, ATCC 43866 and "wild-type" MRSA, CIP 106760, MRSA/VISA) Staphylococcus aureus strains. A vancomycin-resistant Enterococcus faecalis (ATCC 51299, VRE) strain was also used. Both the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were determined on the evaluation of the antibacterial activity of the pure compounds (2.1 - 2.10). Besides the above referred strains, the bacteria panel in these experiments also included a Gram-positive bacillus (Bacillus subtilis ATCC 6633) and two Gram-negative species (Salmonella typhymurium ATCC 13311 and Pseudomonas aeruginosa ATCC 9027). The most active compound was dichamanetin (2.3), displaying broad activity spectra against all the Staphylococcus aureus strains tested (sensitive strain MIC 2 μg mL-1, MBC 4 μg mL-1; methicillin-resistant strain "wild-type" MIC 1 - 4 μg mL-1, MBC 4-8 μg mL-1; vancomicym intermediate strain MIC 2 μg mL-1, MBC 4 μg mL-1). Dichamanetin also showed high activity against the vancomycin-resistant Enterococcus faecalis (MIC 8 μg mL-1) and Bacillus subtilis (MIC 4 μg mL-1) strains. Dichamanetin (2.3) MIC values displayed against Staphylococcus aureus strains were similar to those found for vancomycin (MIC 2 μg mL-1) used as the reference antibiotic. Moreover, cleistenolide (2.7) has also shown a relevant antibacterial activity with MIC values between 8 - 15 μg mL-1 against MRSA strains (ATCC 43300 and "wild type") and 31 μg mL-1 for E. faecalis and B.subtilis. Based on the above results, the most active compounds might be promising hit/leads for the development of new antibiotics against resistant bacterial strains.
The main aim of this work was to contribute to the scientific validation of the traditional use of the specie Cleistochlamys kirkii (Annonaceae) on the treatment of infectious ailments in Mozambique. To achieve this goal, the plant part used by native populations was collected (root bark) and a bioassay-guided phytochemical study was performed on the methanolic extract. From the dichloromethane soluble fraction of the methanol extract (that displayed major antibacterial activity) ten pure compounds were isolated and characterized, namely a new C-benzylated flavanone, named cleistine [5,7-di-hydroxy-6-(4’’’-hydroxybenzyl)-8-(2’’-hydroxybenzyl)-(2S)-phenyl-chroman-4-one] (2.4). Three other known C-benzylated flavanones were also isolated and identified as chamanetin (2.1), isochamanetin (2.2) and dichamanetin (2.3). A dioxopiperazine alkaloid [echinulin (2.5)] was isolated for the first time in the Plantae kingdom. An acetogenin, cis-solamin (2.6), two α,β-unsaturated lactones [cleistenolide (2.7) and acetylmelodorinol (2.8)], a tetracyclic triterpene, polycarpol (2.9) and a phenolic compound identified as benzophenone (2.10) were also isolated. Compounds structures were established by spetroscopic analysis (IR, MS and one- and two dimensional NMR). The evaluation of the antibacterial activity of the extracts/fractions was carried out by using the microdilution method on sensitive (ATCC 6538) and resistant (ATCC 700699, ATCC 43866 and "wild-type" MRSA, CIP 106760, MRSA/VISA) Staphylococcus aureus strains. A vancomycin-resistant Enterococcus faecalis (ATCC 51299, VRE) strain was also used. Both the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were determined on the evaluation of the antibacterial activity of the pure compounds (2.1 - 2.10). Besides the above referred strains, the bacteria panel in these experiments also included a Gram-positive bacillus (Bacillus subtilis ATCC 6633) and two Gram-negative species (Salmonella typhymurium ATCC 13311 and Pseudomonas aeruginosa ATCC 9027). The most active compound was dichamanetin (2.3), displaying broad activity spectra against all the Staphylococcus aureus strains tested (sensitive strain MIC 2 μg mL-1, MBC 4 μg mL-1; methicillin-resistant strain "wild-type" MIC 1 - 4 μg mL-1, MBC 4-8 μg mL-1; vancomicym intermediate strain MIC 2 μg mL-1, MBC 4 μg mL-1). Dichamanetin also showed high activity against the vancomycin-resistant Enterococcus faecalis (MIC 8 μg mL-1) and Bacillus subtilis (MIC 4 μg mL-1) strains. Dichamanetin (2.3) MIC values displayed against Staphylococcus aureus strains were similar to those found for vancomycin (MIC 2 μg mL-1) used as the reference antibiotic. Moreover, cleistenolide (2.7) has also shown a relevant antibacterial activity with MIC values between 8 - 15 μg mL-1 against MRSA strains (ATCC 43300 and "wild type") and 31 μg mL-1 for E. faecalis and B.subtilis. Based on the above results, the most active compounds might be promising hit/leads for the development of new antibiotics against resistant bacterial strains.
Descrição
Tese de mestrado, Química Farmacêutica e Terapêutica, Universidade de Lisboa, Faculdade de Farmácia, 2013
Palavras-chave
Annonaceae Cleistochlamys kirkii Fitoquímica Atividade antibacteriana Teses de mestrado - 2013